US20170335289A1 - Differentiation of Human Embryonic Stem Cells - Google Patents

Differentiation of Human Embryonic Stem Cells Download PDF

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US20170335289A1
US20170335289A1 US15/660,823 US201715660823A US2017335289A1 US 20170335289 A1 US20170335289 A1 US 20170335289A1 US 201715660823 A US201715660823 A US 201715660823A US 2017335289 A1 US2017335289 A1 US 2017335289A1
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Alireza Rezania
Jean Xu
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Janssen Biotech Inc
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LifeScan Inc
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Priority to US16/598,920 priority patent/US11708562B2/en
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention provides methods to promote the differentiation of pluripotent stem cells.
  • the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells.
  • the present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.
  • ⁇ cells insulin-producing cells
  • ⁇ cells appropriate for engraftment.
  • One approach is the generation of functional ⁇ cells from pluripotent stem cells, such as, for example, embryonic stem cells.
  • a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation.
  • Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage.
  • the intermediate stage in this process is the formation of definitive endoderm.
  • Definitive endoderm cells express a number of markers, such as, HNF-3beta, GATA4, Mixl1, CXCR4 and Sox-17.
  • pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm.
  • Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, Pdx1.
  • Pdx1 expression marks a critical step in pancreatic organogenesis.
  • the mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.
  • islet cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse.
  • Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets.
  • Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.
  • Hori et al. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled ⁇ cells.
  • Blyszczuk et al. reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4.
  • retinoic acid can regulate the commitment of embryonic stem cells to form Pdx1 positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdx1 expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).
  • Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdx1. Their results show that exogenous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, P48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).
  • mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.
  • D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (D'Amour K A et al. 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into Pdx1 positive cells after addition of FGF-10 (US 2005/0266554A1).
  • D'Amour et al. states “We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones”.
  • hES human embryonic stem
  • Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1).
  • the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of n-butyrate and activin A. The cells were then cultured with TGF ⁇ antagonists such as Noggin in combination with EGF or betacellulin to generate Pdx1 positive cells. The terminal differentiation was induced by nicotinamide.
  • Benvenistry et al. states: “We conclude that over-expression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo” (Benvenistry et al, Stem Cells 2006; 24:1923-1930).
  • the present invention provides a method for differentiating pluripotent stem cells, comprising the steps of:
  • cells expressing markers characteristic of the definitive endoderm lineage are differentiated from pluripotent stem cells by treating pluripotent stem cells by any one of the following methods:
  • cells expressing markers characteristic of the pancreatic endoderm lineage are differentiated from cells expressing markers characteristic of the definitive endoderm lineage by treating cells expressing markers characteristic of the definitive endoderm lineage by any one of the following methods:
  • cells expressing markers characteristic of the pancreatic endocrine lineage are differentiated from cells expressing markers characteristic of the pancreatic endoderm lineage by treating cells expressing markers characteristic of the pancreatic endoderm lineage by any one of the following methods:
  • the present invention provides a method for treating a patient suffering from diabetes, comprising the steps of:
  • FIG. 1A shows the expression of the definitive endoderm markers CXCR4, GATA4, HNF-3beta, Mixl1, Sox-17 in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for two, five and eight days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIG. 1B shows the expression of the anterior endoderm markers Cerberus, Otx-1 and Hex genes in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for three and five days.
  • FIGS. 2A, 2B and 2C shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for five days. Expression of the definitive endoderm markers was detected by immunohistochemistry.
  • FIG. 2A shows Sox-17 expression.
  • FIG. 2B shows HNF-3beta expression.
  • FIG. 2C shows Oct3/4 expression.
  • FIGS. 3A, 3B, 3C and 3D show the expression of definitive endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIG. 3A shows GATA4 expression.
  • FIG. 3B shows Sox-17 expression.
  • FIG. 3C shows HNF-3beta expression.
  • FIG. 3D shows Mixl1 expression.
  • Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 4A and 4B show the expression of extra-embryonic endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the extraembryonic endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIG. 4A shows the effect of 100 ng/ml activin A on AFP expression.
  • FIG. 4B shows the effect of 100 ng/ml activin A on Sox7 expression.
  • Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 5A and 5B show the expression of mesoderm and ectoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the mesoderm and ectoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIG. 5A shows the effect of 100 ng/ml activin A on Brachyury expression.
  • FIG. 5B shows the effect of 100 ng/ml activin A on Zic1 expression.
  • Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 6A, 6B, 6C, 6D, 6E and 6F show the expression of the definitive endoderm markers Brachyury ( FIG. 6A ) CXCR4 ( FIG. 6B ), Mixl1 ( FIG. 6C ), Sox17 ( FIG. 6D ), HNF-3beta ( FIG. 6E ), Oct4 ( FIG. 6F ) in the human embryonic stem cell line H7 following treatment with 100 ng/ml activin A for one, three, five and seven days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIGS. 7A, 7B, 7C, 7D, 7E and 7F shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the definitive endoderm markers was detected by immunohistochemistry.
  • FIGS. 7A and 7B show Sox-17 expression.
  • FIGS. 7C and 7D show HNF-3beta expression.
  • FIGS. 7E and 7F show GATA4 expression.
  • FIGS. 7B, 7D and 7F show counter staining of the nuclei with DAPI.
  • the columns marked ‘treated’ denote activin A treatment (100 ng/ml) for five days.
  • the columns marked ‘untreated’ denote untreated controls.
  • FIGS. 8A, 8B and 8C shows the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells.
  • FIG. 8A shows Pdx1 expression.
  • FIG. 8B shows GLUT-2 expression.
  • FIG. 8C shows PTF1a expression.
  • FIGS. 9A and 9B show the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was detected by immunohistochemistry.
  • FIG. 9A shows Pdx1 expression in the untreated control
  • FIG. 9B shows Pdx1 expression in the culture treated by the step-wise differentiation protocol.
  • FIGS. 10A, 10B, 10C and 10D show the expression of pancreatic endocrine markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the pancreatic endocrine markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells.
  • FIG. 10A shows NeuroD1 expression.
  • FIG. 10B shows Ngn3 expression.
  • FIG. 10C shows insulin expression.
  • FIG. 10 D shows Hes-1 expression, the expression level is normalized to pancreatic endoderm cells.
  • FIGS. 11A and 11B show the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells.
  • FIG. 11A shows Nkx2.2 expression.
  • FIG. 11B shows Pdx1 expression.
  • FIG. 12 shows the expression of PDX-1 in cells with each passage (P0, P1 and P2) in culture. Expression of the PDX-1 was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells H9.
  • FIGS. 13A and 13B show the expression of hepatocyte markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the hepatocyte markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells.
  • FIG. 13A shows AFP expression.
  • FIG. 13B shows albumin expression.
  • FIGS. 14A and 14B shows the expression of markers of pluripotency in the human embryonic stem cell line H9. Expression of the markers of pluripotency was assayed by immunohistochemistry.
  • FIG. 14A shows Oct-4 expression.
  • FIG. 14B shows alkaline phosphatase expression.
  • FIG. 15 shows the karyotype of the human embryonic cell line H9.
  • the Karyotype was determined on cells at passage number P36 that were cultured on mouse embryonic fibroblast feeder cells.
  • FIG. 16 depicts the outline of a differentiation protocol in this invention, where human embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
  • FIGS. 17A, 17B, 17C, 17D, 17E and 17F depict the FACS profile of human embryonic stem cell line H9 at passage number 44, cultured on varying concentrations of MATRIGEL and exposed to (0.5-2%) low serum and high activin A (100 ng/ml) for 5 days.
  • the expression of definite endoderm marker CXCR4 (CD184) is shown on the Y-axis and the expression of ES marker CD9 is shown on the X-axis.
  • FIG. 18 shows the real-time PCR results for markers of definitive endoderm, from cultures of the human embryonic stem cell line H9 at passage 44 cultured on a 1:10 dilution of MATRIGEL ( ⁇ ), a 1:20 dilution of MATRIGEL ( ⁇ ), or a 1:30 dilution of MATRIGEL ( ⁇ ) and exposed to the differentiation protocol disclosed in Example 14.
  • the fold induction is relative to undifferentiated cells of the human embryonic stem cell line H9, at passage number 44, cultured in medium conditioned using mouse embryonic fibroblasts.
  • FIGS. 19A and 19B show the scatter plots for global gene expression in undifferentiated pluripotent stem cells and definitive endoderm cells obtained from differentiating pluripotent stem cells. Data shown is from cultures of the human embryonic stem cell line H9 cell line at passage 44 cultured on mouse embryonic fibroblasts (right panel) and passage 83 cultured on MATRIGEL (left panel).
  • FIGS. 20A, 20B and 20C depict the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 ( FIG. 20A ), the human embryonic stem cell line H7 ( FIG. 20B ), and the human embryonic stem cell line H9 ( FIG. 20C ) cultured on mouse embryonic fibroblast feeder cells exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
  • FIGS. 21A and 21B show the real-time PCR results of expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 ( FIG. 21A ) and the human embryonic stem cell line H9 ( FIG. 21B ) cultured on mouse embryonic fibroblast feeder cells. Results are expressed as fold increase over undifferentiated cells.
  • FIGS. 22A, 22B and 22C depict the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 ( FIG. 22A ), the human embryonic stem cell line H7 ( FIG. 22B ), and the human embryonic stem cell line H9 ( FIG. 22C ) cultured on MATRIGEL (1:30 dilution) and exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
  • FIGS. 23A, 23B and 23C show the real-time PCR results of the expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 ( FIG. 23A ) and the human embryonic stem cell line H9 ( FIG. 23B ) and the human embryonic stem cell line H1 ( FIG. 23C ). Results are expressed as fold increase over undifferentiated cells. Cells were treated according to the methods disclosed in Example 4.
  • FIGS. 24A and 24B depict phase contrast images of cultures of the human embryonic stem cell line H9 at passage 46 in the presence of 100 ng/ml of activin A ( FIG. 24A ) or 100 ng/ml of activin A+20 ng/ml Wnt-3a ( FIG. 24B ). Cells were treated for five days.
  • FIGS. 25A, 25B, 25C and 25D depict the expression of CXCR4 by FACS in cultures of the human embryonic stem cell line H7 at passage 44 ( FIGS. 25A and 25B ) and H9 at passage 46 ( FIGS. 25C and 25D ), following treatment according to the methods disclosed in Example 4.
  • FIGS. 25B and 25D show the effect of 20 ng/ml of Wnt-3a on CXCR4 expression.
  • FIGS. 25A and 25C show CXCR4 expression in the absence of Wnt-3a. Results were obtained 5 days post treatment.
  • FIGS. 26A and 26B display the real-time PCR data for expression of the genes indicated in cultures of the human embryonic stem cell line H7 ( FIG. 26A ) and H9 ( FIG. 26B ). Cultures were treated with the differentiation protocol disclosed in Example 4. The effects of Wnt agonists Wnt-3a (20 ng/ml), Wnt-5a (20 ng/ml) and Wnt-7a (20 ng/ml) were also tested, as indicated in the panels. Cells were treated for 5 days. Results are expressed as fold increase over undifferentiated cells.
  • FIGS. 27A, 27B, 27C and 27D depict the expression of CXCR4 in cultures of the human embryonic stem cell line H9 at passage 46, by FACS at five days post treatment.
  • FIG. 27A depicts CXCR4 expression in the absence of Wnt-3a.
  • FIG. 27B depicts CXCR4 expression following treatment with 10 ng/ml Wnt-3a.
  • FIG. 27C depicts CXCR4 expression following treatment with 20 ng/ml Wnt-3a
  • FIG. 27D depicts CXCR4 expression following treatment with 50 ng/ml Wnt-3a.
  • FIGS. 28A, 28B and 28C depict the expression of definitive markers indicated in cultures of the human embryonic stem cell line H9 after 5 days of treatment. Results are shown as fold increase in expression over untreated cells, as determined by real-time PCR.
  • FIG. 28A shows the effect of 10, 20 and 50 ng/ml Wnt-3a on the expression of definitive endoderm marker genes indicated.
  • FIG. 28B shows the effect of 1, 5 or 10 ng/ml Wnt-3a (x-axis labels: 10, 5, 1) on the expression on goosecoid ( ⁇ ) and CXCR4 ( ⁇ ) expression, at 2 (2 d) and 5 (5 d) days post treatment.
  • FIG. 28C shows the effect of 1, 5 or 10 ng/ml Wnt-3a on cell number, at 2 days ( ⁇ ) or 5 days ( ⁇ ).
  • FIGS. 29A, 29B, 29C, 29D, 29E, 29F, 29G and 29H depict the expression of CXCR4 in cultures of the human embryonic stem cell line H9 by FACS, following a 5 day treatment with the differentiation protocol disclosed in Example 4.
  • Cells were cultured in the absence of Wnt-3a or GSK-3B inhibitor ( FIG. 29A ), 20 ng/ml Wnt-3a for the entire 5 day period ( FIG. 29B ), 1000 nM GSK-3B inhibitor IX for the entire 5 day period ( FIG. 29C ), 500 nM GSK-3B inhibitor IX for the entire 5 day period ( FIG. 29D ), 100 nM GSK-3B inhibitor IX for the entire 5 day period ( FIG.
  • FIG. 29E 10 nM GSK-3B inhibitor IX for the entire 5 day period ( FIG. 29F ), 100 nM GSK-3B inhibitor IX for days 1-2 ( FIG. 29G ), 10 nM GSK-3B inhibitor IX for days 1-2 ( FIG. 29H ).
  • FIGS. 30A and 30 B depict the gene expression of definitive endoderm markers by real-time PCR. Results are expressed as fold increase over untreated cells.
  • FIG. 30A shows data obtained from the human embryonic cell line H9 at passage number 48, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated.
  • FIG. 30B shows data obtained from the human embryonic cell line H9 at passage number 46, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated.
  • FIGS. 31A, 31B, 31C, 31D, 31E and 31F depict the expression of CXCR4 by FACS for embryonic stem cell lines used in the present invention.
  • FIGS. 31A, 31B, 31C and 31D show data obtained from the human embryonic stem cell line H9 at passage number 49.
  • FIGS. 31E and 31F show data obtained from the human embryonic stem cell line H1 at passage number 46. Data was obtained 5 days post treatment. Cells were treated with the following conditions: FIG. 31A : 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; FIG.
  • FIG. 31B 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days
  • FIG. 31C 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days
  • FIG. 31D 10 ng/ml activin A for all five days plus 100 nM GSK-3B IX inhibitor for the first two days
  • FIG. 31E 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days
  • FIG. 31F 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days.
  • FIGS. 32A and 32B depict the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a:
  • FIG. 32A expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and
  • FIG. 32B SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 33A, 33B, 33C, 33D, 33E and 33F depict the expression of CXCR4 by FACS for the embryonic stem cell line H9 at passage 53. Data was obtained 5 days post treatment. Cells were treated with the following conditions: FIG. 33A : 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days and 25 ng/ml BMP-4 for days 3-5; FIG. 33B : 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; FIG. 33C : 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days; FIG.
  • FIG. 33D 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days
  • FIG. 33E 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a+100 nm GSK-3B inhibitor IX for the first two days
  • FIG. 33F 100 ng/ml activin A+25 ng/ml BMP-4 for all five days.
  • the X-axis represents expression of CD9
  • the Y-axis represents expression of CXCR4 (CD184).
  • FIGS. 34A and 34B depict the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor:
  • FIG. 34A expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4)
  • FIG. 34B SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 35A and 35B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor:
  • FIG. 35A expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and
  • FIG. 35B SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 36A and 36B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 53, treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4, for five days:
  • FIG. 36A expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7
  • FIG. 36B SOX-17, HNF-3B and GATA4.
  • FIG. 37 depicts the percentage of CXCR4 expression, determined by FACS, in cultures of the human embryonic stem cell line H9, treated with the conditions listed in Example 22.
  • FIGS. 38A and 38B depict the expression of definitive endoderm markers as determined by FACS in cultures of the human embryonic stem cell line H9, cultured on fibronectin ( FIG. 38A ) or MATRIGELTM ( FIG. 38B ).
  • FIG. 39 depicts the expression of definitive endoderm markers as determined by real-time PCR in cultures of the human embryonic stem cell line H9, cultured on fibronectin ( ⁇ ) or a 1:10 dilution of growth factor reduced MATRIGEL ( ⁇ ).
  • FIG. 40 depicts the effect of various concentrations of MATRIGEL in the presence of low serum, 100 ng/ml of activin A and 20 ng/ml of Wnt-3a on differentiating human embryonic stem cells into definitive endoderm.
  • Cells were treated according to the methods disclosed in Example 4. Results shown are the expression levels of the genes indicated, as determined by real-time PCR.
  • FIGS. 41A, 41B, 41C, 41D, 41E-1, 41E-2, 41F-1, 41F-2, 41G-1, 41G-2 depicts the role of Wnt-3a in definitive endoderm formation by human embryonic stem cells maintained on MATRIGEL, but differentiated on mouse embryonic fibroblasts.
  • FIGS. 41A, 41B, 41C and 41D show real-time PCR data for the genes indicated.
  • FIGS. 41E-1, 41E-2, 41F-1, 41F-2, 41G-1, 41G-2 show FACS data for the conditions indicated.
  • FIG. 42 shows the differentiation of human embryonic stem cells cultured on tissue culture substrate coated with MATRIGELTM to definitive endoderm following treatment with the Wnt Inhibitor DKK-1. Results shown are the expression of the genes indicated, as determined by real-time PCR in H9 cells treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3A plus 100 ng/ml of DKK1 (DE+DKK1), or in the absence of DKK1 (DE).
  • FIGS. 43A and 43B show immunofluorescence staining of definitive endoderm markers in cultures of the human embryonic stem cell line H9 cultured on tissue culture substrate coated with MATRIGEL and differentiated in low serum plus 100 ng/ml of activin-A without ( FIG. 43A ), or with ( FIG. 43B ) 20 ng/ml of Wnt-3a.
  • Ecad E-cadherin
  • NCAM N-cadherin
  • FIGS. 44A and 44B show the differentiation of the human embryonic stem cell line SA002 at passage 38 into definitive endoderm. Cells were treated for five days with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in FIGS. 44A and 44B .
  • FIGS. 45A, 45B and 45C show the expression of CXCR4 by FACS in the human embryonic stem cell line SA002 at passage 38, following treatment with 100 ng/ml activin A treatment ( FIG. 45A ), 100 ng/ml activin A+20 ng/ml Wnt-3a ( FIG. 45B ), or 100 ng/ml activin A+100 nM GSK-3B inhibitor IX ( FIG. 45C ). Cells were treated for five days.
  • FIGS. 46A and 46B show the differentiation of the human embryonic stem cell line H1 at passage 55 into definitive endoderm on tissue culture substrate coated with human serum. Cells were treated with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in FIGS. 46A and 46B .
  • FIGS. 47A and 47B show the differentiation of cultures of the human embryonic stem cell line H1 at P54, on tissue culture substrate coated with MATRIGELTM to definitive endoderm.
  • the effects of various GSK-B inhibitors were tested following a five-day DE protocol.
  • the following GSK-3B inhibitors were evaluated at 100 nM for the first two days of treatment: GSK-3B VIII, IX, XI, and XII.
  • FIGS. 48A, 48B, 48C and 48D show the expression of AFP ( FIG. 48A ), Pdx-1 ( FIG. 48B ), Cdx-2 and Glut-2 ( FIG. 48C ) and HNF-3beta, HNF-6 and somatostatin ( FIG. 48D ) in cultures of the human embryonic stem cell line H9 at passage 49, cultured and treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3a for the first two days of treatment.
  • the cells were treated for three additional days with 2% FBS plus 1 ⁇ M retinoic acid, 0.1 to 1 ⁇ M TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid), or 0.1-10 ⁇ M AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid).
  • the cells were next treated for three additional days in 2% FBS plus 20 ng/ml of bFGF.
  • FIGS. 49A and 49B show the real-time PCR results of the expression of the definitive endoderm markers indicated in FIGS. 49A and 49B . in cultures of the human embryonic stem cell line H1 treated with activin A and Wnt-1 for the times and concentrations indicated.
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell.
  • the term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • AFP or “alpha-fetoprotein protein” as used herein, refers to an antigen produced at the onset of liver development. AFP may also be expressed in extraembryonic cells.
  • Albumin is a soluble monomeric protein that makes up about half of all serum proteins in adults.
  • ⁇ -cell lineage refer to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6.
  • Cells expressing markers characteristic of the ⁇ cell lineage include ⁇ cells.
  • Brain is a T-box gene family member. It is the marker for primitive streak and mesoderm cells.
  • Cells expressing markers characteristic of the definitive endoderm lineage refer to cells expressing at least one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2.
  • Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
  • c-Kit and CD117 both refer to a cell surface receptor tyrosine kinase having a sequence disclosed in Genbank Accession No. X06182, or a naturally occurring variant sequence thereof (e.g., allelic variant).
  • CD99 refers to the protein encoded by the gene with the accession number NM_002414.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage refer to cells expressing at least one of the following markers: PDX-1, HNF-1beta, PTF-1 alpha, HNF-6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, Ngn-3, or PTF-1 alpha.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the ⁇ -cell lineage.
  • Cystein1 or “Cerebrus” as used herein is a member of the cysteine knot superfamily of proteins.
  • CXCR4 refers to the stromal cell-derived factor 1 (SDF-1) receptor, also known as “LESTR” or “fusin”. In the gastrulating mouse embryo, CXCR4 is expressed in the definitive endoderm and mesoderm but not in extraembryonic endoderm.
  • SDF-1 stromal cell-derived factor 1
  • Definitive endoderm refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF-3 beta, GATA-4, SOX-17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and Mixl1.
  • Extraembryonic endoderm refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, and SPARC.
  • FGF-2 “FGF-4” “FGF-8” “FGF-10”, and “FGF-17” as used herein, are members of the fibroblast growth factor family.
  • GATA-4 and GATA-6 are members of the GATA transcription factor family. This family of transcription factors is induced by TGF-3 signaling, and contribute to the maintenance of early endoderm markers.
  • GLUT-2 refers to the glucose transporter molecule that is expressed in numerous fetal and adult tissues, including pancreas, liver, intestine, brain, and kidney.
  • GSC homeodomain transcription factor expressed in the dorsal lip of the blastopore.
  • HB9 refers to the homeobox gene 9.
  • HNF-1 alpha “HNF-1 beta”, “HNF-3 beta”, and “HNF-6” belong to the hepatic nuclear factor family of transcription factors, which is characterized by a highly conserved DNA binding domain and two short carboxy-terminal domains.
  • Islet-1 or “Isl-1” as used herein is a member of the LIM/homeodomain family of transcription factors, and is expressed in the developing pancreas.
  • MicrofA as used herein is a transcription factor expressed in the pancreas, and controls the expression of genes involved in insulin biosynthesis and secretion.
  • Markers are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest.
  • differential expression means an increased level for a positive marker and a decreased level for a negative marker.
  • the detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • Mesendoderm cell refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta, GSC, FGF17, GATA-6.
  • Matl1 refers to a homeobox gene, which is marker for the cells in the primitive streak, mesoderm, and endoderm.
  • NeuroD as used herein is basic helix-loop-helix (bHLH) transcription factor implicated in neurogenesis.
  • NTN-3 as used herein, is a member of the neurogenin family of basic loop-helix-loop transcription factors.
  • Nkx-2.2 and Nkx-6.1 are members of the Nkx transcription factor family.
  • Nodal as used herein, is a member of the TGF beta superfamily of proteins.
  • Oct-4 is a member of the POU-domain transcription factor and is widely regarded as a hallmark of pluripotent stem cells.
  • the relationship of Oct-4 to pluripotent stem cells is indicated by its tightly restricted expression to undifferentiated pluripotent stem cells. Upon differentiation to somatic lineages, the expression of Oct-4 disappears rapidly.
  • Pantendocrine cell or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Pantix hormone secreting cell refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Pant-4 and “Pax-6” as used herein are pancreatic 13 cell specific transcription factors that are implicated in islet development.
  • PDX-1 refers to a homeodomain transcription factor implicated in pancreas development.
  • Pre-primitive streak cell refers to a cell expressing at least one of the following markers: Nodal, or FGF8
  • Primary streak cell refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
  • PTF-1 alpha refers to a basic helix-loop-helix protein of 48 kD that is a sequence-specific DNA-binding subunit of the trimeric pancreas transcription factor-1 (PTF1).
  • SOX-1 SOX-2
  • SOX-7 SOX-17
  • SPARC as used herein is also known as “secreted protein acidic and rich in cysteine”.
  • SSEA-1 Stage Specific Embryonic Antigen-1 is a glycolipid surface antigen present on the surface of murine teratocarcinoma stem cells (EC), murine and human embryonic germ cells (EG), and murine embryonic stem cells (ES).
  • SSEA-3 Stage Specific Embryonic Antigen-3 is a glycolipid surface antigen present on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • SSEA-4 Stage Specific Embryonic Antigen-4 is a glycolipid surface antigen present on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • TRA1-60 is a keratin sulfate related antigen that is expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • TRA1-81 is a keratin sulfate related antigen that is expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • TRA2-49 is an alkaline phosphatase isozyme expressed on the surface of human teratocarcinoma stem cells (EC) and human embryonic stem cells (ES).
  • UTF-1 refers a transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.
  • Zic1 as used herein is a member of the Zic transcription factor family. Zic1 regulates the expression of neural and neural crest-specific genes and is expressed in the cells of the dorsal neural tube and the premigratory neural crest.
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1.
  • SSEA stage-specific embryonic antigens
  • Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
  • pluripotent stem cells Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues.
  • Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers.
  • SCID severe combined immunodeficient
  • pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
  • pluripotent stem cells include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • pre-embryonic tissue such as, for example, a blastocyst
  • embryonic tissue or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and H9 (WiCell).
  • the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues.
  • cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells are also suitable are mutant human embryonic stem cell lines, such as,
  • human embryonic stem cells are prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).
  • pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways.
  • pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
  • Reubinoff et al (Nature Biotechnology 18: 399-404 (2000)) and Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391, pp. 1145-1147) disclose the culture of pluripotent stem cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer.
  • Richards et al (Stem Cells 21: 546-556, 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture.
  • Richards et al states: “human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripotent”.
  • US20020072117 discloses cell lines that produce media that support the growth of primate pluripotent stem cells in feeder-free culture.
  • the cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells.
  • US20020072117 also discloses the use of the cell lines as a primary feeder cell layer.
  • Wang et al discloses methods for the long-term growth of human pluripotent stem cells on feeder cell layers derived from human embryonic stem cells.
  • Stojkovic et al (Stem Cells 2005 23: 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic stem cells.
  • Miyamoto et al (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta.
  • Amit et al discloses a feeder cell layer derived from human foreskin.
  • Inzunza et al (Stem Cells 23: 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts.
  • U.S. Pat. No. 6,642,048 discloses media that support the growth of primate pluripotent stem (pPS) cells in feeder-free culture, and cell lines useful for production of such media.
  • U.S. Pat. No. 6,642,048 states: “This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure.”
  • WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells.
  • the culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte).”
  • Xu et al (Stem Cells 22: 972-980, 2004) discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase.
  • US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells.
  • An alternative culture system employs serum-free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells.
  • SR unconditioned serum replacement
  • Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF.
  • US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
  • US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells.
  • the media is substantially isotonic as compared to the stem cells being cultured.
  • the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
  • U.S. Pat. No. 6,800,480 states “In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells.
  • the basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells.
  • the medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.”
  • US20050244962 states: “In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor.”
  • WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of bFGF sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
  • WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGF ⁇ ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
  • TGF ⁇ transforming growth factor-beta
  • FGF fibroblast growth factor
  • NIC nicotinamide
  • the pluripotent stem cells may be plated onto a suitable culture substrate.
  • the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings.
  • a the suitable culture substrate is Matrigel® (Becton Dickenson). Matrigel® is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
  • extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
  • the pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
  • Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
  • DMEM Dulbecco's modified Eagle's medium
  • KO DMEM Knockout Dulbecco's modified Eagle's medium
  • Ham's F12/50% DMEM basal medium 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma
  • Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line SA002 (Cellartis, Sweden). Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cells: ABCG2, cripto, CD9, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.
  • Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, Hnf-3beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
  • Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of Pdx1, HNF-1beta, PTF1a, HNF-6, HB9 and PROX1.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage.
  • a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
  • a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage.
  • a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell.
  • the pancreatic endocrine cell may be a pancreatic hormone expressing cell.
  • the pancreatic endocrine cell may be a pancreatic hormone secreting cell.
  • the pancreatic endocrine cell is a cell expressing markers characteristic of the ⁇ cell lineage.
  • a cell expressing markers characteristic of the ⁇ cell lineage expresses Pdx1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6.
  • a cell expressing markers characteristic of the ⁇ cell lineage is a ⁇ cell.
  • Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534-1541 (2005).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651-1662 (2004).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29-38 (2007).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration.
  • An example of this method is disclosed in Nature Biotechnology 23, 1534-1541 (2005).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration.
  • An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2005.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for a period of time, and then culturing the pluripotent stem cells with activin A in a second culture medium containing a greater concentration of serum for about another period of time.
  • the concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days.
  • the concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for about a period of time, and then culturing the pluripotent stem cells with activin A and a Wnt ligand in a second culture medium containing a greater concentration of serum for another period of time.
  • the concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days.
  • the concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
  • the present invention provides a method for differentiating pluripotent stem cells expressing markers characteristic of the definitive endoderm lineage, comprising the steps of:
  • Culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed in a single culture medium. Alternatively, culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed separately or together in more than one culture media. In one embodiment, culturing the pluripotent stem cells with activin A and a Wnt ligand is performed in two culture media.
  • the pluripotent stem cells are cultured and differentiated on a tissue culture substrate coated with an extracellular matrix.
  • the extracellular matrix may be a solubilized basement membrane preparation extracted from mouse sarcoma cells (which is sold by BD Biosciences under the trade name MATRIGEL).
  • the extracellular matrix may be growth factor-reduced MATRIGEL.
  • the extracellular matrix may fibronectin.
  • the pluripotent stem cells are cultured and differentiated on tissue culture substrate coated with human serum.
  • the extracellular matrix may be diluted prior to coating the tissue culture substrate.
  • suitable methods for diluting the extracellular matrix and for coating the tissue culture substrate may be found in Kleinman, H. K., et al., Biochemistry 25:312 (1986), and Hadley, M. A., et al., J. Cell. Biol. 101:1511 (1985).
  • the extracellular matrix is MATRIGEL.
  • the tissue culture substrate is coated with MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:60 dilution.
  • the extracellular matrix is growth factor-reduced MATRIGEL.
  • the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:60 dilution.
  • a single culture medium When a single culture medium is used, it should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells to definitive endoderm, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1): 241-8.).
  • the culture medium may have a serum concentration in the range of about 0% to about 10%. In an alternate embodiment, the concentration may be in the range of about 0% to about 5%. In an alternate embodiment, the concentration may be in the range of about 0% to about 2%. In an alternate embodiment, the concentration may be about 2%.
  • the time of culturing with activin A and a Wnt ligand may range from about 1 day to about 7 days. In an alternate embodiment, the time of culturing may range from about 1 day to about 3 days. In an alternate embodiment, the time of culturing may be about 3 days.
  • Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells.
  • the concentration maybe from about 1 pg/ml to about 100 ⁇ g/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 ⁇ g/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • the choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process.
  • the Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.
  • the Wnt ligand is Wnt-1.
  • the Wnt ligand is Wnt-3a.
  • the Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • the single culture medium may also contain a GSK-3B inhibitor.
  • the GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI.
  • the GSK-3B inhibitor is GSK-3B inhibitor IX. I did not see the definitions of IX and XI inhibitors. I did have these in the original WNT patent application.
  • the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM.
  • the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • the single culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells.
  • the at least one other additional factor may enhance the proliferation of the ells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention.
  • the at least one other additional factor may enhance the ability of the ells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • the at least one additional factor may be, for example, nicotinamide, members of the TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,
  • the at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241), and primary or transformed endothelial cells.
  • pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), intestinal cell lines such as, for example, FHs
  • Differentiation of pluripotent stem cells into cells of a definitive endoderm lineage may be accomplished by culturing the pluripotent stem cells with activin A and a Wnt ligand using two culture media.
  • the differentiation of the pluripotent stem cells may be accomplished as follows:
  • the first culture medium may contain serum at a low concentration
  • the second culture medium may contain serum at a higher concentration than the first culture medium
  • the second culture medium may contain a Wnt ligand.
  • the first culture medium should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
  • the first culture medium there may be a lower concentration of serum, relative to the second culture medium.
  • Increasing the serum concentration in the second culture medium increases the survival of the cells, or, alternatively, may enhance the proliferation of the cells.
  • the serum concentration of the first medium may be in the range of about 0% to about 10%.
  • the serum concentration of the first medium may be in the range of about 0% to about 2%.
  • the serum concentration of the first medium may be in the range of about 0% to about 1%.
  • the serum concentration of the first medium may be about 0.5%.
  • the time of culturing in the first culture medium may range from about 1 day to about 3 days.
  • Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells.
  • the concentration maybe from about 1 pg/ml to about 100 ⁇ g/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 pg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • the choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process.
  • the Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.
  • the Wnt ligand is Wnt-1.
  • the Wnt ligand is Wnt-3a.
  • the Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • the first culture medium may also contain a GSK-3B inhibitor.
  • the GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
  • the GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
  • the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM.
  • the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • the first culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells.
  • the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention.
  • the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • the at least one additional factor may be, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, tric
  • the at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • the second culture medium should contain certain factors, such as, for example, insulin and IGF (as disclosed in WO2006020919), at a sufficient concentration to promote the survival of the cultured cells. This may be achieved by increasing the serum concentration, or, alternatively, by using chemically defined media where the concentrations of insulin and IGF are increased relative to the first culture medium. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
  • the serum concentration of the second culture medium may be in the range about 0.5% to about 10%.
  • the serum concentration of the second culture medium may be in the range of about 0.5% to about 5%.
  • the serum concentration of the second culture medium may be in the range of about 0.5% to about 2%.
  • the serum concentration of the second culture medium may be about 2%.
  • the time of culturing may range from about 1 day to about 4 days.
  • Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells.
  • the concentration maybe from about 1 pg/ml to about 100 ⁇ g/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 ⁇ g/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • the Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • the Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
  • the second culture medium may also contain a GSK-3B inhibitor.
  • the GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
  • the GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
  • the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM.
  • the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • the second culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells.
  • the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention.
  • the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • the at least one additional factor may be, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, tric
  • the at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
  • an agent such as an antibody
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
  • pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.
  • the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage.
  • an agent such as an antibody
  • a protein marker such as CXCR4
  • Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention.
  • cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine.
  • a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine an example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time. That period of time may be from about one to about six days.
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells with retinoic acid for a period of time. That period of time maybe from about one to about three days. The retinoic acid is subsequently removed and the cells are treated with at least one fibroblast growth factor for another period of time. That period of time may be from about one to about three days.
  • the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about one to about six days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about six days.
  • the at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about three days.
  • the at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid.
  • the cells expressing markers characteristic of the definitive endoderm lineage are treated with FGF-2, or alternatively FGF-4, or alternatively FGF-10.
  • the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one of the following factors: retinoic acid, FGF-2, FGF-4 or FGF-10.
  • the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and at least one of the following fibroblast growth factors: FGF-2, FGF-4 or FGF-10.
  • the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-2. In another embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-4. In a further embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-10.
  • Retinoic acid may be used at a concentration from about 1 nM to about 1 mM. In one embodiment, retinoic acid is used at a concentration of 1 ⁇ M.
  • FGF-2 may be used at a concentration from about 50 pg/ml to about 50 ⁇ g/ml. In one embodiment, FGF-2 is used at a concentration of 50 ng/ml.
  • FGF-4 may be used at a concentration from about 50 pg/ml to about 50 ⁇ g/ml. In one embodiment, FGF-4 is used at a concentration of 50 ng/ml.
  • FGF-10 may be used at a concentration from about 50 pg/ml to about 50 ⁇ g/ml. In one embodiment, FGF-10 is used at a concentration of 50 ng/ml.
  • Cells expressing markers characteristic of the definitive endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endoderm lineage.
  • the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention.
  • the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • the at least one additional factor may be, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, tric
  • the at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1beta.
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
  • an agent such as an antibody
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method disclosed in this invention.
  • cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF.
  • An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4.
  • An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4.
  • An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway.
  • the factor that inhibits the Notch signaling pathway may be an antagonist for the Notch extracellular receptor.
  • the factor may inhibit the biological activity of the Notch receptor.
  • the factor may inhibit or be an antagonist of an element in the Notch signal transduction pathway within a cell.
  • the factor that inhibits the Notch signaling pathway is a ⁇ -secretase inhibitor.
  • the ⁇ -secretase inhibitor is 1SBenzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-5 2Rhydrozy-5 phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • L-685,458 may be used at a concentration from about 0.1 ⁇ M to about 100 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 90 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 80 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 70 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 60 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 50 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 40 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 30 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 20 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 10 ⁇ M.
  • the present invention provides a method for differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:
  • Any cell expressing markers characteristic of the pancreatic endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endocrine lineage using this method.
  • factor that inhibits the Notch signaling pathway is a ⁇ -secretase inhibitor.
  • the ⁇ -secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about one to about five days.
  • the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about three to about five days.
  • the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about five days.
  • factor that inhibits the Notch signaling pathway is a ⁇ -secretase inhibitor.
  • the ⁇ -secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • L-685,458 may be used at a concentration from about 0.1 ⁇ M to about 100 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 90 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 80 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 70 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 60 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 50 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 40 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 30 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 20 ⁇ M. In one embodiment, L-685,458 is used at a concentration of about 10 ⁇ M.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endocrine lineage.
  • the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention.
  • the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • the at least one additional factor may be, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, tric
  • the at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage.
  • Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN-3, NeuroD, Islet-1.
  • ⁇ cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, Pdx1 (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Isl1, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3beta, and MafA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage.
  • an agent such as an antibody
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the ⁇ cell lineage.
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
  • the present invention provides a method for treating a patient suffering from, or at risk of developing, Type1 diabetes. This method involves culturing pluripotent stem cells, differentiating the pluripotent stem cells in vitro into a ⁇ -cell lineage, and implanting the cells of a ⁇ -cell lineage into a patient.
  • this invention provides a method for treating a patient suffering from, or at risk of developing, Type 2 diabetes. This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a ⁇ -cell lineage, and implanting the cells of a ⁇ -cell lineage into the patient.
  • the patient can be further treated with pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells.
  • agents may include, for example, insulin, members of the TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -7, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others.
  • TGF- ⁇ family including TGF- ⁇ 1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA
  • Other pharmaceutical compounds can include, for example, nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2 mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • GLP-1 glucagon like peptide-I
  • GLP-1 and 2 mimetibody GLP-1 and 2 mimetibody
  • Exendin-4 retinoic acid
  • parathyroid hormone retinoic acid
  • MAPK inhibitors such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • the pluripotent stem cells may be differentiated into an insulin-producing cell prior to transplantation into a recipient.
  • the pluripotent stem cells are fully differentiated into ⁇ -cells, prior to transplantation into a recipient.
  • the pluripotent stem cells may be transplanted into a recipient in an undifferentiated or partially differentiated state. Further differentiation may take place in the recipient.
  • Definitive endoderm cells or, alternatively, pancreatic endoderm cells, or, alternatively, ⁇ cells may be implanted as dispersed cells or formed into clusters that may be infused into the hepatic portal vein.
  • cells may be provided in biocompatible degradable polymeric supports, porous non-degradable devices or encapsulated to protect from host immune response.
  • Cells may be implanted into an appropriate site in a recipient. The implantation sites include, for example, the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach, or a subcutaneous pocket.
  • additional factors such as growth factors, antioxidants or anti-inflammatory agents, can be administered before, simultaneously with, or after the administration of the cells.
  • growth factors are utilized to differentiate the administered cells in vivo. These factors can be secreted by endogenous cells and exposed to the administered cells in situ. Implanted cells can be induced to differentiate by any combination of endogenous and exogenously administered growth factors known in the art.
  • the amount of cells used in implantation depends on a number of various factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art.
  • this invention provides a method for treating a patient suffering from, or at risk of developing diabetes.
  • This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a ⁇ -cell lineage, and incorporating the cells into a three-dimensional support.
  • the cells can be maintained in vitro on this support prior to implantation into the patient.
  • the support containing the cells can be directly implanted in the patient without additional in vitro culturing.
  • the support can optionally be incorporated with at least one pharmaceutical agent that facilitates the survival and function of the transplanted cells.
  • Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair.
  • synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No. 5,759,830, U.S. Pat. No.
  • the pharmaceutical agent can be mixed with the polymer solution prior to forming the support.
  • a pharmaceutical agent could be coated onto a fabricated support, preferably in the presence of a pharmaceutical carrier.
  • the pharmaceutical agent may be present as a liquid, a finely divided solid, or any other appropriate physical form.
  • excipients may be added to the support to alter the release rate of the pharmaceutical agent.
  • the support is incorporated with at least one pharmaceutical compound that is an anti-inflammatory compound, such as, for example compounds disclosed in U.S. Pat. No. 6,509,369.
  • the support may be incorporated with at least one pharmaceutical compound that is an anti-apoptotic compound, such as, for example, compounds disclosed in U.S. Pat. No. 6,793,945.
  • the support may also be incorporated with at least one pharmaceutical compound that is an inhibitor of fibrosis, such as, for example, compounds disclosed in U.S. Pat. No. 6,331,298.
  • the support may also be incorporated with at least one pharmaceutical compound that is capable of enhancing angiogenesis, such as, for example, compounds disclosed in U.S. Published Application 2004/0220393 and U.S. Published Application 2004/0209901.
  • the support may also be incorporated with at least one pharmaceutical compound that is an immunosuppressive compound, such as, for example, compounds disclosed in U.S. Published Application 2004/0171623.
  • an immunosuppressive compound such as, for example, compounds disclosed in U.S. Published Application 2004/0171623.
  • the support may also be incorporated with at least one pharmaceutical compound that is a growth factor, such as, for example, members of the TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others.
  • a growth factor such as, for example, members of the TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA
  • Other pharmaceutical compounds can include, for example, nicotinamide, hypoxia inducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 and GLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptides, cathelicidins, defensins, laminin, biological peptides containing cell- and heparin-binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • MAPK inhibitors such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • the incorporation of the cells of the present invention into a scaffold can be achieved by the simple depositing of cells onto the scaffold.
  • Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)).
  • Several other approaches have been developed to enhance the efficiency of cell seeding.
  • spinner flasks have been used in seeding of chondrocytes onto polyglycolic acid scaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)).
  • Another approach for seeding cells is the use of centrifugation, which yields minimum stress to the seeded cells and enhances seeding efficiency.
  • Yang et al. developed a cell seeding method (J. Biomed. Mater. Res. 55(3): 379-86 (2001)), referred to as Centrifugational Cell Immobilization (CCI).
  • CCI Centrifugational Cell Immobilization
  • the human embryonic stem cell lines H1, H7 and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River.
  • MEF mouse embryonic fibroblast
  • MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 ⁇ g/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2 ⁇ 10 4 /cm 2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO 2 /within a humidified tissue culture incubator.
  • FBS Hyclone
  • MEM nonessential amino acids 100 mM MEM nonessential amino acids.
  • Sub-confluent MEF cell cultures were treated with 10 ⁇ g/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and
  • human embryonic stem cells When confluent (approximately 5-7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surface using a 5-ml pipette. Cells were spun at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells in fresh culture medium.
  • collagenase type IV Invitrogen/GIBCO
  • activin A The effects of activin A on the expression of markers of definitive endoderm were examined.
  • Activin A 100 ng/ml was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts. Cells were cultured continuously in the presence of activin A and harvested at the times indicated.
  • the level of expression of definitive endoderm markers was examined by PCR ( FIGS. 1A and 1B ), FACS (results summarized in Table II), and immunohistochemistry ( FIGS. 2A, 2B and 2C ).
  • Activin A evoked a time-dependent increase in the expression of CXCR4, GATA4, HNF-3beta, Mixl1 and Sox-17 mRNA in the H9 line ( FIG. 1A ).
  • a significant up regulation of anterior endoderm markers, Cerberus, Otx-1 and Hex genes was also observed ( FIG. 1B ).
  • An increase in CXCR4 protein was observed by FACS analysis following activin A treatment.
  • the expression of E-cadherin and N-cadherin did not change following activin A treatment (Table HA).
  • CXCR4 positive cells were also highly positive for C-kit, EPCAM, CD99, and negative for CD9. The expression pattern for these markers was consistent among all three hES cell lines examined (Table IIB for H7 and Table IIC for H1).
  • pancreatic endoderm Growth factors known to induce the differentiation of human embryonic stem cells to pancreatic endoderm were added to cell cultures.
  • activin A, bFGF, and retinoic acid known to induce the formation of pancreatic endoderm, were added to cell cultures.
  • activin A was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts for up to seven days in DMEM/F12 supplemented with 0% to 2% serum and Activin A (100 ng/ml). Cells were harvested at the time points indicated in FIGS. 3A, 3B, 3C and 3D and assayed by PCR for the expression of genes shown ( FIGS. 3A, 3B, 3C, 3D, 4A, 4B, 5A and 5B ). In FIGS. 3A, 3B, 3C and 3D , PCR analysis indicated that activin treated cells expressed a broad spectrum of genes associated with endoderm development, including GATA4 ( FIG. 3A ), Sox-17 ( FIG.
  • Activin A evoked a time-dependent decrease in the expression of the extraembryonic endoderm markers Sox7 ( FIG. 4A ) and AFP ( FIG. 4B ).
  • Activin A decreased the expression of Brachyury ( FIG. 5A ) but had no effect on expression of the neuronal marker Zic1 ( FIG. 5B ).
  • cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After the cells were 70-80% confluent, the medium was changed to DMEM/F12 with 0 to 2% FBS with addition of activin A at 100 ng/ml and cultured in the presence of activin A for either three, five, or seven days. After this time interval, the cells were then further treated for five to six days with combinations of retinoic acid and bFGF as shown in FIGS. 8A, 8B and 8C . Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
  • Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 3-4 days according to the methods described in Example 1. After the cells were 50-60% confluent, the medium was changed to DMEM/F12 without FBS, containing activin A at 100 ng/ml, and the cells were cultured in this medium for one day. Following the one day culture, the medium was removed and replaced with medium containing 0.5% FBS with 100 ng/ml activin A, and the cells were cultured for one day. Following the second one-day culture, the medium was removed and replaced with medium containing 2% FBS with 100 ng/ml activin A, and the cells were cultured for one day.
  • the cells were then treated for six days with combinations of retinoic acid and FGF as outlined in Example 2, then the culture medium was removed and replaced with medium comprising DMEM/F12 with 2% FBS, containing the ⁇ -secretase inhibitors L-685,458 at 10 ⁇ M for three days. Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
  • FIGS. 10A and 10C Gene expression analysis revealed that activin A alone or in combination with retinoic acid and FGFs did not induce the expression of Ngn3 or Insulin.
  • a decrease in the expression of Hes-1 was also observed following treatment with L-685,458. The maximal inhibition was observed on day three post treatment ( FIG. 10D ).
  • treatment of cells with L-685,458 induced the expression of Ngn3 to a level approximately 50-fold higher than that observed in samples treated with activin A alone or retinoic acid with FGFs in combination.
  • a 70-fold increase of insulin expression was observed in samples treated with the ⁇ -secretase inhibitor.
  • NeuroD1 expression was also increased further by the L-685,458 treatment ( FIG. 10A ).
  • Definitive endoderm cells obtained according to the methods outlined in Example 2 were treated as follows: Cells were cultured in basal medium, comprising DMEM/F12 with 2% FBS plus 50 ng/ml activin A, 50 ng/ml basic FGF and 1 ⁇ M of Retinoic Acid for 3 to 5 days. Cells were continuously cultured for another 3 to 5 days in basal medium with retinoic acid at 1 ⁇ M, alone or with bFGF. RNA samples were harvested from cells at various time points along this process to help evaluate the directed differentiation of the cells. Furthermore, culture medium and factors were regularly removed and replenished throughout the differentiation protocol. Addition of activin A showed an increase of Nkx2.2 expression about 35-fold compared to samples without activin A.
  • pancreatic endoderm cells derived from human embryonic stem cells herein can be maintained in cell culture and passaged without further differentiation.
  • Pancreatic endoderm cells were differentiated in the presence of 100 ng/ml activin A in low serum DMEM/F12.
  • the low serum DMEM/F12 contained 0% (v/v) fetal bovine serum (FBS) on day 1, 0.5% (v/v) FBS on day two and 2% (v/v) FBS on each day thereafter.
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • 2% (v/v) FBS on each day thereafter.
  • the cells were cultured in low serum DMEM/F12 contained 2% (v/v) FBS, 1 ⁇ M retinoic acid and 50 ng/ml bFGF for a total of six more days.
  • FIG. 12 shows that cells grown in the presence of 50 ng/ml FGF10 maintained expression of the pancreatic endoderm marker Pdx1 during the 6 day culture period subsequent to their derivation.
  • Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After cells were 70-80% confluent, the medium was changed to DMEM/F12 with 2% FBS containing activin A at 100 ng/ml, and cells were cultured in the presence of activin A for seven days. After 7 days treatment with activin A, the cells were then treated for five days with the conditions shown in FIGS. 13A and 13B . After this time, the cells were harvested, and samples of mRNA were collected for analysis.
  • FIG. 13A An increase in the expression of ⁇ -fetoprotein (AFP) and albumin was observed ( FIG. 13A ) for cells cultured in the absence of activin A. This was further increased by retinoic acid and FGF-4 ( FIG. 13B ).
  • AFP ⁇ -fetoprotein
  • FIG. 13B An increase in the expression of ⁇ -fetoprotein (AFP) and albumin was observed ( FIG. 13A ) for cells cultured in the absence of activin A. This was further increased by retinoic acid and FGF-4 ( FIG. 13B ).
  • H9 cells The quality of H9 cells was monitored over time by evaluating expression of several markers expressed by undifferentiated ES cells (Carpenter et al., 2001; Reubinoff et al., 2000; Thomson et al., 1998a). H9 cells exhibited reciprocal expression of stage-specific embryonic antigens (Table IH). H9 cells play strong immunoreactivity for SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, AP and CD9 antigens, all of which are characteristic of undifferentiated human embryonic stem cells.
  • RT-PCR was performed to assess the expression of genes characteristic of embryonic stem cells, such as, for example, OCT3/4, SOX-2, UTF-1, REX-1, Cx43, Cx45, ABCG-2 and TERT, confirming that the cells grown in this example appeared similar to previously described undifferentiated embryonic stem cells (Table IH).
  • OCT3/4 protein expression and Alkaline Phosphatase activity were confirmed by immunostaining.
  • a majority of H9 cells were positive for OCT3/4 and AP ( FIGS. 14A and 14B ). Overall, these results demonstrate that the H9 cells used in this example were not significantly different in morphology, antigen immunostaining, or pluripotency marker expression when compared to reports from other laboratories.
  • Adhered cells were removed from culture plates by five-minute incubation with TrypLETM Express solution (Invitrogen, CA). Released cells were resuspended in human embryonic stem cell culture medium and recovered by centrifugation, followed by washing and resuspending the cells in a staining buffer consisting of 2% BSA, 0.05% sodium azide in PBS (Sigma, MO). As appropriate, the cells were Fc-receptor blocked for 15 minutes using a 0.1% ⁇ -globulin (Sigma) solution.
  • Control samples included reactions where the primary antibody was omitted or where the primary antibody was replaced with corresponding matched negative control immunoglobulins at the same concentration as the primary antibodies. Stained samples were rinsed; a drop of PROLONG® (Invitrogen, CA) containing diamidino-2-phenylindole, dihydrochloride (DAPI) was added to each sample to counter-stain the nucleus and to function as an anti-fade reagent. Images were acquired using a Nikon Confocal Eclipse C-1 inverted microscope (Nikon, Japan) and a 10-60 ⁇ objective.
  • RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants.
  • the RNA was further purified using a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer.
  • cDNA copies were made from purified RNA using an ABI (ABI, CA) high capacity cDNA archive kit.
  • reagents were purchased from Applied Biosystems. Real-time PCR reactions were performed using the ABI PRISM® 7900 Sequence Detection System. TAQMAN® UNIVERSAL PCR MASTER MIX® (ABI, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 ⁇ l. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN®probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by Applied Biosystem.
  • GPDH human glyceraldehyde-3-phosphate dehydrogenase
  • FIG. 15 depicts a typical karyotype obtained from the human embryonic stem cell line H9.
  • the human embryonic stem cell lines H1, H7, and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River.
  • MEF mouse embryonic fibroblast
  • MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 ⁇ g/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2 ⁇ 104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO 2 within a humidified tissue culture incubator.
  • FBS Hyclone
  • MEM nonessential amino acids 100 mM MEM nonessential amino acids.
  • Sub-confluent MEF cell cultures were treated with 10 ⁇ g/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2 ⁇
  • human embryonic stem cells When confluent (approximately 5 to 7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5 to 10 min and then gently scraped off the surface using a 5 ml glass pipette. Cells were centrifuged at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells on plates coated with a 1:30 dilution of growth factor reduced MATRIGELTM (BD Biosciences).
  • 1 mg/ml collagenase type IV Invitrogen/GIBCO
  • Cells were subsequently cultured in MEF-conditioned media supplemented with 8 ng/ml bFGF and collagenase passaged on MATRIGEL coated plates for at least five passages.
  • the cells cultured on MATRIGELTM were routinely passaged with collagenase IV (Invitrogen/GIBCO), Dispase (BD Biosciences) or Liberase enzyme (Roche, IN).
  • H9 cultures at approximately 60 to 70% confluency were exposed to DMEM:/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days, followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days.
  • H9 cells were cultured on plates coated with growth factor reduced MATRIGEL at a 1:30 to 1:10 dilution or on regular MATRIGEL at a 1:30 to 1:10 dilution The plates were coated with MATRIGEL for 1 hr at room temperature.
  • FIGS. 17A, 17B, 17C, 17D, 17E and 17F depicts the expression of CXCR4 by FACS.
  • FIG. 18 shows the real-time PCR results verifying that cells cultured on plates coated with a 1:10 dilution of MATRIGEL showed a significant upregulation of definitive endoderm markers as compared to cells cultured on a 1:30 dilution of MATRIGEL.
  • Total RNA was isolated from the following human embryonic stem cell cultures using an RNeasy mini kit (Qiagen): H9P83 cells cultured on MATRIGEL-coated plates and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA) for an additional three days; H9P44 cells cultured on MEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A for an additional three days. Controls for each group included cells plated on MATRIGEL-coated dishes and cultured in MEF-conditioned medium or cells plated on MEFs and cultured in ES medium.
  • H9, H7, or H1 cells seeded on growth factor reduced MATRIGELTM (1:30 dilution) cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A (R&D Systems, MN)) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days.
  • this treatment regimen will be referred to as the definite endoderm (DE) protocol.
  • H9, H7, or H1 cells cultured on MEF feeders were also exposed to the same DE protocol outlined above.
  • the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9, CD99, and N-cadherin (CD56) expression and by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, and GATA4.
  • AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak.
  • FIGS. 20A, 20B and 20C H-lines cultured on mouse feeders and exposed to the DE protocol resulted in a robust expression of DE markers and expression of CXCR4 by FACS ( FIGS. 20A, 20B and 20C ). There was also a significant decrease in expression of E-cadherin following treatment with the DE protocol. Lastly, the CXCR4+population also stained positive for CD117.
  • FIGS. 21A and 21B shows a significant upregulation of definitive endoderm markers as compared to untreated H7 ( FIG. 21A ) and H9 cells ( FIG. 21B ).
  • H-lines cultured on MATRIGELTM Unlike H-lines cultured on MEF feeders, H-lines cultured on MATRIGELTM (1:30 dilution) and treated with the definitive endoderm protocol failed to show robust expression of definitive endoderm markers.
  • the expression of CXCR4 by FACS and by real-time PCR was significantly lower for cells cultured on MATRIGELTM as compared to cells cultured on mouse embryonic fibroblasts.
  • Expression of definitive endoderm markers follows a general response pattern with H1 being greater than H9, which is greater than H7 ( FIGS. 22A, 22B, 22C, 23A, 23B and 23C ). From FIGS. 22A, 22B and 22C , H1 cells showed a significant increase in CXCR4 expression as compared to H7 and H9 lines.
  • FIGS. 23A , 23 B, and 23 C shows the real-time PCR results showing that there was modest increase in upregulation of definitive endoderm markers in H7 ( FIG. 23A ) and H9 ( FIG. 23B ) lines.
  • H1 ( FIG. 23C ) line showed a more robust upregulation of definitive endoderm markers as compared to H7 and H9 lines.
  • H7P44 and H9P46 embryonic stem cells were cultured on MATRIGELTM (1:10 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days.
  • FIGS. 24A and 24B depicts phase contrast images of H9P46 definitive endoderm culture in the presence of high concentration of (a) AA or (b) AA+20 ng/ml Wnt-3a.
  • FIGS. 25A, 25B, 25C and 25D depicts the expression of CXCR4 by FACS at day 5 for H7P44, and H9P46 lines cultured on MATRIGELTM (1:30 dilution) and exposed to the DE protocol+Wnt-3a ( FIGS. 25B and 25D ) and ⁇ Wnt-3a ( FIGS. 25A and C). Presence of Wnt-3a in DE cultures resulted in robust expression of CXCR4 (CD184) as compared to DE cultures treated with low serum plus high concentration of AA.
  • FIGS. 26A and 26B displays the real-time PCR data for a) H7 and b) H9 cultures treated with low serum+AA+/ ⁇ Wnt ligands. For both H-lines, addition of WNT-3a resulted in significant upregulation of definitive endoderm markers. In contrast, Wnt 5a, Wnt-5b and Wnt-7a had minimal impact on expression of definitive endoderm markers.
  • H9P46 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml Activin A (AA), and 10-50 ng/ml WNt-3a (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 10-50 ng/ml Wnt-3a for an additional three days. Control cultures were not treated with Wnt-3a.
  • FIG. 27A depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a, b) 10 ng/ml Wnt-3a, c) 20 ng/ml Wnt-3a and d) 50 ng/ml Wnt-3a.
  • the expression of CXCR4 was very low.
  • addition of 10-50 ng/ml of Wnt-3a significantly increased the number of CXCR4 positive cells.
  • addition of 10 ng/ml of Wnt-3a was as effective as addition of 50 ng/ml of Wnt-3a.
  • Real-time PCR results ( FIG. 28A ) also confirm this finding.
  • FIG. 28B shows PCR analysis of the DE markers after 5 days of treatment. The number of cells at the end of the experiment is noted in FIG. 28C . This indicates that cells are proliferating when higher doses of Wnt-3a are used. Extension to 5 days of Wnt3a treatment (5 D) had little effect on DE markers by PCR and did not significantly increase cell numbers ( FIG. 28C ). These data indicate that 10 ng/ml Wnt3a for 2 days is sufficient to reach optimal cell expansion and definitive endoderm differentiation.
  • a GSK-3 inhibitor was used to activate the downstream targets of Wnt, such as beta catenin.
  • H9P46-P48 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin-A (AA), and 10-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 0-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for an additional three days.
  • FIG. 29A depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a or GSK-3B inhibitor, b)+20 ng/ml Wnt-3a, c)+1000 nM GSK-3B inhibitor IX, d)+500 nM GSK-3B inhibitor IX, e)+100 nM GSK-3B inhibitor IX, f)+10 nM GSK-3B inhibitor IX, g)+100 nM GSK-3B inhibitor IX for days 1-2, and h)+10 nM GSK-3B inhibitor IX for days 1-2.
  • FIGS. 30A and 30B depicts the gene expression of definitive endoderm markers for ( FIG. 30A ) H9P48 cells and ( FIG. 30B ) H9P46 cells.
  • FIG. 16 depicts the outline of a differentiation protocol in this invention, where embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
  • H9P49 and H1P46 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 10-100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) or 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus 100 ng/ml of activin A.
  • 31A, 31B, 31C, 31D, 31E and 31F depicts the expression of CXCR4 by FACS for H9P49 and H1P46 at day 5 with a) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 10 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, and f) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days.
  • FIGS. 31A, 31B, 31C and 31D is for H9P49 cells and FIGS. 31E and 31F is for H1P46 cells.
  • FIGS. 32A and 32B depicts the gene expression of definitive endoderm markers for H9P49 cultures treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a:
  • FIG. 32A expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4)
  • FIG. 32B SOX-17 and GATA4. This does not add up—It appears to be a problem with figure numbers or it is a cut and paste problem.
  • H9P53 embryonic stem cells were cultured on MATRIGELTM coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) +/ ⁇ 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin-A (AA) for an additional three days.
  • DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) +/ ⁇ 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin-A (AA) for an additional three days
  • H9P53 cultures were treated with 25 ng/ml BMP-4 (Catalog#314-BP-010, R&D Systems, MN) +/ ⁇ 20 ng/ml Wnt-3A+/ ⁇ 100 ng/ml activin A.
  • Control cultures were treated with low serum plus 100 ng/ml of activin A.
  • 33A, 33B, 33C, 33D, 33E , and 33 F depicts the expression of CXCR4 by FACS at day 5 with a) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days and 25 ng/ml BMP-4 for days 3-5, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A+100 nm GSK-3B inhibitor IX for the first two days, and f) 100 ng/ml activin A+25 ng/ml BMP-4 for all five
  • FIGS. 34A and 34B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and panel (b): SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 34A and 34B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct
  • 35A and 35B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
  • 36A and 36B depicts the gene expression of definitive endoderm markers for H9P53 culture treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4: a) expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and b) SOX-17, HNF-3B and GATA4. Addition of BMP-4 to the DE protocol appears to induce formation of mesoderm marker BRY and combination of Wnt-3A and GSK-4B inhibitor do not lead to significant upregulation of definitive endoderm markers as compared to addition of each agent by itself in the presence of activin A.
  • H9P44 cells were plated onto 6 well plates previously coated with mitomycin treated mouse embryonic fibroblasts (MEF). Cells were grown until 70 to 80% confluency in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine (all from Invitrogen/GIBCO) and 8 ng/ml human basic fibroblast growth factor (bFGF) (R&D Systems).
  • DMEM/F12 Invitrogen/GIBCO
  • bFGF human basic fibroblast growth factor
  • Activin A 100 ng/ml
  • growth factors were added to increasing concentration of FBS in a stepwise manner as indicated in the following regimen:
  • H9P55 cells were grown and differentiated on human fibronectin or regular growth factor MATRIGELTM (BD Biosciences). 1 ml of DMEM/F12 (Invitrogen/GIBCO) containing 1 ug/ml of human fibronectin (R&D systems, MN) was added to each well of 6 well tissue culture treated dish. Alternatively, regular growth factor MATRIGELTM was diluted 1:10 in DMEM/F12 and 1 ml of diluted MATRIGELTM was added to each well of 6 well tissue culture treated dish. Cells were passed with collagenase.
  • FIGS. 38A and 38B depict the expression of CXCR4 by embryonic stem cells cultured on fibronectin and MATRIGEL, respectively. Real-time PCR results ( FIG. 39 ) confirm that formation of definitive endoderm was equivalent on fibronectin and MATRIGELTM coated plates.
  • H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A (AA) for an additional three days.
  • H9 cells were cultured on plates coated with regular MATRIGEL at a 1:60 to 1:10 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
  • Cells from the human embryonic stem cell line H9 cultured on MATRIGELTM for at least five passages were seeded onto MEF feeders in ES media. When the cells reached 60 to 70% confluency they were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. Additional treatment groups include Wnt-3a at 20 ng/ml for all five days+10-100 ng/ml of activin A.
  • AFP expression levels of cells cultured in 100 ng/ml Activin A are similar to those seen in untreated controls. However, with the addition of Wnt3a to cells cultured in 100 ng/ml activin A, there is an increase in the expression of AFP that increases over time. When a lower concentration of Activin A is used, AFP expression is very high, regardless of the presence of Wnt3a ( FIG. 41A ). This suggests that a high concentration of Activin A is necessary to keep the cells from differentiating to extra-embryonic tissues.
  • CXCR4 positive cells ranged from 32-42% of the population in samples treated with a high concentration of Activin A but not treated with Wnt3a as compared to 23-33% of the population in samples treated with a high concentration of Activin A and Wnt3a at day 3 ( FIG. 41E ).
  • day 5 of treatment 28-32% of cells treated with a high concentration of activin A but not treated with Wnt-3a expressed CXCR4 as compared to 43-51% of cells treated with a high concentration of Activin A and Wnt3a ( FIG. 41F ).
  • Wnt-3a does not appear to play a significant role in differentiation of human embryonic stem cells, cultured on MEFs, to definitive endoderm. This suggests that the feeder layer is probably secreting sufficient Wnt-3a or analogous ligand to enhance activin A induced definitive endoderm formation.
  • H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt3a, 100 ng/ml Dikkopf-1 (DKK-1) and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days.
  • H9 cells were cultured on plates coated with Growth Factor Reduced MATRIGEL at a 1:30 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
  • the cultures were analyzed by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, GATA4, hTERT and Oct4.
  • AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak.
  • hTERT and Oct-4 are markers for self renewal and pluripotency respectively. Results are shown in FIG. 42 .
  • FIGS. 45A, 45B and 45C a) AA treatment b) AA+Wnt-3a c) AA+GSK-3B inhibitor.
  • FIGS. 46A and 46B Cultures were then analyzed by real-time PCR ( FIGS. 46A and 46B ). Robust expression of definitive endoderm markers were noted for cells treated with activin A+GSK-3B inhibitor or Wnt-3A as compared to cells treated with activin A only. These findings parallel our findings for human embryonic stem cells cultured on MATRIGELTM or human fibronectin coated plates.
  • GSK-3B inhibitors were evaluated at 100 nM: GSK-3B inhibitor VIII (Catalog#361549, Calbiochem, CA), GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA), GSK-3B inhibitor XI (Catalog#361553, Calbiochem, CA), GSK-3B inhibitor XII (Catalog#361554, Calbiochem, CA).
  • H1P54 ES cells were cultured on MATRIGELTM coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA)+/ ⁇ various GSK-3B inhibitors for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus high dose of AA.
  • FIGS. 47A and 47B depicts the gene expression of definitive endoderm markers at day 5.
  • GSK-3B inhibitor IX and XI were both effective in inducing DE formation as compared to GSK-3B inhibitor VIII and XII.
  • H9P49 embryonic stem cells were cultured on MATRIGELTM (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a (Catalog#1324-WN-002, R&D Systems, MN), and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. At day 5, cells were collected for evaluation by FACS and real-time PCR. As indicated in previous examples, this protocol resulted in robust upregulation of definitive endoderm markers, such as CXCR4 and SOX-17.
  • the resulting definitive endoderm cells at day 5 were exposed to the following media conditions to induce pancreatic endoderm formation: culturing in DMEM/F12 media supplemented with 2% FBS and 1 ⁇ M all-trans retinoic acid (RA) (Catalog#R2625, Sigma, MO), or 0.1-10 ⁇ M AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid, Catalog#A8843, Sigma, MO), or 0.1-1 ⁇ M TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid, Catalog#T3757, Sigma, MO) for 3 days.
  • RA all-trans retinoic acid
  • AM-580 and TTNPB are retinoic acid analogs with affinity for retinoic acid receptors.
  • RA treatment was followed by additional three day treatment in DMEM/F12 media supplemented with 2% FBS and 20-50 ng/ml bFGF (Catalog#F0291, Sigma, MO). Cultures were harvested and samples of mRNA were collected for analysis.
  • FIGS. 48A, 48B, 48C and 48D Gene expression analysis revealed that ( FIGS. 48A, 48B, 48C and 48D ) addition of 1 ⁇ M RA followed by exposure to bFGF significantly upregulates pancreatic endoderm markers, such as PDX-1. Furthermore, this protocol resulted in robust expression of foregut endoderm markers, such as CDX-2 and AFP. At 1 ⁇ M concentration, addition of RA analogs resulted in equivalent pancreatic endoderm and foregut markers. However, addition of 1 ⁇ M RA analogs resulted in more robust expression of AFP as compared to all-trans retinoic acid. However, addition of 10 ⁇ M AM-580 suppressed AFP and CDX-2 expression while maintaining a high expression of PDX-1.
  • Cell lysates were prepared from cells of each treatment group, with a mammalian cell lysis kit (Sigma-Aldrich, MO). Conditioned media from each treatment group was collected and concentrated. Cytokine array analysis was completed using Cytokine Array panels provided by RayBiotech, GA (http://www.raybiotech.com/). Table VII lists cytokine, growth factor, and receptor expression following normalization of the data and background subtraction. For each panel, positive and negative controls are also included. The data shown are two independent samples per cell treatment group (1, 2).
  • H1P55 ES cells were cultured on MATRIGELTM (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A+/ ⁇ 10-20 ng/ml of WNT-1 (PeproTech, NJ, Catalogue#120-17) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) and +/ ⁇ 10 or 20 ng/ml of WNT-1 for an additional three days.
  • the following combinations of WNT1+AA were tested:
  • FIGS. 49A and 49B displays the real-time PCR data for definitive endoderm markers following treatment of the H1 cells with low serum, AA and Wnt-1. Addition of 20 ng/ml of Wnt1 in the present of 100 ng/ml of AA resulted in significant upregulation of definitive endoderm markers (Bry, CXCR4, GSC, SOX17, HNF-3B, and GATA-4).
  • CST1 cystatin SN
  • /FL gb: AF240634.1 gb: NM_018440.1 NM_022642 Homo — ⁇ 2.24 0.12 2.97 0.42 ⁇ 3.78 0.07 2.51 0.44 sapiens chorionic somatomammotropin hormone 1 (placentallactogen) (CSH1), transcript variant 4, mRNA.
  • CSH1 chorionic somatomammotropin hormone 1
  • CSH1 chorionic somatomammotropin hormone 1 (placentallactogen) (CSH1), transcript variant 1, mRNA.
  • /PROD chorionic somatomammotropin hormone 1 (placentallactogen)
  • CSH2 chorionic somatomammotropin hormone 1
  • NPPB natriuretic peptide precursor B
  • /PROD natriuretic peptide precursor
  • B /FL gb: NM_002521.1 gb: M25296.1 AA352113 ESTs ST8SIA4 ⁇ 4.01 1.24 ⁇ 0.99 2.04 ⁇ 4.79 1.00 1.05 1.62 BM128432 Homo IGFBP5 ⁇ 2.73 1.11 2.31 2.30 ⁇ 3.48 0.56 4.45 2.02 sapiens full length insert cDNA clone YA81B05 NM_002770 Homo PRSS1 ⁇ 2.77 0.33 1.59 2.68 ⁇ 3.13 0.48 3.88 2.95 sapiens protease, serine, 2 (trypsin 2) (PRSS2), mRNA.
  • PRSS2 trypsin 2
  • /PROD protease, serine, 2 (trypsin 2)
  • /FL gb: M27602.1
  • Homo CSH1 ⁇ 1.58 0.91 2.48 0.38 ⁇ 3.33 0.13 1.77 0.49 sapiens chorionic somatomammotropin hormone-like 1 (CSHL1), transcript variant 3, mRNA.
  • /PROD chorionic somatomammotropin hormone 2
  • isoform 3precursor /FL gb: NM_022645.1 AI821586 ESTs, LOC440981 ⁇ 3.22 0.97 0.66 3.19 ⁇ 2.97 0.13 4.22 2.76 Moderately similar to JE0284 Mm- 1 cell derived transplantability- associated protein 1b ( H. sapiens ) AL121722 Human DNA — ⁇ 2.95 0.36 ⁇ 0.01 2.69 ⁇ 3.43 0.38 2.95 2.66 sequence from clone RP4-788L20 on chromosome 20 Contains the HNF3B (hepatocyte nuclear factor 3, beta) gene.
  • HNF3B hepatocyte nuclear factor 3, beta
  • CSH2 chorionic somatomammotropin hormone 2
  • /PROD chorionic somatomammotropin hormone 2
  • isoform4 /FL gb: NM_022646.1 AW007532 Human IGFBP5 0.31 0.25 4.59 1.53 0.72 0.09 6.19 1.58 insulin-like growth factor binding protein 5 (IGFBP5) mRNA NM_002160 Homo TNC ⁇ 0.24 0.29 2.23 0.80 ⁇ 0.81 0.81 2.85 0.82 sapiens hexabrachion (tenascin C, cytotactin) (HXB), mRNA.
  • PDGFB platelet- derived growth factor beta polypeptide
  • /PROD platelet- derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)
  • /FL gb: M12783.1 gb: NM_002 AW444761 ESTs CDKN2B ⁇ 3.35 0.90 0.39 0.52 ⁇ 3.11 0.88 0.80 0.25 BF223214 ESTs — ⁇ 0.48 0.08 1.69 2.04 ⁇ 1.51 0.25 4.09 1.98 AF154054 Homo GREM1 1.68 0.26 4.61 0.74 0.62 0.04 3.58 0.87 sapiens DRM (DRM) mRNA, complete cds.
  • DRM sapiens DRM
  • G protein- coupled receptor 87 /FL gb: NM_023915.1
  • WT1 Wilms tumor 1 isoform
  • D /FL gb: NM_024424.1
  • NM_033136 Homo FGF1 ⁇ 3.10 1.42 0.09 0.83 ⁇ 3.16 0.63 ⁇ 0.78 1.21 sapiens fibroblast growth factor 1 (acidic) (FGF1), transcript variant 2, mRNA.
  • GH2 sapiens growth hormone 2
  • CSHL1 chorionic somatomammotropin hormone-like 1
  • /PROD chorionic somatomammotropin hormone-like 1, isoform 4
  • /FL gb: NM_022580.1 J03580 Human, PTHLH ⁇ 2.72 0.33 ⁇ 0.80 0.40 ⁇ 4.05 0.41 ⁇ 1.42 0.73 parathyroid- like protein (associated with humoral hypercalcemia of malignancy) mRNA, complete cds.
  • /FL gb: J03580.1 BC029835 Homo LOC646867 ⁇ 2.66 1.12 1.01 1.85 ⁇ 1.40 0.41 3.03 1.61 sapiens , clone IMAGE: 5169759, mRNA.
  • AI452798 ESTs MYOCD 0.98 0.13 3.31 0.66 ⁇ 0.07 0.28 2.80 0.98 NM_022559 Homo CSH1 ⁇ 1.56 0.38 2.00 0.32 ⁇ 2.07 0.42 1.47 0.28 sapiens growth hormone 1 (GH1), transcript variant 2, mRNA.
  • /PROD growth hormone 1, isoform 2 precursor
  • /FL gb: NM_022559.1 NM_001318
  • CSHL1 sapiens chorionic somatomammotropin hormone-like 1
  • IGFBP-5 insulin-like growth factor binding protein 5
  • GH1 sapiens growth hormone 1
  • PRO2032 /FL gb: AF116683.1 gb: NM_018615.1 NM_022469
  • CSHL1 chorionic somatomammotropin hormone-like 1
  • /PROD cysteine knot superfamily 1
  • BMP antagonist 1 /FL gb: NM_013372.1 gb: AF110137.2 gb: AF045800.1 gb: AF154054.1 NM_022561 Homo CSH1 ⁇ 1.32 0.60 1.91 0.25 ⁇ 1.66 0.46 1.57 0.22 sapiens growth hormone 1 (GH1), transcript variant 4, mRNA.
  • PITX2 paired-like homeodomain transcription factor 2
  • /PROD serine (or cysteine) proteinase inhibitor, cladeE (nexin, plasminogen activator inhibitor type 1), membe NM_001480 Homo GALR1 ⁇ 1.90 0.68 0.04 0.73 ⁇ 2.42 0.72 ⁇ 0.34 0.73 sapiens galanin receptor 1 (GALR1), mRNA.
  • CKLF colon Kruppel-like factor
  • EBAF transforming growth factor beta superfamily
  • /PROD transforming growth factor
  • beta 4 /FL gb: U81523.1 gb: NM_003240.1
  • member B /FL gb: NM_004040.1 NM_001792
  • SRCL C-type lectin
  • IGFBP5 insulin-like growth factor binding protein 5
  • /PROD KIAA1389 protein NM_001394 Homo DUSP4 0.22 0.37 2.88 1.09 0.50 0.14 4.12 0.99 sapiens dual specificity phosphatase 4 (DUSP4), mRNA.
  • MBD2 methyl-CpG binding protein MBD2
  • /PROD methyl- CpG binding protein
  • MBD2 /FL gb: NM_003927.2
  • MILD1 Mix- like homeobox protein 1
  • TIMP3 tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory)
  • NM_001828 Homo CLC 2.17 0.21 3.53 1.07 0.45 0.08 2.11 1.27 sapiens Charot- Leyden crystal protein (CLC), mRNA.
  • CLC Charot- Leyden crystal protein
  • beta 1 /FL gb: M38449.1 gb: BC001180.1 gb: BC000125.1 gb: NM_000660.1 NM_016931 Homo NOX4 1.83 0.06 3.31 1.29 1.20 0.14 2.28 1.27 sapiens NADPH oxidase 4 (NOX4), mRNA.
  • ADM adrenomedullin
  • /PROD neuropilin- 2b(5)
  • /FL gb: AF280544.1 gb: AF280545.1 NM_014624 Homo S100A6 3.08 0.38 3.10 0.18 ⁇ 0.57 0.37 3.24 0.19 sapiens S100 calcium- binding protein A6 (calcyclin) (S100A6), mRNA.
  • NM_001553 Homo IGFBP7 3.41 0.19 4.63 1.07 1.65 0.10 3.34 1.16 sapiens insulin-like growth factor binding protein 7 (IGFBP7), mRNA.
  • IGFBP7 insulin-like growth factor binding protein 7
  • /FL gb: NM_001553.1 gb: L19182.1 BG285011 Homo ARID5B 0.05 0.22 1.79 0.21 ⁇ 0.95 0.42 1.16 0.55 sapiens mRNA; cDNA DKFZp586N012 (from clone DKFZp586N012) BE967311 Homo — 1.99 0.06 4.08 0.92 2.28 0.17 5.01 0.87 sapiens mRNA; cDNA DKFZp762O1615 (from clone DKFZp762O1615) BC005047 Homo DUSP6 2.50 0.32 4.04 0.67 1.56 0.16 2.94 0.84 sapiens , clone MGC: 12852,
  • CST4 cystatin S
  • Rho GDP dissociation inhibitor beta /FL gb: AF498927.1 AF329092 Homo DOC1 ⁇ 2.38 0.45 1.00 0.19 ⁇ 1.62 0.74 0.75 0.11 sapiens GPBP- interacting protein 90 mRNA, complete cds.
  • NTS neurotensin
  • QPCT glutaminyl- peptide cyclotransferase
  • NM_014942 Homo ANKRD6 0.62 0.04 2.17 1.11 0.59 0.23 3.30 1.07 sapiens KIAA0957 protein (KIAA0957), mRNA.
  • gb: NM_014942.1 AF260333 Homo C4orf18 ⁇ 1.58 0.10 0.21 1.21 ⁇ 3.19 1.07 1.60 1.12 sapiens AD036 mRNA, complete cds.
  • NM_006763 Homo BTG2 2.30 0.10 3.28 0.62 0.85 0.25 3.84 0.74 sapiens BTG family, member 2 (BTG2), mRNA.
  • transgelin 2 /FL gb: BC002616.1 AF078077 Homo GADD45B 1.30 0.17 3.41 0.31 0.61 0.12 3.05 0.29 sapiens growth arrest and DNA- damage- inducible protein GADD45beta mRNA, complete cds.
  • PRO2841 /FL gb: AF130095.1 H92988 tyrosine 3- C9orf19 2.00 0.06 3.54 0.91 1.93 0.14 4.61 0.81 monooxygenasetryptophan 5- monooxygenase activation protein, eta polypeptide X02761 Human FN1 5.67 0.17 6.65 0.54 4.52 0.21 7.21 0.42 mRNA for fibronectin (FN precursor).
  • /PROD fibronectin precursor AI016316 ESTs — 0.24 0.18 1.19 1.17 ⁇ 0.33 0.18 0.25 1.18 NM_006622 Homo PLK2 4.64 0.14 5.88 0.45 3.50 0.06 5.19 0.47 sapiens serum- inducible kinase (SNK), mRNA.
  • SNK serum- inducible kinase
  • NM_001458 Homo FLNC 3.28 0.17 4.17 0.70 2.11 0.17 3.39 0.64 sapiens filamin C, gamma (actin- binding protein-280) (FLNC), mRNA.
  • /FL gb: AF151909.1 gb: AF077041.1 gb: NM_016081.1 BC005858 Homo FN1 5.86 0.10 6.86 0.49 4.70 0.19 7.37 0.36 sapiens , clone MGC: 3255, mRNA, complete cds.
  • /PROD sialyltransferase 4A (beta- galactosidase alpha-2,3- sialytransferase)
  • /FL gb: L13972.1 gb: L29555.1
  • gb: NM_003033.1 AI222435 ESTs — ⁇ 4.14 1.52 ⁇ 0.73 0.32 ⁇ 2.33 0.62 ⁇ 0.33 0.12 NM_001924 Homo GADD45A 3.59 0.09 5.04 0.44 3.06 0.17 5.38 0.48 sapiens growth arrest and DNA- damage- inducible, alpha (GADD45A), mRNA.
  • /PROD growth arrest and DNA- damage- inducible
  • alpha /FL gb: M60974.1 gb: NM_001924.2 NM_001425
  • EMP3 epithelial membrane protein 3
  • gb U87947.1 AB017493
  • Homo KLF6 1.69 0.20 4.02 1.01 1.80 0.08 4.87 0.87 sapiens mRNA for DNA-binding zinc finger(GBF), complete cds.
  • /PROD microfibril- associated glycoprotein- 2
  • MAGP-2 /FL gb: NM_003480.1
  • gb: U37283.1 AI819043 ESTs CREB5 0.92 0.26 2.63 0.73 0.65 0.15 1.80 0.79 NM_001511
  • /FL gb: M36172.1 gb: M24121.1 gb: NM_002476.1 AB033831 Homo PDGFC 0.48 0.21 0.63 0.15 ⁇ 1.87 0.96 0.21 0.16 sapiens hSCDGF mRNA for spinal cord- derived growth factor, complete cds.
  • IGSF4 immunoglobulin superfamily
  • member 4 /FL gb: NM_014333.1 gb: AF132811.1 AF345910 Homo TTC29 0.35 0.26 1.53 0.93 ⁇ 0.51 0.52 0.91 0.72 sapiens NYD-SP14 mRNA, complete cds.
  • /PROD hypothetical protein AB051826 Homo RHOU 0.55 0.23 2.18 0.61 0.17 0.29 2.73 0.65 sapiens hG28K mRNA for GTP-binding protein like 1, complete cds.
  • HP protein (HP) mRNA complete cds.
  • /PROD cAMP response element- binding protein
  • CRE- BPa /FL gb: NM_004904.1
  • /FL gb: NM_018534.1 BE542563 ESTs LOC643277 2.20 0.10 0.54 1.49 ⁇ 3.73 0.40 ⁇ 1.74 1.96 AW005572 putative 47 kDa ANKS1B ⁇ 1.21 0.52 ⁇ 0.24 0.82 ⁇ 1.19 0.55 1.05 0.64 protein AW665892 paternally MFAP5 ⁇ 3.87 0.82 ⁇ 1.56 1.24 ⁇ 2.40 0.24 ⁇ 3.49 2.10 expressed 3 NM_006206 Homo PDGFRA 0.92 0.18 2.34 0.74 0.72 0.09 3.18 0.62 sapiens platelet- derived growth factor receptor, alpha polypeptide (PDGFRA), mRNA.
  • PDGFRA platelet- derived growth factor receptor, alpha polypeptide
  • /PROD platelet- derived growth factor receptor
  • alphapolypeptide /FL gb: NM_006206.1
  • NM_002425 Homo MMP10 ⁇ 1.21 0.70 1.21 0.88 ⁇ 0.37 0.28 0.52 0.45 sapiens matrix metalloproteinase 10 (stromelysin 2) (MMP10), mRNA.
  • MMP10 matrix metalloproteinase 10
  • TLE2 2.65 0.07 4.05 0.66 1.95 0.13 4.76 0.61
  • APR-1 APR-1 protein
  • NM_013959 Homo NRG1 1.50 0.20 3.22 0.68 1.34 0.13 3.78 0.75 sapiens neuregulin 1 (NRG1), transcript variant SMDF, mRNA.
  • SMDF /FL gb: L41827.1 gb: NM_013959.1 NM_004887 Homo CXCL14 0.70 0.07 1.70 0.37 ⁇ 0.15 0.11 2.36 0.63 sapiens small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK) (SCYB14), mRNA.
  • BA108L7.2 rat tricarboxylate carrier-like protein
  • /PROD acyl- Coenzyme A oxidase 3
  • pristanoyl /FL gb: NM_003501.1
  • AF144103 Homo CXCL14 1.69 0.13 2.16 0.55 0.40 0.21 2.73 0.69 sapiens NJAC protein (NJAC) mRNA, complete cds.
  • NM_004815 Homo ARHGAP29 2.18 0.02 3.18 0.82 1.69 0.03 4.15 0.69 sapiens PTPL1- associated RhoGAP 1 (PARG1), mRNA.
  • PARG1 PTPL1- associated RhoGAP 1
  • /FL gb: U90920.1 gb: NM_004815.1
  • BG028597 ESTs COL11A1 0.19 0.02 1.61 1.02 ⁇ 0.11 0.38 0.48 1.09 AB019562
  • Homo SPP1 0.58 0.14 2.79 1.07 1.10 0.02 1.21 1.06 sapiens mRNA expressed only in placental villi, clone SMAP41.
  • NM_002346 Homo LY6E 3.54 0.06 3.92 1.16 1.81 0.23 4.93 1.39 sapiens lymphocyte antigen 6 complex, locus E (LY6E), mRNA.
  • /PROD lymphocyte antigen 6 complex
  • locus E /FL gb: U42376.1 gb: NM_002346.1
  • BF344237 Homo — ⁇ 2.82 0.20 ⁇ 0.92 0.64 ⁇ 0.73 0.67 ⁇ 1.25 1.03 sapiens mRNA; cDNA DKFZp564N1116 (from clone DKFZp564N1116) NM_014481 Homo APEX2 2.76 0.15 3.18 0.80 1.57 0.11 3.91 0.93 sapiens apurinicapyrimidinic endonuclease (APEX nuclease)- like 2 protein (APEXL2), mRNA.
  • APEX nuclease apurinicapyrimidinic endonuclease
  • HE4 putative ovarian carcinoma marker
  • DKK1 Xenopus laevis
  • ANGPT1 angiopoietin 1
  • PCSK5 proprotein convertase subtilisinkexin type 5
  • MME membrane metallo- endopeptidase
  • SARP2 apoptosis related protein 2
  • NM_003182 Homo TAC1 1.89 0.20 1.65 0.13 2.46 0.09 1.48 0.26 sapiens tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma) (TAC1), transcript variant beta, mRNA.
  • /FL gb: NM_153262.1 AA156873 albumin PELO 0.77 0.12 ⁇ 0.23 0.93 0.65 0.14 ⁇ 2.33 1.01 BC012375 Homo ARSG ⁇ 0.46 0.48 ⁇ 0.78 0.42 0.57 0.22 ⁇ 0.41 0.55 sapiens , Similar to KIAA1001 protein, clone MGC: 8996 IMAGE: 3882163, mRNA, complete cds.
  • /PROD LYST- interacting protein LIP3 BF435123 bromodomain — 2.28 0.17 1.31 0.51 2.70 0.12 1.30 1.00 and PHD finger containing, 3 AK056212 Homo — 1.03 0.14 0.14 0.27 1.10 0.07 ⁇ 1.41 0.51 sapiens cDNA FLJ31650 fis, clone NT2RI2004079. NM_001446 Homo FABP7 2.07 0.15 0.74 0.29 2.20 0.04 1.08 0.16 sapiens fatty acid binding protein 7, brain (FABP7), mRNA.
  • FABP7 sapiens fatty acid binding protein 7, brain
  • /PROD fatty acid binding protein 7
  • brain /FL gb: U81235.1 gb: D88648.1 gb: U51338.1 gb: NM_001446.1 gb: D50373.1 AW051591 ESTs, RNF175 1.41 0.32 0.22 0.10 2.60 0.06 0.96 0.20 Moderately similar to unnamed protein product ( H. sapiens ) BC041970 Homo C9orf122 0.74 0.03 ⁇ 0.35 0.49 0.76 0.06 ⁇ 2.31 1.25 sapiens , clone IMAGE: 5302687, mRNA.
  • AI654224 ESTs — 0.46 0.36 0.14 0.44 1.03 0.07 ⁇ 0.81 0.85 AA167449 nuclear XIST ⁇ 3.08 0.10 ⁇ 0.47 2.59 4.59 0.07 2.80 1.86 receptor subfamily 1, group I, member 3 BF977837 KIAA0527 SUSD5 1.64 0.02 0.50 0.68 1.96 0.16 ⁇ 1.52 1.66 protein BC029425 Homo FILIP1 ⁇ 0.83 0.45 ⁇ 0.03 0.62 0.76 0.20 ⁇ 0.29 0.44 sapiens , Similar to KIAA1275 protein, clone IMAGE: 4616553, mRNA.
  • TRHDE thyrotropin- releasing hormone degrading ectoenzyme
  • /FL gb: M31213.1 Z92546 Human DNA — 0.58 0.13 ⁇ 1.16 0.54 1.07 0.12 ⁇ 1.27 0.14 sequence from clone CTA-65A6 on chromosome 22q11-12 Contains the 3 part of the gene for the ortholog of rat CAIN (KIAA0330), the gene for a novel Sushi domain (SCR repeat) containing protein similar to Mucins, ESTs, an STS, GSSs and two . . .
  • /PROD proteolipid protein (Pelizaeus- Merzbacherdisease, spastic paraplegia 2, uncomplicated)
  • /FL gb: BC002665.1 NM_001243
  • /FL gb: NM_152647.1 BC036917 Homo C6orf141 ⁇ 0.82 0.30 ⁇ 1.18 0.40 0.88 0.11 ⁇ 2.18 0.62 sapiens , clone MGC: 46457 IMAGE: 5201433, mRNA, complete cds.
  • ODC1 oxodicarboxylate carrier
  • /PROD fatty acid binding protein 7
  • brain /FL gb: U81235.1 gb: D88648.1 gb: U51338.1 gb: NM_001446.1 gb: D50373.1 AW004016 ESTs ST6GAL2 1.57 0.14 0.89 0.32 3.30 0.07 1.48 0.43 AW072790 contactin 1 CNTN1 0.50 0.20 0.18 0.98 1.84 0.10 1.01 1.39 AL512686 Homo GNAO1 2.16 0.50 ⁇ 0.28 1.03 1.67 0.18 ⁇ 1.12 0.37 sapiens mRNA; cDNA DKFZp761I177 (from clone DKFZp761I177).
  • AI638063 ESTs CBX5 0.49 0.16 ⁇ 0.34 0.34 0.98 0.11 ⁇ 2.71 1.22 AU157049 Homo LOC153346 2.24 0.20 0.34 0.47 2.24 0.09 ⁇ 0.44 0.96 sapiens cDNA FLJ14284 fis, clone PLACE1005898 NM_002738 Homo PRKCB1 0.99 0.28 ⁇ 0.15 0.45 1.42 0.08 ⁇ 1.02 1.04 sapiens protein kinase C, beta 1 (PRKCB1), mRNA.
  • /PROD protein kinase C
  • beta 1 /FL gb: NM_002738.1
  • AI185136 ESTs DYDC2 0.12 0.31 ⁇ 0.80 0.08 0.72 0.08 ⁇ 0.60 0.09
  • AI928037 ESTs RPIB9 ⁇ 0.55 0.55 ⁇ 2.71 0.74 0.49 0.12 ⁇ 0.89 0.27 NM_016179
  • Homo TRPC4 ⁇ 1.51 0.85 ⁇ 1.87 1.42 1.71 0.11 ⁇ 0.13 1.23 sapiens transient receptor potential channel 4 (TRPC4), mRNA.
  • TRPC4 transient receptor potential channel 4
  • NM_005375 Homo MYB 2.11 0.17 ⁇ 0.52 0.18 2.24 0.03 0.10 0.08 sapiens v- myb avian myeloblastosis viral oncogene homolog (MYB), mRNA.
  • MYB myb avian myeloblastosis viral oncogene homolog
  • /PROD v- myb avian myeloblastosis viral oncogenehomolog
  • /FL gb: NM_005375.1
  • NM_017671 Homo C20orf42 3.19 0.12 0.97 0.70 3.26 0.07 0.16 0.94 sapiens hypothetical protein FLJ20116 (FLJ20116), mRNA.
  • GALNAC-4- ST1 N- acetylgalactosamine- 4-O- sulfotransferase
  • KCNG3 potassium voltage- gated channel, subfamily G, member 3
  • FEZ1 fasciculation and elongation protein zeta 1 (zygin I) (FEZ1), transcript variant 1, mRNA.
  • PAH phenylalanine hydroxylase
  • /PROD proteasome (prosome, macropain) subunit, betatype, 9 (large multifunctional protease 2)
  • /FL gb: U01025.1 gb: NM_002800.1 BE972639 ESTs LOC646326 0.00 0.15 ⁇ 2.71 0.55 0.33 0.22 ⁇ 2.24 0.61 BC044830 Homo C10orf96 1.38 0.12 ⁇ 0.32 0.82 2.28 0.06 ⁇ 1.39 0.90 sapiens , Similar to RIKEN cDNA 1700011F14 gene, clone MGC: 35062 IMAGE: 5166167, mRNA, complete cds.
  • PAK5 /FL gb: AB040812.1 AI985987 ESTs, SCNN1G ⁇ 0.28 0.31 ⁇ 0.99 0.35 0.12 0.19 ⁇ 2.70 0.59 Moderately similar to ALU1_HUMAN ALU SUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY ( H. sapiens ) NM_001877 Homo CR2 1.50 0.13 ⁇ 0.43 1.05 2.35 0.14 ⁇ 1.44 1.51 sapiens complement component (3dEpstein Barr virus) receptor 2 (CR2), mRNA.
  • complement component (3dEpstein Barr virus) receptor 2 (CR2)
  • MDA5 melanoma differentiation associated protein-5
  • AI056877 Human DNA LOC200230 0.11 0.30 ⁇ 2.37 0.94 1.15 0.18 ⁇ 1.35 0.75 sequence from clone RP4-530I15 on chromosome 20.
  • PTPN1 protein tyrosine phosphatase, non- receptor type 1 (EC 3.1.3.48), the gene for a novel protein similar to placental protein DIFF40, an RPL36 (60S Ribos NM_002522 Homo NPTX1 2.35 0.25 ⁇ 0.48 0.19 2.17 0.09 ⁇ 1.19 0.83 sapiens neuronal pentraxin I (NPTX1), mRNA.
  • Hip Mus musculus hedgehog- interacting protein
  • AV715309 ESTs, C20orf118 4.28 0.13 1.89 0.39 4.76 0.10 1.48 0.42 Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCE CONTAMINATION WARNING ENTRY ( H.
  • /FL gb: NM_002164.1
  • Homo SMOC2 1.25 0.12 ⁇ 2.04 0.48 1.94 0.06 ⁇ 2.27 1.12 sapiens mRNA for SMAP-2b, complete cds.
  • MCP-1 monocyte chemotactic protein ⁇ 2.27571 5.13499 2.95543 2.92E ⁇ 02 (human, aortic endothelial cells, mRNA, 661 nt).
  • /PROD MCP-1 Homo sapiens Mix-like homeobox ⁇ 1.54648 4.47601 0.921971 2.01E ⁇ 02 protein 1 (MILD1) mRNA, complete cds.
  • /PROD glutamate decarboxylase 1
  • isoform GAD25 /FL gb: NM_013445.1
  • /PROD cardiac ventricular troponin
  • C /FL gb: NM_003280.1
  • gb: AF020769.1 ESTs ⁇ 2.89554 3.42817 0.926036 2.62E ⁇ 02
  • Homo sapiens fibroblast growth factor 8 ⁇ 4.32791 2.19561 0.015827 5.91E ⁇ 03 (androgen-induced) (FGF8), mRNA.
  • /PROD preproapolipoprotein AI
  • MLL septin-like fusion /FL gb: AF123052.1 gb: NM_006640.1 Homo sapiens cystatin S (CST4), 0.92448 6.48842 3.87036 7.20E ⁇ 03 mRNA.
  • /PROD glutamate decarboxylase 1
  • GAD67 /FL gb: NM_000817.1
  • L16888.1 ESTs ⁇ 0.731491 5.43249 3.52168 1.60E ⁇ 02 ESTs ⁇ 2.03591 3.38924 0.760984 2.34E ⁇ 03
  • HP Homo sapiens haptoglobin
  • TM transmembrane domain
  • SEMA5A short cytoplasmic domain
  • /PROD sema domain, seven thrombospondin repeats (type1 and type 1-like), transmem Homo sapiens mRNA; cDNA ⁇ 0.835182 5.22406 3.69882 2.08E ⁇ 02 DKFZp586J0624 (from clone DKFZp586J0624); complete cds.
  • /PROD glutamate decarboxylase 1
  • isoform GAD25 /FL gb: NM_013445.1
  • Homo sapiens clone 23736 mRNA ⁇ 0.047182 5.79006 4.50744 1.74E ⁇ 02 sequence
  • Homo sapiens glycophorin E (GYPE) ⁇ 2.01601 1.79002 ⁇ 1.50134 1.97E ⁇ 02 mRNA.
  • Ig immunoglobulin domain
  • SEMA3E short basic domain
  • mRNA short basic domain
  • Kell blood group antigen /FL gb: BC003135.1 gb: NM_000420.1 splicing factor, arginineserine-rich 5 0.7481 5.68934 4.27169 2.97E ⁇ 03 Human prostatic secretory protein 57 ⁇ 3.01313 1.46338 ⁇ 0.40767 1.97E ⁇ 02 mRNA, complete cds.
  • /PROD KIAA1409 protein Homo sapiens mRNA; cDNA ⁇ 2.63427 1.64513 ⁇ 0.3056 2.52E ⁇ 02 DKFZp434D0818 (from clone DKFZp434D0818) ESTs 0.35393 5.14775 3.74875 3.16E ⁇ 02 Homo sapiens TBX3-iso protein (TBX3- ⁇ 2.34566 2.45238 1.07038 1.31E ⁇ 02 iso), mRNA.
  • GATA-6 /FL gb: U66075.1 gb: NM_005257.1 gb: D87811.1 Homo sapiens ankyrin-like with ⁇ 1.10879 3.93484 2.81939 1.29E ⁇ 02 transmembrane domains 1 (ANKTM1), mRNA G protein-coupled receptor 49 0.265509 4.46257 2.50537 2.19E ⁇ 02 Homo sapiens growth differentiation 1.67253 5.34944 2.87486 8.62E ⁇ 05 factor 3 (GDF3), mRNA.
  • ANKTM1 transmembrane domains 1
  • G protein-coupled receptor 49 0.265509 4.46257 2.50537 2.19E ⁇ 02
  • GDF3 8.62E ⁇ 05 factor 3
  • /PROD glycophorin HeP2 Homo sapiens cDNA FLJ12993 fis, 0.147259 4.12241 2.06949 6.84E ⁇ 03 clone NT2RP3000197 Homo sapiens presenilin stabilization ⁇ 0.86173 2.85614 0.56745 6.09E ⁇ 03 factor b (PSF) mRNA, complete cds; alternatively spliced.
  • Homo sapiens LYST-interacting protein ⁇ 0.682149 2.34271 ⁇ 0.31253 9.87E ⁇ 03 LIP3 mRNA, partial cds.
  • /PROD LYST- interacting protein LIP3 putative 47 kDa protein ⁇ 0.123937 3.2914 1.05247 3.72E ⁇ 03 Homo sapiens protein S (alpha) 0.577604 4.00035 1.7883 7.89E ⁇ 03 (PROS1), mRNA.
  • FGFR4 soluble-form splice variant
  • /PROD fibroblast growth factor receptor 4
  • soluble-form splice variant /FL gb: NM_022963.1 gb: AF202063.1 X75208
  • /FEATURE cds ⁇ 1.36729 2.60456 1.08204 1.58E ⁇ 02
  • DEFINITION HSPTKR H. sapiens HEK2 mRNA for protein tyrosine kinase receptor Homo sapiens chemokine receptor 2.30557 6.72969 5.66933 3.31E ⁇ 02 CXCR4 mRNA, complete cds.
  • ALU4_HUMAN ALU SUBFAMILY SB2 SEQUENCE CONTAMINATION WARNING ENTRY ( H. sapiens ) Homo sapiens arrestin, beta 1 (ARRB1), ⁇ 0.158844 2.41791 ⁇ 0.40915 1.12E ⁇ 02 transcript variant 1, mRNA.
  • DKK1 homolog 1
  • BMP2 bone morphogenetic 2.04506 6.19114 5.02927 4.98E ⁇ 02 protein 2
  • gb U65932.1
  • gb U68186.1
  • /FL gb: BC016043.1 ESTs ⁇ 2.01523 2.16773 1.12403 1.99E ⁇ 02 Homo sapiens oxoglutarate ⁇ 0.032595 2.3997 ⁇ 0.38533 1.45E ⁇ 02 dehydrogenase (lipoamide) (OGDH), mRNA.
  • /PROD oxoglutarate dehydrogenase (lipoamide)
  • /FL gb: D10523.1 gb: BC004964.1 gb: NM_002541.1 ESTs, Weakly similar to T32252 2.20966 5.35974 3.33255 1.12E ⁇ 04 hypothetical protein T15B7.2- Caenorhabditis elegans ( C.
  • BNIP3 kD-interacting protein 3
  • /PROD hypothetical protein Homo sapiens cystatin C (amyloid 2.08601 5.30525 3.51965 2.20E ⁇ 03 angiopathy and cerebral hemorrhage) (CST3), mRNA.
  • /PROD cystatin C (amyloid angiopathy and cerebralhemorrhage)
  • /FL gb: NM_000099.1 ESTs, Moderately similar to NFY-C ⁇ 0.749407 2.00148 ⁇ 0.24268 2.34E ⁇ 03 ( H. sapiens ) ESTs 2.54243 5.75201 3.97307 2.60E ⁇ 02 Homo sapiens sialyltransferase (STHM), ⁇ 0.68709 2.89586 1.50348 3.70E ⁇ 02 mRNA.
  • /PROD SG2NA beta isoform
  • MAZ purine-binding transcription factor
  • CARP 3.44E ⁇ 02 protein
  • porcupine /FL gb: AF317059.1 gb: AF317058.1 gb: NM_022825.1 collagen, type V, alpha 1 1.5875 3.98844 1.66569 1.17E ⁇ 03
  • /FL gb: D90279.1 gb: NM_000093.1 gb: M76729.1 Homo sapiens forkhead box F2 ⁇ 1.54469 1.02305 ⁇ 1.11517 6.95E ⁇ 03 (FOXF2), mRNA.
  • /PROD forkhead box
  • F2 /FL gb: U13220.1
  • FKBP38 binding protein homologue
  • putative sterol reductase 1.66857 4.3767 2.47833 5.37E ⁇ 04 SR-1 (TM7SF2) mRNA, complete cds.
  • alpha /FL gb
  • MERTK tyrosine kinase
  • /FL gb: M36172.1 gb: M24121.1 gb: NM_002476.1 Homo sapiens phosphatidylinositol-4- 1.34876 4.12788 2.46657 3.66E ⁇ 03 phosphate 5-kinase, type I, beta (PIP5K1B), mRNA.
  • /PROD dystrophin Dp427p2 isoform
  • /FL gb: NM_004010.1 Homo sapiens , clone MGC: 14801, 2.03928 3.96694 1.51059 5.65E ⁇ 04 mRNA, complete cds.
  • /PROD Unknown (protein for MGC: 14801)
  • /FL gb: BC005997.1
  • /PROD enolase 2, (gamma, neuronal)
  • /FL gb: NM_001975.1 gb: BC002745.1 gb: M22349.1 Homo sapiens AD036 mRNA, complete ⁇ 0.503334 2.65787 1.60278 3.61E ⁇ 02 cds.
  • AD036 /FL gb: AF260333.1 ESTs ⁇ 2.07933 1.00157 ⁇ 0.1307 2.39E ⁇ 02 Homo sapiens keratin 19 (KRT19), 3.80582 6.96535 5.91937 2.11E ⁇ 02 mRNA.
  • MAPK10 mitogen-activated protein
  • /PROD KIAA1161 protein Homo sapiens , Similar to B9 protein, 1.43881 3.80457 2.29205 8.62E ⁇ 05 clone MGC: 11339, mRNA, complete cds.
  • Human DNA sequence from clone RP4- 2.82768 5.54517 4.43909 2.04E ⁇ 03 781L3 on chromosome 1p34.3-36.11 Contains a pseudogene similar to IFITM3 (interferon inducedntransmembrane protein 3 (1- 8U)), STSs and GSSs Homo sapiens KIAA0127 gene product 2.05236 4.43794 3.00216 3.12E ⁇ 02 (KIAA0127), mRNA.
  • IFITM3 interferon inducedntransmembrane protein 3 (1- 8U)
  • STSs and GSSs Homo sapiens KIAA0127 gene product 2.05236 4.43794 3.00216 3.12E ⁇ 02 (KIAA0127), mRNA.
  • TCF2 hepatic nuclear factor
  • /PROD transcription factor 2
  • isoform a /FL gb: NM_000458.1 H2A histone family, member X 1.37085 2.48515 ⁇ 0.04057 1.34E ⁇ 02 Homo sapiens H1 histone family, 3.21701 5.68105 4.50969 4.29E ⁇ 03 member X (H1FX), mRNA.
  • /PROD adaptor-related protein complex 3
  • beta 2subunit /FL gb: AF022152.1
  • Homo sapiens Similar to RIKEN cDNA 0.087468 2.45867 1.42701 3.35E ⁇ 03 C330013D18 gene, clone MGC: 11226, mRNA, complete cds Homo sapiens cDNA: FLJ22731 fis, ⁇ 2.30948 1.62817 2.24143 4.15E ⁇ 02 clone HSI15841.
  • testican 3 /FL gb: NM_016950.1 gb: BC000460.1 gb: BC003017.1 Homo sapiens gelsolin (amyloidosis, 1.6198 4.54047 4.45008 1.07E ⁇ 02 Finnish type) (GSN), mRNA.
  • SARP2 3.31E ⁇ 02 related protein 2
  • /FL gb: NM_173648.1 Homo sapiens renal tumor antigen ⁇ 0.305406 2.4556 2.09136 3.30E ⁇ 02 (RAGE), mRNA.
  • MAP3K8 kinase kinase kinase 8
  • EHHADH hydratase3-hydroxyacyl Coenzyme A dehydrogenase
  • SFRP1 protein 1
  • triadin /FL gb: U18985.1 ⁇ 1.61408 0.713083 0.566175 1.87E ⁇ 02 gb: NM_006073.1 Homo sapiens adlican mRNA, complete 0.872786 3.20404 3.02406 4.21E ⁇ 02 cds.
  • /PROD synovial sarcoma
  • X breakpoint 4 /FL gb: BC005325.1 ESTs ⁇ 3.61193 ⁇ 0.9408 ⁇ 1.53439 2.43E ⁇ 03 ESTs ⁇ 0.619714 1.93617 1.44843 4.14E ⁇ 02 ESTs 0.446983 3.14315 2.48676 1.98E ⁇ 02 Homo sapiens flavin containing ⁇ 0.476203 1.99948 1.55491 3.73E ⁇ 02 monooxygenase 5 (FMO5), mRNA.
  • FMO5 monooxygenase 5
  • /PROD sialyltransferase 8 (alpha-2,8-polysialytransferase)
  • D /FL gb: NM_005668.1 gb: L41680.1
  • hypothetical protein MGC4342 ⁇ 0.403018 2.01722 1.41263 1.97E ⁇ 02 /FL gb: NM_024329.1 gb: BC003033.1 Homo sapiens PHD finger protein 1 0.620171 2.95191 2.42296 2.32E ⁇ 02 (PHF1), transcript variant 2, mRNA.
  • /PROD Unknown (protein for IMAGE: 3840937) Homo sapiens phosphoglucomutase 1 4.74364 7.09524 6.43056 2.21E ⁇ 02 (PGM1), mRNA.
  • ASM Human acid sphingomyelinase
  • /PROD KIAA1758 protein prostaglandin E receptor 4 (subtype 2.58497 0.196846 0.527172 1.42E ⁇ 02 EP4)
  • /FL gb: L25124.1 gb: D28472.1 gb: NM_000958.1 gb: L28175.1
  • HRC10126 Homo sapiens heparan sulfate 3.18462 0.571052 0.709206 4.54E ⁇ 02 (glucosamine) 3-O-sulfotransferase 2 (HS3ST2), mRNA.
  • SCHIP-1 protein 1
  • /FL gb: AB002384.1 ESTs 3.22146 0.851355 0.404487 3.41E ⁇ 03 Human glioma pathogenesis-related 3.20187 0.502872 0.348681 1.34E ⁇ 02 protein (GliPR) mRNA, complete cds.
  • Homo sapiens mRNA cDNA 6.06894 3.31919 3.4798 4.97E ⁇ 02 DKFZp761I1912 (from clone DKFZp761I1912) ESTs 4.12419 1.53997 1.88239 5.68E ⁇ 03 Homo sapiens fibroblast growth factor 2 5.49297 2.64534 2.76054 4.71E ⁇ 02 (basic) (FGF2), mRNA.
  • /PROD KIAA0559 protein Human 65-kilodalton phosphoprotein 3.25522 0.620942 1.15317 2.88E ⁇ 02 (p65) mRNA, complete cds.
  • SGK regulated kinase
  • a kinase (PRKA) anchor protein 2 6.03149 3.65135 4.63469 1.17E ⁇ 02 ESTs 3.8522 1.17894 0.483367 3.08E ⁇ 02 Homo sapiens similar to rat 1.74077 ⁇ 1.45032 ⁇ 1.24979 5.69E ⁇ 04 tricarboxylate carrier-like protein (BA108L7.2), mRNA.
  • LOC51339 novel gene-3 protein
  • DRM Homo sapiens DRM
  • /PROD bicarbonate transporter- related protein
  • BTR1 /FL gb: AF336127.1 ESTs 1.08377 ⁇ 1.27197 0.228305 4.76E ⁇ 02 ESTs 2.94657 0.127188 1.20032 1.43E ⁇ 02 Homo sapiens mRNA; cDNA 2.47798 ⁇ 0.26439 0.894731 4.51E ⁇ 03 DKFZp434B2016 (from clone DKFZp434B2016).
  • /PROD synaptotagmin interacting protein STIP1 Homo sapiens connexin 26 (GJB2) 2.86049 ⁇ 0.57377 ⁇ 0.09328 4.27E ⁇ 04 mRNA, complete cds.
  • BDNF neurotrophic factor
  • Homo sapiens clone IMAGE 451939, 5.15776 2.22786 3.40438 2.60E ⁇ 02 mRNA sequence
  • proprotein convertase subtilisinkexin type 5 /FL gb: U56387.2 gb: NM_006200.1 Cluster Incl.
  • /FL gb: AB017269.1 gb: NM_016192.2 gb: AF179274.2 gb: AF242222.1 Homo sapiens , clone MGC: 3328, 3.54625 0.772486 2.30168 4.16E ⁇ 02 mRNA, complete cds.
  • PCAF p300CBP-associated factor
  • /PROD Similar to TAF5-like RNA polymerase II, p300CBP- associated factor (PCAF)-associat Homo sapiens mRNA; cDNA 1.42404 ⁇ 1.99222 ⁇ 0.77126 3.21E ⁇ 02 DKFZp761J1324 (from clone DKFZp761J1324) solute carrier family 30 (zinc 2.82535 0.288493 2.38889 0.00E+00 transporter), member 1 antizyme inhibitor 3.65887 0.37346 1.74912 1.86E ⁇ 02 Homo sapiens GRO1 oncogene 2.51473 0.107263 2.38874 7.16E ⁇ 04 (melanoma growth stimulating activity, alpha) (GRO1), mRNA.
  • PCAF p300CBP- associated factor
  • /PROD GRO1 oncogene (melanoma growth stimulatingactivity, alpha)
  • /FL gb: NM_001511.1 Homo sapiens proprotein convertase 4.90999 0.871309 1.52441 1.71E ⁇ 02 subtilisinkexin type 5 (PCSK5), mRNA.
  • INAM1 pro
  • RODH hydroxysteroid epimerase
  • /PROD regulator of G-protein signalling 2
  • 24 kD /FL gb: L13463.1 gb: NM_002923.1 ESTs 4.49987 1.01623 2.35659 1.86E ⁇ 03 ESTs 0.623422 ⁇ 1.94907 0.317138 8.95E ⁇ 04 ESTs 0.326084 ⁇ 1.69499 1.13586 1.10E ⁇ 02 ESTs 3.82952 0.673296 2.37112 3.92E ⁇ 02
  • /PROD KIAA0930 protein Homo sapiens forkhead transcription 1.51993 ⁇ 2.14186 ⁇ 0.86243 3.75E ⁇ 02 factor FOXL2 (BPES), mRNA.
  • MBD2 protein MBD2
  • K- cadherin (fetalkidney) /FL gb: BC000019.1 ESTs 0.133612 ⁇ 3.77953 ⁇ 1.56636 1.40E ⁇ 02 paternally expressed 3 2.87581 ⁇ 1.67669 ⁇ 0.0118 2.17E ⁇ 02 Homo sapiens Charot-Leyden crystal 3.30086 ⁇ 0.41644 2.1096 2.08E ⁇ 02 protein (CLC), mRNA.
  • TAC1 transcript variant beta
  • PDGFRA alpha-receptor
  • CSH2 somatomammotropin hormone 2
  • /PROD chorionic somatomammotropin hormone 2
  • isoform4 /FL gb: NM_022646.1
  • HERP1 /FL HES-related repressor protein 1
  • HERP1 /FL gb: NM_012259.1
  • SCCA2b /FL gb: AB046400.1 Homo sapiens KIAA0469 gene product 4.03097 ⁇ 1.94826 2.56518 2.49E ⁇ 04 (KIAA0469), mRNA.

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Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Description

    FIELD OF THE INVENTION
  • The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.
    • BACKGROUND
  • Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent stem cells, such as, for example, embryonic stem cells.
  • In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF-3beta, GATA4, Mixl1, CXCR4 and Sox-17.
  • Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, Pdx1. In the absence of Pdx1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, Pdx1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.
  • Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.
  • In one example, Hori et al. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled β cells.
  • In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4.
  • Micallef et al. reports that retinoic acid can regulate the commitment of embryonic stem cells to form Pdx1 positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdx1 expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).
  • Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdx1. Their results show that exogenous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, P48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).
  • Skoudy et al. reports that activin A (a member of the TGFβ superfamily) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouse embryonic stem cells. The maximal effect was observed using 1 nM activin A. They also observed that the expression level of insulin and Pdx1 mRNA was not affected by retinoic acid; however, 3 nM FGF7 treatment resulted in an increased level of the transcript for Pdx1 (Biochem. J. 379: 749, 2004).
  • Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into Pdx1 positive cells. They observed that TGFβ2 reproducibly yielded a higher proportion of Pdx1 positive cells (Genes Cells. 2005 June; 10(6): 503-16.).
  • Gordon et al. demonstrated the induction of brachyury+/HNF-3beta+ endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A1).
  • Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the anterior primitive streak”.
  • However, the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.
  • Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).
  • D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (D'Amour K A et al. 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into Pdx1 positive cells after addition of FGF-10 (US 2005/0266554A1).
  • D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states “We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones”.
  • In another example, Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of n-butyrate and activin A. The cells were then cultured with TGFβ antagonists such as Noggin in combination with EGF or betacellulin to generate Pdx1 positive cells. The terminal differentiation was induced by nicotinamide.
  • In one example, Benvenistry et al. states: “We conclude that over-expression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo” (Benvenistry et al, Stem Cells 2006; 24:1923-1930).
  • Therefore, there still remains a significant need to develop conditions for establishing pluripotent stem cell lines that can be expanded to address the current clinical needs, while retaining the potential to differentiate into pancreatic endocrine cells, pancreatic hormone expressing cells, or pancreatic hormone secreting cells. We have taken an alternative approach to improve the efficiency of differentiating human embryonic stem cells toward pancreatic endocrine cells.
    • SUMMARY
  • In one embodiment, the present invention provides a method for differentiating pluripotent stem cells, comprising the steps of:
      • a. Culturing the pluripotent stem cells, and
      • b. Differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage,
      • c. Differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage, and
      • d. Differentiating the cells expressing markers characteristics of the pancreatic endoderm lineage into cells expressing markers characteristics of the pancreatic endocrine lineage.
  • In one embodiment, cells expressing markers characteristic of the definitive endoderm lineage are differentiated from pluripotent stem cells by treating pluripotent stem cells by any one of the following methods:
      • a. Culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration, or
      • b. Culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration, or
      • c. Culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum, or
      • d. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and culturing the pluripotent stem cells with activin A and a Wnt ligand, or
      • e. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum, then culturing the pluripotent stem cells with activin A in a second culture medium containing serum, or
      • f. Culturing the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a second culture medium containing serum of a different concentration.
  • In one embodiment, cells expressing markers characteristic of the pancreatic endoderm lineage are differentiated from cells expressing markers characteristic of the definitive endoderm lineage by treating cells expressing markers characteristic of the definitive endoderm lineage by any one of the following methods:
      • a. Treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and a hedgehog signaling pathway inhibitor, then removing the medium containing the fibroblast growth factor and the hedgehog signaling pathway inhibitor and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and the hedgehog signaling pathway inhibitor, or
      • b. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor, or
      • c. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid, then removing the retinoic acid and subsequently treating the cells with at least one fibroblast growth factor.
  • In one embodiment, cells expressing markers characteristic of the pancreatic endocrine lineage are differentiated from cells expressing markers characteristic of the pancreatic endoderm lineage by treating cells expressing markers characteristic of the pancreatic endoderm lineage by any one of the following methods:
      • a. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF, or
      • b. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF, or
      • c. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, or
      • d. Culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, or
      • e. Treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway.
  • In one embodiment, the present invention provides a method for treating a patient suffering from diabetes, comprising the steps of:
      • a. Culturing pluripotent stem cells,
      • b. Differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage,
      • c. Differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage,
      • d. Differentiating the cells expressing markers characteristics of the pancreatic endoderm lineage into cells of a β-cell lineage, and
      • e. Implanting the cells of a β-cell lineage into the patient.
    BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A shows the expression of the definitive endoderm markers CXCR4, GATA4, HNF-3beta, Mixl1, Sox-17 in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for two, five and eight days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. FIG. 1B shows the expression of the anterior endoderm markers Cerberus, Otx-1 and Hex genes in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for three and five days.
  • FIGS. 2A, 2B and 2C shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following treatment with 100 ng/ml activin A for five days. Expression of the definitive endoderm markers was detected by immunohistochemistry. FIG. 2A shows Sox-17 expression. FIG. 2B shows HNF-3beta expression. FIG. 2C shows Oct3/4 expression.
  • FIGS. 3A, 3B, 3C and 3D show the expression of definitive endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. FIG. 3A shows GATA4 expression. FIG. 3B shows Sox-17 expression. FIG. 3C shows HNF-3beta expression. FIG. 3D shows Mixl1 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 4A and 4B show the expression of extra-embryonic endoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the extraembryonic endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. FIG. 4A shows the effect of 100 ng/ml activin A on AFP expression. FIG. 4B shows the effect of 100 ng/ml activin A on Sox7 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 5A and 5B show the expression of mesoderm and ectoderm markers in the human embryonic stem cell line H9 following a step-wise differentiation protocol. Expression of the mesoderm and ectoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells. FIG. 5A shows the effect of 100 ng/ml activin A on Brachyury expression. FIG. 5B shows the effect of 100 ng/ml activin A on Zic1 expression. Data points marked ‘AA’ denote activin A treatment for one (1 d), three (3 d), five (5 d), or seven days (7 d). Data points marked ‘UT’ denote untreated controls cultured for one (1 d), three (3 d), five (5 d), or seven days (7 d).
  • FIGS. 6A, 6B, 6C, 6D, 6E and 6F show the expression of the definitive endoderm markers Brachyury (FIG. 6A) CXCR4 (FIG. 6B), Mixl1 (FIG. 6C), Sox17 (FIG. 6D), HNF-3beta (FIG. 6E), Oct4 (FIG. 6F) in the human embryonic stem cell line H7 following treatment with 100 ng/ml activin A for one, three, five and seven days. Expression of definitive endoderm markers was assayed at the mRNA level and normalized to expression levels in untreated human embryonic stem cells.
  • FIGS. 7A, 7B, 7C, 7D, 7E and 7F shows the expression of definitive endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the definitive endoderm markers was detected by immunohistochemistry. FIGS. 7A and 7B show Sox-17 expression. FIGS. 7C and 7D show HNF-3beta expression.
  • FIGS. 7E and 7F show GATA4 expression. FIGS. 7B, 7D and 7F show counter staining of the nuclei with DAPI. The columns marked ‘treated’ denote activin A treatment (100 ng/ml) for five days. The columns marked ‘untreated’ denote untreated controls.
  • FIGS. 8A, 8B and 8C shows the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. FIG. 8A shows Pdx1 expression. FIG. 8B shows GLUT-2 expression. FIG. 8C shows PTF1a expression.
  • FIGS. 9A and 9B show the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a second differentiation protocol. Expression of the pancreatic endoderm markers was detected by immunohistochemistry. FIG. 9A shows Pdx1 expression in the untreated control, and FIG. 9B shows Pdx1 expression in the culture treated by the step-wise differentiation protocol.
  • FIGS. 10A, 10B, 10C and 10D show the expression of pancreatic endocrine markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the pancreatic endocrine markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. FIG. 10A shows NeuroD1 expression. FIG. 10B shows Ngn3 expression. FIG. 10C shows insulin expression. FIG. 10D shows Hes-1 expression, the expression level is normalized to pancreatic endoderm cells.
  • FIGS. 11A and 11B show the expression of pancreatic endoderm markers in the human embryonic stem cell line H9 following application of a differentiation protocol. Expression of the pancreatic endoderm markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. FIG. 11A shows Nkx2.2 expression. FIG. 11B shows Pdx1 expression.
  • FIG. 12 shows the expression of PDX-1 in cells with each passage (P0, P1 and P2) in culture. Expression of the PDX-1 was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells H9.
  • FIGS. 13A and 13B show the expression of hepatocyte markers in the human embryonic stem cell line H9 following application of a third differentiation protocol. Expression of the hepatocyte markers was assayed by PCR and normalized to expression levels in activin A treated human embryonic stem cells. FIG. 13A shows AFP expression. FIG. 13B shows albumin expression.
  • FIGS. 14A and 14B shows the expression of markers of pluripotency in the human embryonic stem cell line H9. Expression of the markers of pluripotency was assayed by immunohistochemistry. FIG. 14A shows Oct-4 expression. FIG. 14B shows alkaline phosphatase expression.
  • FIG. 15 shows the karyotype of the human embryonic cell line H9. The Karyotype was determined on cells at passage number P36 that were cultured on mouse embryonic fibroblast feeder cells.
  • FIG. 16 depicts the outline of a differentiation protocol in this invention, where human embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
  • FIGS. 17A, 17B, 17C, 17D, 17E and 17F depict the FACS profile of human embryonic stem cell line H9 at passage number 44, cultured on varying concentrations of MATRIGEL and exposed to (0.5-2%) low serum and high activin A (100 ng/ml) for 5 days. The expression of definite endoderm marker CXCR4 (CD184) is shown on the Y-axis and the expression of ES marker CD9 is shown on the X-axis.
  • FIG. 18 shows the real-time PCR results for markers of definitive endoderm, from cultures of the human embryonic stem cell line H9 at passage 44 cultured on a 1:10 dilution of MATRIGEL (▪), a 1:20 dilution of MATRIGEL (▪), or a 1:30 dilution of MATRIGEL (□) and exposed to the differentiation protocol disclosed in Example 14. The fold induction is relative to undifferentiated cells of the human embryonic stem cell line H9, at passage number 44, cultured in medium conditioned using mouse embryonic fibroblasts.
  • FIGS. 19A and 19B show the scatter plots for global gene expression in undifferentiated pluripotent stem cells and definitive endoderm cells obtained from differentiating pluripotent stem cells. Data shown is from cultures of the human embryonic stem cell line H9 cell line at passage 44 cultured on mouse embryonic fibroblasts (right panel) and passage 83 cultured on MATRIGEL (left panel).
  • FIGS. 20A, 20B and 20C depict the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 (FIG. 20A), the human embryonic stem cell line H7 (FIG. 20B), and the human embryonic stem cell line H9 (FIG. 20C) cultured on mouse embryonic fibroblast feeder cells exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
  • FIGS. 21A and 21B show the real-time PCR results of expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 (FIG. 21A) and the human embryonic stem cell line H9 (FIG. 21B) cultured on mouse embryonic fibroblast feeder cells. Results are expressed as fold increase over undifferentiated cells.
  • FIGS. 22A, 22B and 22C depict the expression of CXCR4 by FACS at day 5 for the human embryonic stem cell line H1 (FIG. 22A), the human embryonic stem cell line H7 (FIG. 22B), and the human embryonic stem cell line H9 (FIG. 22C) cultured on MATRIGEL (1:30 dilution) and exposed to the definitive endoderm differentiation protocol disclosed in Example 4.
  • FIGS. 23A, 23B and 23C show the real-time PCR results of the expression of the indicated definitive endoderm markers in cultures of the human embryonic stem cell line H7 (FIG. 23A) and the human embryonic stem cell line H9 (FIG. 23B) and the human embryonic stem cell line H1 (FIG. 23C). Results are expressed as fold increase over undifferentiated cells. Cells were treated according to the methods disclosed in Example 4.
  • FIGS. 24A and 24B depict phase contrast images of cultures of the human embryonic stem cell line H9 at passage 46 in the presence of 100 ng/ml of activin A (FIG. 24A) or 100 ng/ml of activin A+20 ng/ml Wnt-3a (FIG. 24B). Cells were treated for five days.
  • FIGS. 25A, 25B, 25C and 25D depict the expression of CXCR4 by FACS in cultures of the human embryonic stem cell line H7 at passage 44 (FIGS. 25A and 25B) and H9 at passage 46 (FIGS. 25C and 25D), following treatment according to the methods disclosed in Example 4. FIGS. 25B and 25D show the effect of 20 ng/ml of Wnt-3a on CXCR4 expression. FIGS. 25A and 25C show CXCR4 expression in the absence of Wnt-3a. Results were obtained 5 days post treatment.
  • FIGS. 26A and 26B display the real-time PCR data for expression of the genes indicated in cultures of the human embryonic stem cell line H7 (FIG. 26A) and H9 (FIG. 26B). Cultures were treated with the differentiation protocol disclosed in Example 4. The effects of Wnt agonists Wnt-3a (20 ng/ml), Wnt-5a (20 ng/ml) and Wnt-7a (20 ng/ml) were also tested, as indicated in the panels. Cells were treated for 5 days. Results are expressed as fold increase over undifferentiated cells.
  • FIGS. 27A, 27B, 27C and 27D depict the expression of CXCR4 in cultures of the human embryonic stem cell line H9 at passage 46, by FACS at five days post treatment. FIG. 27A depicts CXCR4 expression in the absence of Wnt-3a. FIG. 27B depicts CXCR4 expression following treatment with 10 ng/ml Wnt-3a. FIG. 27C depicts CXCR4 expression following treatment with 20 ng/ml Wnt-3a, and FIG. 27D depicts CXCR4 expression following treatment with 50 ng/ml Wnt-3a.
  • FIGS. 28A, 28B and 28C depict the expression of definitive markers indicated in cultures of the human embryonic stem cell line H9 after 5 days of treatment. Results are shown as fold increase in expression over untreated cells, as determined by real-time PCR. FIG. 28A shows the effect of 10, 20 and 50 ng/ml Wnt-3a on the expression of definitive endoderm marker genes indicated. FIG. 28B shows the effect of 1, 5 or 10 ng/ml Wnt-3a (x-axis labels: 10, 5, 1) on the expression on goosecoid (▪) and CXCR4 (□) expression, at 2 (2 d) and 5 (5 d) days post treatment. FIG. 28C shows the effect of 1, 5 or 10 ng/ml Wnt-3a on cell number, at 2 days (□) or 5 days (▪).
  • FIGS. 29A, 29B, 29C, 29D, 29E, 29F, 29G and 29H depict the expression of CXCR4 in cultures of the human embryonic stem cell line H9 by FACS, following a 5 day treatment with the differentiation protocol disclosed in Example 4. Cells were cultured in the absence of Wnt-3a or GSK-3B inhibitor (FIG. 29A), 20 ng/ml Wnt-3a for the entire 5 day period (FIG. 29B), 1000 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29C), 500 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29D), 100 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29E), 10 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29F), 100 nM GSK-3B inhibitor IX for days 1-2 (FIG. 29G), 10 nM GSK-3B inhibitor IX for days 1-2 (FIG. 29H).
  • FIGS. 30A and 30 B depict the gene expression of definitive endoderm markers by real-time PCR. Results are expressed as fold increase over untreated cells. FIG. 30A shows data obtained from the human embryonic cell line H9 at passage number 48, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated. FIG. 30B shows data obtained from the human embryonic cell line H9 at passage number 46, treated to the definitive endoderm protocol disclosed in Example 4, containing the Wnt-3a or GSK-3B inhibitor at the concentrations and the times indicated.
  • FIGS. 31A, 31B, 31C, 31D, 31E and 31F depict the expression of CXCR4 by FACS for embryonic stem cell lines used in the present invention. FIGS. 31A, 31B, 31C and 31D show data obtained from the human embryonic stem cell line H9 at passage number 49. FIGS. 31E and 31F show data obtained from the human embryonic stem cell line H1 at passage number 46. Data was obtained 5 days post treatment. Cells were treated with the following conditions: FIG. 31A: 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; FIG. 31B: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; FIG. 31C: 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days; FIG. 31D: 10 ng/ml activin A for all five days plus 100 nM GSK-3B IX inhibitor for the first two days, FIG. 31E: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days, and FIG. 31F: 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days.
  • FIGS. 32A and 32B depict the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a: FIG. 32A: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and FIG. 32B: SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 33A, 33B, 33C, 33D, 33E and 33F depict the expression of CXCR4 by FACS for the embryonic stem cell line H9 at passage 53. Data was obtained 5 days post treatment. Cells were treated with the following conditions: FIG. 33A: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days and 25 ng/ml BMP-4 for days 3-5; FIG. 33B: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a for the first two days; FIG. 33C: 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days; FIG. 33D: 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days; FIG. 33E: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3a+100 nm GSK-3B inhibitor IX for the first two days, and FIG. 33F: 100 ng/ml activin A+25 ng/ml BMP-4 for all five days. For all the panels, the X-axis represents expression of CD9 and the Y-axis represents expression of CXCR4 (CD184).
  • FIGS. 34A and 34B depict the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: FIG. 34A: expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and FIG. 34B: SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 35A and 35B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor: FIG. 35A: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and FIG. 35B: SOX-17 and GATA4. Results are expressed as fold increase over untreated cells.
  • FIGS. 36A and 36B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 53, treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4, for five days: FIG. 36A: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and FIG. 36B: SOX-17, HNF-3B and GATA4.
  • FIG. 37 depicts the percentage of CXCR4 expression, determined by FACS, in cultures of the human embryonic stem cell line H9, treated with the conditions listed in Example 22.
  • FIGS. 38A and 38B depict the expression of definitive endoderm markers as determined by FACS in cultures of the human embryonic stem cell line H9, cultured on fibronectin (FIG. 38A) or MATRIGEL™ (FIG. 38B).
  • FIG. 39 depicts the expression of definitive endoderm markers as determined by real-time PCR in cultures of the human embryonic stem cell line H9, cultured on fibronectin (□) or a 1:10 dilution of growth factor reduced MATRIGEL (▪).
  • FIG. 40 depicts the effect of various concentrations of MATRIGEL in the presence of low serum, 100 ng/ml of activin A and 20 ng/ml of Wnt-3a on differentiating human embryonic stem cells into definitive endoderm. Cells were treated according to the methods disclosed in Example 4. Results shown are the expression levels of the genes indicated, as determined by real-time PCR.
  • FIGS. 41A, 41B, 41C, 41D, 41E-1, 41E-2, 41F-1, 41F-2, 41G-1, 41G-2 depicts the role of Wnt-3a in definitive endoderm formation by human embryonic stem cells maintained on MATRIGEL, but differentiated on mouse embryonic fibroblasts. FIGS. 41A, 41B, 41C and 41D show real-time PCR data for the genes indicated. FIGS. 41E-1, 41E-2, 41F-1, 41F-2, 41G-1, 41G-2 show FACS data for the conditions indicated.
  • FIG. 42 shows the differentiation of human embryonic stem cells cultured on tissue culture substrate coated with MATRIGEL™ to definitive endoderm following treatment with the Wnt Inhibitor DKK-1. Results shown are the expression of the genes indicated, as determined by real-time PCR in H9 cells treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3A plus 100 ng/ml of DKK1 (DE+DKK1), or in the absence of DKK1 (DE).
  • FIGS. 43A and 43B show immunofluorescence staining of definitive endoderm markers in cultures of the human embryonic stem cell line H9 cultured on tissue culture substrate coated with MATRIGEL and differentiated in low serum plus 100 ng/ml of activin-A without (FIG. 43A), or with (FIG. 43B) 20 ng/ml of Wnt-3a. Ecad=E-cadherin, NCAM=N-cadherin,
  • FIGS. 44A and 44B show the differentiation of the human embryonic stem cell line SA002 at passage 38 into definitive endoderm. Cells were treated for five days with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in FIGS. 44A and 44B.
  • FIGS. 45A, 45B and 45C show the expression of CXCR4 by FACS in the human embryonic stem cell line SA002 at passage 38, following treatment with 100 ng/ml activin A treatment (FIG. 45A), 100 ng/ml activin A+20 ng/ml Wnt-3a (FIG. 45B), or 100 ng/ml activin A+100 nM GSK-3B inhibitor IX (FIG. 45C). Cells were treated for five days.
  • FIGS. 46A and 46B show the differentiation of the human embryonic stem cell line H1 at passage 55 into definitive endoderm on tissue culture substrate coated with human serum. Cells were treated with the conditions indicated and gene expression was determined by real-time PCR, for the genes indicated in FIGS. 46A and 46B.
  • FIGS. 47A and 47B show the differentiation of cultures of the human embryonic stem cell line H1 at P54, on tissue culture substrate coated with MATRIGEL™ to definitive endoderm. The effects of various GSK-B inhibitors were tested following a five-day DE protocol. The following GSK-3B inhibitors were evaluated at 100 nM for the first two days of treatment: GSK-3B VIII, IX, XI, and XII.
  • FIGS. 48A, 48B, 48C and 48D show the expression of AFP (FIG. 48A), Pdx-1 (FIG. 48B), Cdx-2 and Glut-2 (FIG. 48C) and HNF-3beta, HNF-6 and somatostatin (FIG. 48D) in cultures of the human embryonic stem cell line H9 at passage 49, cultured and treated according to the methods disclosed in Example 4 in the presence of 20 ng/ml of Wnt-3a for the first two days of treatment. Following the treatment, the cells were treated for three additional days with 2% FBS plus 1 μM retinoic acid, 0.1 to 1 μM TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid), or 0.1-10 μM AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid). The cells were next treated for three additional days in 2% FBS plus 20 ng/ml of bFGF.
  • FIGS. 49A and 49B show the real-time PCR results of the expression of the definitive endoderm markers indicated in FIGS. 49A and 49B. in cultures of the human embryonic stem cell line H1 treated with activin A and Wnt-1 for the times and concentrations indicated.
    •  DETAILED DESCRIPTION
  • For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.
    • Definitions
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • “AFP” or “alpha-fetoprotein protein” as used herein, refers to an antigen produced at the onset of liver development. AFP may also be expressed in extraembryonic cells.
  • “Albumin” is a soluble monomeric protein that makes up about half of all serum proteins in adults.
  • “β-cell lineage” refer to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6. Cells expressing markers characteristic of the β cell lineage include β cells.
  • “Brachyury”, as used herein, is a T-box gene family member. It is the marker for primitive streak and mesoderm cells.
  • “Cells expressing markers characteristic of the definitive endoderm lineage” as used herein refer to cells expressing at least one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
  • “c-Kit” and “CD117” both refer to a cell surface receptor tyrosine kinase having a sequence disclosed in Genbank Accession No. X06182, or a naturally occurring variant sequence thereof (e.g., allelic variant).
  • “CD99” as used herein refers to the protein encoded by the gene with the accession number NM_002414.
  • “Cells expressing markers characteristic of the pancreatic endoderm lineage” as used herein refer to cells expressing at least one of the following markers: PDX-1, HNF-1beta, PTF-1 alpha, HNF-6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.
  • “Cells expressing markers characteristic of the pancreatic endocrine lineage” as used herein refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, Ngn-3, or PTF-1 alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.
  • “Cer1” or “Cerebrus” as used herein is a member of the cysteine knot superfamily of proteins.
  • “CXCR4” as used herein refers to the stromal cell-derived factor 1 (SDF-1) receptor, also known as “LESTR” or “fusin”. In the gastrulating mouse embryo, CXCR4 is expressed in the definitive endoderm and mesoderm but not in extraembryonic endoderm.
  • “Definitive endoderm” as used herein refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF-3 beta, GATA-4, SOX-17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and Mixl1.
  • “Extraembryonic endoderm” as used herein refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, and SPARC.
  • “FGF-2”, “FGF-4” “FGF-8” “FGF-10”, and “FGF-17” as used herein, are members of the fibroblast growth factor family.
  • “GATA-4” and “GATA-6” are members of the GATA transcription factor family. This family of transcription factors is induced by TGF-3 signaling, and contribute to the maintenance of early endoderm markers.
  • “GLUT-2”, as used herein, refers to the glucose transporter molecule that is expressed in numerous fetal and adult tissues, including pancreas, liver, intestine, brain, and kidney.
  • “Goosecoid” or “GSC” as used herein, refers to a homeodomain transcription factor expressed in the dorsal lip of the blastopore.
  • “HB9” as used herein, refers to the homeobox gene 9.
  • “HNF-1 alpha”, “HNF-1 beta”, “HNF-3 beta”, and “HNF-6” belong to the hepatic nuclear factor family of transcription factors, which is characterized by a highly conserved DNA binding domain and two short carboxy-terminal domains.
  • “Islet-1” or “Isl-1” as used herein is a member of the LIM/homeodomain family of transcription factors, and is expressed in the developing pancreas.
  • “MafA” as used herein is a transcription factor expressed in the pancreas, and controls the expression of genes involved in insulin biosynthesis and secretion.
  • “Markers” as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • “Mesendoderm cell” as used herein refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta, GSC, FGF17, GATA-6.
  • “Mixl1” as used herein refers to a homeobox gene, which is marker for the cells in the primitive streak, mesoderm, and endoderm.
  • “NeuroD” as used herein is basic helix-loop-helix (bHLH) transcription factor implicated in neurogenesis.
  • “NGN-3” as used herein, is a member of the neurogenin family of basic loop-helix-loop transcription factors.
  • “Nkx-2.2” and “Nkx-6.1” as used herein are members of the Nkx transcription factor family.
  • “Nodal” as used herein, is a member of the TGF beta superfamily of proteins.
  • “Oct-4” is a member of the POU-domain transcription factor and is widely regarded as a hallmark of pluripotent stem cells. The relationship of Oct-4 to pluripotent stem cells is indicated by its tightly restricted expression to undifferentiated pluripotent stem cells. Upon differentiation to somatic lineages, the expression of Oct-4 disappears rapidly.
  • “Pancreatic endocrine cell”, or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • “Pancreatic hormone secreting cell” as used herein refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • “Pax-4” and “Pax-6” as used herein are pancreatic 13 cell specific transcription factors that are implicated in islet development.
  • “PDX-1” as used herein refers to a homeodomain transcription factor implicated in pancreas development.
  • “Pre-primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Nodal, or FGF8
  • “Primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
  • “PTF-1 alpha” as used herein refers to a basic helix-loop-helix protein of 48 kD that is a sequence-specific DNA-binding subunit of the trimeric pancreas transcription factor-1 (PTF1).
  • “SOX-1”, “SOX-2”, “SOX-7”, and “SOX-17” as used herein, are a members of the SOX transcription factor family, and are implicated in embryogenesis.
  • “SPARC” as used herein is also known as “secreted protein acidic and rich in cysteine”.
  • “SSEA-1” (Stage Specific Embryonic Antigen-1) is a glycolipid surface antigen present on the surface of murine teratocarcinoma stem cells (EC), murine and human embryonic germ cells (EG), and murine embryonic stem cells (ES).
  • “SSEA-3” (Stage Specific Embryonic Antigen-3) is a glycolipid surface antigen present on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • “SSEA-4” (Stage Specific Embryonic Antigen-4) is a glycolipid surface antigen present on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • “TRA1-60” is a keratin sulfate related antigen that is expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • “TRA1-81” is a keratin sulfate related antigen that is expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG), and human embryonic stem cells (ES).
  • “TRA2-49” is an alkaline phosphatase isozyme expressed on the surface of human teratocarcinoma stem cells (EC) and human embryonic stem cells (ES).
  • “UTF-1” as used herein, refers a transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.
  • “Zic1” as used herein is a member of the Zic transcription factor family. Zic1 regulates the expression of neural and neural crest-specific genes and is expressed in the cells of the dorsal neural tube and the premigratory neural crest.
    • Isolation, Expansion and Culture of Pluripotent Stem Cells Characterization of Pluripotent Stem Cells
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
  • Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
    • Sources of Pluripotent Stem Cells
  • The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BG01v (BresaGen, Athens, Ga.).
  • In one embodiment, human embryonic stem cells are prepared as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).
    • Culture of Pluripotent Stem Cells
  • In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways. Alternatively, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation. The growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type. Alternatively, the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
  • For example, Reubinoff et al (Nature Biotechnology 18: 399-404 (2000)) and Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391, pp. 1145-1147) disclose the culture of pluripotent stem cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer.
  • Richards et al, (Stem Cells 21: 546-556, 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture. Richards et al, states: “human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripotent”.
  • US20020072117 discloses cell lines that produce media that support the growth of primate pluripotent stem cells in feeder-free culture. The cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. US20020072117 also discloses the use of the cell lines as a primary feeder cell layer.
  • In another example, Wang et al (Stem Cells 23: 1221-1227, 2005) discloses methods for the long-term growth of human pluripotent stem cells on feeder cell layers derived from human embryonic stem cells.
  • In another example, Stojkovic et al (Stem Cells 2005 23: 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic stem cells.
  • In a further example, Miyamoto et al (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta.
  • Amit et al (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder cell layer derived from human foreskin.
  • In another example, Inzunza et al (Stem Cells 23: 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts.
  • U.S. Pat. No. 6,642,048 discloses media that support the growth of primate pluripotent stem (pPS) cells in feeder-free culture, and cell lines useful for production of such media. U.S. Pat. No. 6,642,048 states: “This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure.”
  • In another example, WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. WO2005014799 states: “The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte).”
  • In another example, Xu et al (Stem Cells 22: 972-980, 2004) discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase.
  • In another example, US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells.
  • An alternative culture system employs serum-free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells. For example, Cheon et al (BioReprod DOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose a feeder-free, serum-free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.
  • In another example, Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF.
  • In another example, US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
  • In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
  • In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.”
  • In another example, US20050244962 states: “In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor.”
  • In a further example, WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of bFGF sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
  • In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGFβ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
  • The pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a the suitable culture substrate is Matrigel® (Becton Dickenson). Matrigel® is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
  • Other extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
  • The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
  • Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
    • Differentiation of Pluripotent Stem Cells into Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
  • Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line SA002 (Cellartis, Sweden). Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cells: ABCG2, cripto, CD9, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.
  • Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, Hnf-3beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
  • Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of Pdx1, HNF-1beta, PTF1a, HNF-6, HB9 and PROX1. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
  • Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1, Pax-4, Ngn-3, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.
  • In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdx1 and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.
    • Formation of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
  • Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention.
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534-1541 (2005).
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651-1662 (2004).
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29-38 (2007).
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration. An example of this method is disclosed in Nature Biotechnology 23, 1534-1541 (2005).
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2005.
  • For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • In one aspect of the present invention, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for a period of time, and then culturing the pluripotent stem cells with activin A in a second culture medium containing a greater concentration of serum for about another period of time.
  • The concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days. The concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
  • In an alternate embodiment of the present invention, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, then culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium containing serum for about a period of time, and then culturing the pluripotent stem cells with activin A and a Wnt ligand in a second culture medium containing a greater concentration of serum for another period of time.
  • The concentration of serum in the first culture medium disclosed above may be from about zero to about 0.5 percent, and the culture time may be from about one to about three days. The concentration of serum in the second culture medium disclosed above may be from about 0.5 percent to about two percent, and the culture time may be from about one to about four days.
  • In one embodiment, the present invention provides a method for differentiating pluripotent stem cells expressing markers characteristic of the definitive endoderm lineage, comprising the steps of:
      • a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and
      • b. Culturing the pluripotent stem cells with activin A and a Wnt ligand.
  • Culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed in a single culture medium. Alternatively, culturing the pluripotent stem cells with activin A and a Wnt ligand may be performed separately or together in more than one culture media. In one embodiment, culturing the pluripotent stem cells with activin A and a Wnt ligand is performed in two culture media.
    • Extracellular Matrix
  • In one aspect of the present invention, the pluripotent stem cells are cultured and differentiated on a tissue culture substrate coated with an extracellular matrix. The extracellular matrix may be a solubilized basement membrane preparation extracted from mouse sarcoma cells (which is sold by BD Biosciences under the trade name MATRIGEL). Alternatively, the extracellular matrix may be growth factor-reduced MATRIGEL. Alternatively, the extracellular matrix may fibronectin. In an alternate embodiment, the pluripotent stem cells are cultured and differentiated on tissue culture substrate coated with human serum.
  • The extracellular matrix may be diluted prior to coating the tissue culture substrate. Examples of suitable methods for diluting the extracellular matrix and for coating the tissue culture substrate may be found in Kleinman, H. K., et al., Biochemistry 25:312 (1986), and Hadley, M. A., et al., J. Cell. Biol. 101:1511 (1985).
  • In one embodiment, the extracellular matrix is MATRIGEL. In one embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with MATRIGEL at a 1:60 dilution.
  • In one embodiment, the extracellular matrix is growth factor-reduced MATRIGEL. In one embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:10 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:15 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissue culture substrate is coated with growth factor-reduced MATRIGEL at a 1:60 dilution.
    • Differentiation of Pluripotent Stem Cells into Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage on an Extracellular Matrix, Using a Single Culture Medium
  • When a single culture medium is used, it should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells to definitive endoderm, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1): 241-8.).
  • The culture medium may have a serum concentration in the range of about 0% to about 10%. In an alternate embodiment, the concentration may be in the range of about 0% to about 5%. In an alternate embodiment, the concentration may be in the range of about 0% to about 2%. In an alternate embodiment, the concentration may be about 2%.
  • The time of culturing with activin A and a Wnt ligand may range from about 1 day to about 7 days. In an alternate embodiment, the time of culturing may range from about 1 day to about 3 days. In an alternate embodiment, the time of culturing may be about 3 days.
  • Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 μg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • The choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process. The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
  • The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • The single culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX. I did not see the definitions of IX and XI inhibitors. I did have these in the original WNT patent application.
  • When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • The single culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the ells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the ells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • The at least one additional factor may be, for example, nicotinamide, members of the TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine 1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
  • The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241), and primary or transformed endothelial cells.
    • Differentiation of Pluripotent Stem Cells into Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage on an Extracellular Matrix, Using Two Culture Media
  • Differentiation of pluripotent stem cells into cells of a definitive endoderm lineage may be accomplished by culturing the pluripotent stem cells with activin A and a Wnt ligand using two culture media. Thus, the differentiation of the pluripotent stem cells may be accomplished as follows:
      • a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix,
      • b. Culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium, and
      • c. Culturing the pluripotent stem cells with activin A in a second culture medium.
  • The first culture medium may contain serum at a low concentration, and the second culture medium may contain serum at a higher concentration than the first culture medium.
  • The second culture medium may contain a Wnt ligand.
  • First Culture Medium:
  • The first culture medium should contain sufficiently low concentrations of certain factors to allow the differentiation of pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, such as, for example insulin and IGF (as disclosed in WO2006020919). This may be achieved by lowing the serum concentration, or alternatively, by using chemically defined media that lacks insulin and IGF. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
  • In the first culture medium there may be a lower concentration of serum, relative to the second culture medium. Increasing the serum concentration in the second culture medium increases the survival of the cells, or, alternatively, may enhance the proliferation of the cells. The serum concentration of the first medium may be in the range of about 0% to about 10%. Alternatively, the serum concentration of the first medium may be in the range of about 0% to about 2%. Alternatively, the serum concentration of the first medium may be in the range of about 0% to about 1%. Alternatively, the serum concentration of the first medium may be about 0.5%.
  • When culturing the pluripotent stem cells with activin A and a Wnt ligand using at least two culture media, the time of culturing in the first culture medium may range from about 1 day to about 3 days.
  • Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 pg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • The choice of the Wnt ligand may be optimized to improve the efficiency of the differentiation process. The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
  • The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • The first culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
  • The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
  • When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • The first culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
  • The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
  • Second Culture Medium:
  • The second culture medium should contain certain factors, such as, for example, insulin and IGF (as disclosed in WO2006020919), at a sufficient concentration to promote the survival of the cultured cells. This may be achieved by increasing the serum concentration, or, alternatively, by using chemically defined media where the concentrations of insulin and IGF are increased relative to the first culture medium. Examples of chemically defined media are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).
  • In a second culture medium having higher concentrations of serum, the serum concentration of the second culture medium may be in the range about 0.5% to about 10%. Alternatively, the serum concentration of the second culture medium may be in the range of about 0.5% to about 5%. Alternatively, the serum concentration of the second culture medium may be in the range of about 0.5% to about 2%. Alternatively, the serum concentration of the second culture medium may be about 2%. When culturing pluripotent stem cells with the second culture medium, the time of culturing may range from about 1 day to about 4 days.
  • Similar to the first culture medium, Activin A may be used at any concentration suitable to cause differentiation of the pluripotent stem cells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 μg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 50 ng/ml to about 100 ng/ml. In another alternate embodiment, the concentration may be about 100 ng/ml.
  • The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000 ng/ml. In an alternate embodiment, the concentration may be about 10 ng/ml to about 100 ng/ml.
  • The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.
  • The second culture medium may also contain a GSK-3B inhibitor. The GSK-3B inhibitor may be added to the first culture medium, to the second culture medium, or to both the first and second culture media.
  • The GSK-3B inhibitor may be selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3B inhibitor is GSK-3B inhibitor IX.
  • When culturing pluripotent stem cells with a GSK-3B inhibitor, the concentration of the GSK-3B inhibitor may be from about 1 nM to about 1000 nM. In an alternate embodiment, the pluripotent stem cells are cultured with the GSK-3B inhibitor at a concentration of about 10 nM to about 100 nM.
  • Similar to the first culture medium, the second culture medium may also contain at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the definitive endoderm lineage from pluripotent stem cells. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the definitive endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
  • The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
    • Detection of Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage
  • Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
  • The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
  • Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
  • For example, characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.
  • After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage.
    • Formation of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
  • Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention.
  • For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time. That period of time may be from about one to about six days.
  • In an alternate aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells with retinoic acid for a period of time. That period of time maybe from about one to about three days. The retinoic acid is subsequently removed and the cells are treated with at least one fibroblast growth factor for another period of time. That period of time may be from about one to about three days.
  • In one embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:
      • a. Culturing cells expressing markers characteristic of the definitive endoderm lineage, and
      • b. Treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about one to about six days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid and at least one fibroblast growth factor for about six days.
  • The at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • In an alternate embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:
      • a. Culturing cells expressing markers characteristic of the definitive endoderm lineage,
      • b. Treating the cells expressing markers characteristic of the definitive endoderm lineage treating the cells with retinoic acid, and
      • c. Removing the retinoic acid and subsequently treating the cells with at least one fibroblast growth factor.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid for about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about one to about three days. In one embodiment, the cells expressing markers characteristic of the definitive endoderm are treated with at least one fibroblast growth factor for about three days.
  • The at least one fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
  • Any cell expressing markers characteristic of the definitive endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endoderm lineage using this method.
  • In one embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid. Alternatively, the cells expressing markers characteristic of the definitive endoderm lineage are treated with FGF-2, or alternatively FGF-4, or alternatively FGF-10. In an alternate embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one of the following factors: retinoic acid, FGF-2, FGF-4 or FGF-10. In an alternate embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and at least one of the following fibroblast growth factors: FGF-2, FGF-4 or FGF-10. In one embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-2. In another embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-4. In a further embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and FGF-10.
  • Retinoic acid may be used at a concentration from about 1 nM to about 1 mM. In one embodiment, retinoic acid is used at a concentration of 1 μM.
  • FGF-2 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-2 is used at a concentration of 50 ng/ml.
  • FGF-4 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-4 is used at a concentration of 50 ng/ml.
  • FGF-10 may be used at a concentration from about 50 pg/ml to about 50 μg/ml. In one embodiment, FGF-10 is used at a concentration of 50 ng/ml.
  • Cells expressing markers characteristic of the definitive endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endoderm lineage. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endoderm lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
  • The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
    • Detection of Cells Expressing Markers Characteristic of the Pancreatic Endoderm Lineage
  • Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endoderm lineage. Pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1beta.
  • The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
  • Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
    • Formation of Cells Expressing Markers of the Pancreatic Endocrine Lineage
  • Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method disclosed in this invention.
  • For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401 (2006).
  • For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF. An example of this method is disclosed in Nature Biotechnology 24, 1392-1401 (2006).
  • For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4. An example of this method is disclosed in D'Amour et al, Nature Biotechnology, 2006.
  • In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway. The factor that inhibits the Notch signaling pathway may be an antagonist for the Notch extracellular receptor. Alternatively, the factor may inhibit the biological activity of the Notch receptor. Alternatively, the factor may inhibit or be an antagonist of an element in the Notch signal transduction pathway within a cell.
  • In one embodiment the factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1SBenzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-5 2Rhydrozy-5 phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • L-685,458 may be used at a concentration from about 0.1 μM to about 100 μM. In one embodiment, L-685,458 is used at a concentration of about 90 μM. In one embodiment, L-685,458 is used at a concentration of about 80 μM. In one embodiment, L-685,458 is used at a concentration of about 70 μM. In one embodiment, L-685,458 is used at a concentration of about 60 μM. In one embodiment, L-685,458 is used at a concentration of about 50 μM. In one embodiment, L-685,458 is used at a concentration of about 40 μM. In one embodiment, L-685,458 is used at a concentration of about 30 μM. In one embodiment, L-685,458 is used at a concentration of about 20 μM. In one embodiment, L-685,458 is used at a concentration of about 10 μM.
  • In one embodiment, the present invention provides a method for differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:
      • a. Culturing cells expressing markers characteristic of the pancreatic endoderm lineage, and
      • b. Treating the cells with a factor that inhibits the Notch signaling pathway.
  • Any cell expressing markers characteristic of the pancreatic endoderm lineage is suitable for differentiating into a cell expressing markers characteristic of the pancreatic endocrine lineage using this method.
  • In one embodiment, factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • The cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about one to about five days. Alternatively, the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about three to about five days. Alternatively, the cells expressing markers characteristic of the pancreatic endoderm lineage are treated with the factor that inhibits the Notch signaling pathway for about five days.
  • In one embodiment, factor that inhibits the Notch signaling pathway is a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is 1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl] carbamic Acid tert-butyl Ester, also known as L-685,458.
  • L-685,458 may be used at a concentration from about 0.1 μM to about 100 μM. In one embodiment, L-685,458 is used at a concentration of about 90 μM. In one embodiment, L-685,458 is used at a concentration of about 80 μM. In one embodiment, L-685,458 is used at a concentration of about 70 μM. In one embodiment, L-685,458 is used at a concentration of about 60 μM. In one embodiment, L-685,458 is used at a concentration of about 50 μM. In one embodiment, L-685,458 is used at a concentration of about 40 μM. In one embodiment, L-685,458 is used at a concentration of about 30 μM. In one embodiment, L-685,458 is used at a concentration of about 20 μM. In one embodiment, L-685,458 is used at a concentration of about 10 μM.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage may be treated with at least one other additional factor that may enhance the formation of cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the at least one other additional factor may enhance the proliferation of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention. Further, the at least one other additional factor may enhance the ability of the cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention to form other cell types, or improve the efficiency of any other additional differentiation steps.
  • The at least one additional factor may be, for example, nicotinamide, members of TGF-β family, including TGF-β1, 2, and 3, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway inhibitors, sonic hedgehog inhibitors, or combinations thereof.
  • The at least one other additional factor may be supplied by conditioned media obtained from pancreatic cells lines such as, for example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as, for example, FHs 74 (ATCC No: CCL-241).
    • Detection of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
  • Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage. Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN-3, NeuroD, Islet-1.
  • Markers characteristic of cells of the β cell lineage are well known to those skilled in the art, and additional markers characteristic of the β cell lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the β-cell lineage. β cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, Pdx1 (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Isl1, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3beta, and MafA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
  • The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the β cell lineage.
  • Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.
    • Therapies
  • In one aspect, the present invention provides a method for treating a patient suffering from, or at risk of developing, Type1 diabetes. This method involves culturing pluripotent stem cells, differentiating the pluripotent stem cells in vitro into a β-cell lineage, and implanting the cells of a β-cell lineage into a patient.
  • In yet another aspect, this invention provides a method for treating a patient suffering from, or at risk of developing, Type 2 diabetes. This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a β-cell lineage, and implanting the cells of aβ-cell lineage into the patient.
  • If appropriate, the patient can be further treated with pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells. These agents may include, for example, insulin, members of the TGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -7, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others. Other pharmaceutical compounds can include, for example, nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2 mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • The pluripotent stem cells may be differentiated into an insulin-producing cell prior to transplantation into a recipient. In a specific embodiment, the pluripotent stem cells are fully differentiated into β-cells, prior to transplantation into a recipient. Alternatively, the pluripotent stem cells may be transplanted into a recipient in an undifferentiated or partially differentiated state. Further differentiation may take place in the recipient.
  • Definitive endoderm cells or, alternatively, pancreatic endoderm cells, or, alternatively, β cells, may be implanted as dispersed cells or formed into clusters that may be infused into the hepatic portal vein. Alternatively, cells may be provided in biocompatible degradable polymeric supports, porous non-degradable devices or encapsulated to protect from host immune response. Cells may be implanted into an appropriate site in a recipient. The implantation sites include, for example, the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach, or a subcutaneous pocket.
  • To enhance further differentiation, survival or activity of the implanted cells, additional factors, such as growth factors, antioxidants or anti-inflammatory agents, can be administered before, simultaneously with, or after the administration of the cells. In certain embodiments, growth factors are utilized to differentiate the administered cells in vivo. These factors can be secreted by endogenous cells and exposed to the administered cells in situ. Implanted cells can be induced to differentiate by any combination of endogenous and exogenously administered growth factors known in the art.
  • The amount of cells used in implantation depends on a number of various factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art.
  • In one aspect, this invention provides a method for treating a patient suffering from, or at risk of developing diabetes. This method involves culturing pluripotent stem cells, differentiating the cultured cells in vitro into a β-cell lineage, and incorporating the cells into a three-dimensional support. The cells can be maintained in vitro on this support prior to implantation into the patient. Alternatively, the support containing the cells can be directly implanted in the patient without additional in vitro culturing. The support can optionally be incorporated with at least one pharmaceutical agent that facilitates the survival and function of the transplanted cells.
  • Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures, which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No. 5,759,830, U.S. Pat. No. 6,626,950, U.S. Pat. No. 6,534,084, U.S. Pat. No. 6,306,424, U.S. Pat. No. 6,365,149, U.S. Pat. No. 6,599,323, U.S. Pat. No. 6,656,488, U.S. Published Application 2004/0062753 A1, U.S. Pat. No. 4,557,264 and U.S. Pat. No. 6,333,029.
  • To form a support incorporated with a pharmaceutical agent, the pharmaceutical agent can be mixed with the polymer solution prior to forming the support. Alternatively, a pharmaceutical agent could be coated onto a fabricated support, preferably in the presence of a pharmaceutical carrier. The pharmaceutical agent may be present as a liquid, a finely divided solid, or any other appropriate physical form. Alternatively, excipients may be added to the support to alter the release rate of the pharmaceutical agent. In an alternate embodiment, the support is incorporated with at least one pharmaceutical compound that is an anti-inflammatory compound, such as, for example compounds disclosed in U.S. Pat. No. 6,509,369.
  • The support may be incorporated with at least one pharmaceutical compound that is an anti-apoptotic compound, such as, for example, compounds disclosed in U.S. Pat. No. 6,793,945.
  • The support may also be incorporated with at least one pharmaceutical compound that is an inhibitor of fibrosis, such as, for example, compounds disclosed in U.S. Pat. No. 6,331,298.
  • The support may also be incorporated with at least one pharmaceutical compound that is capable of enhancing angiogenesis, such as, for example, compounds disclosed in U.S. Published Application 2004/0220393 and U.S. Published Application 2004/0209901.
  • The support may also be incorporated with at least one pharmaceutical compound that is an immunosuppressive compound, such as, for example, compounds disclosed in U.S. Published Application 2004/0171623.
  • The support may also be incorporated with at least one pharmaceutical compound that is a growth factor, such as, for example, members of the TGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin, among others. Other pharmaceutical compounds can include, for example, nicotinamide, hypoxia inducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 and GLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptides, cathelicidins, defensins, laminin, biological peptides containing cell- and heparin-binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin, MAPK inhibitors, such as, for example, compounds disclosed in U.S. Published Application 2004/0209901 and U.S. Published Application 2004/0132729.
  • The incorporation of the cells of the present invention into a scaffold can be achieved by the simple depositing of cells onto the scaffold. Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developed to enhance the efficiency of cell seeding. For example, spinner flasks have been used in seeding of chondrocytes onto polyglycolic acid scaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approach for seeding cells is the use of centrifugation, which yields minimum stress to the seeded cells and enhances seeding efficiency. For example, Yang et al. developed a cell seeding method (J. Biomed. Mater. Res. 55(3): 379-86 (2001)), referred to as Centrifugational Cell Immobilization (CCI).
  • The present invention is further illustrated, but not limited by, the following examples.
    • EXAMPLES Example 1 Human Embryonic Stem Cell Culture
  • The human embryonic stem cell lines H1, H7 and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2×104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO2/within a humidified tissue culture incubator. When confluent (approximately 5-7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surface using a 5-ml pipette. Cells were spun at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells in fresh culture medium.
    • Example 2 Formation of Definitive Endoderm
  • The effects of activin A on the expression of markers of definitive endoderm were examined. Activin A (100 ng/ml) was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts. Cells were cultured continuously in the presence of activin A and harvested at the times indicated. The level of expression of definitive endoderm markers was examined by PCR (FIGS. 1A and 1B), FACS (results summarized in Table II), and immunohistochemistry (FIGS. 2A, 2B and 2C).
  • Activin A evoked a time-dependent increase in the expression of CXCR4, GATA4, HNF-3beta, Mixl1 and Sox-17 mRNA in the H9 line (FIG. 1A). A significant up regulation of anterior endoderm markers, Cerberus, Otx-1 and Hex genes was also observed (FIG. 1B). An increase in CXCR4 protein was observed by FACS analysis following activin A treatment. The expression of E-cadherin and N-cadherin did not change following activin A treatment (Table HA). CXCR4 positive cells were also highly positive for C-kit, EPCAM, CD99, and negative for CD9. The expression pattern for these markers was consistent among all three hES cell lines examined (Table IIB for H7 and Table IIC for H1). Immunocytochemistry conducted on cells treated with activin A for five days revealed that 30-40% cells in the treated culture were positive for Sox17 and HNF-3beta. In parallel, almost 100% of the differentiated cells were still Oct4 positive (FIGS. 2A, 2B and 2C). With the decrease in expression of surface markers of pluripotency, combined with an increase in the expression of definitive endoderm markers, these data suggest that activin A promotes the differentiation of human embryonic stem cells to definitive endoderm.
    • Example 3 Formation of Pancreatic Endoderm
  • Growth factors known to induce the differentiation of human embryonic stem cells to pancreatic endoderm were added to cell cultures. In particular, activin A, bFGF, and retinoic acid, known to induce the formation of pancreatic endoderm, were added to cell cultures.
  • In a first series of experiments, activin A, was added to populations of human embryonic stem cells cultured on mouse embryonic fibroblasts for up to seven days in DMEM/F12 supplemented with 0% to 2% serum and Activin A (100 ng/ml). Cells were harvested at the time points indicated in FIGS. 3A, 3B, 3C and 3D and assayed by PCR for the expression of genes shown (FIGS. 3A, 3B, 3C, 3D, 4A, 4B, 5A and 5B). In FIGS. 3A, 3B, 3C and 3D, PCR analysis indicated that activin treated cells expressed a broad spectrum of genes associated with endoderm development, including GATA4 (FIG. 3A), Sox-17 (FIG. 3B), HNF-3beta (FIG. 3C), and Mixl-1 (FIG. 3D). However, no Pdx1 gene expression was observed. The same expression pattern of endoderm lineage markers was observed in Activin A treated H7 cells (FIGS. 6A, 6B, 6C, 6D, 6E and 6F). At this stage, there was no significant decrease of Oct4 expression.
  • Activin A evoked a time-dependent decrease in the expression of the extraembryonic endoderm markers Sox7 (FIG. 4A) and AFP (FIG. 4B). Activin A decreased the expression of Brachyury (FIG. 5A) but had no effect on expression of the neuronal marker Zic1 (FIG. 5B).
  • Taken together, these data suggest that the increased expression of Sox-17, Mixl1, Gata4, and HNF-3beta together with the upregulation of anterior endoderm markers Otx1, Cer1 and Hex genes, corresponds to the formation of definitive endoderm in response to activin A treatment. Analysis of definitive endoderm markers by immunocytochemistry revealed that protein expression for these genes also reflected the trends observed in mRNA expression. Levels of expression for HNF-3beta, Sox-17, and GATA4 were low in untreated cells, approximately 10 to 20% of all cells. Activin A (100 ng/ml) treatment for five days increased the expression of HNF-3beta, Sox-17, and GATA4 to approximately 50% to 90% of all cells (FIGS. 7A, 7B, 7C, 7D, 7E and 7F).
  • In a second series of experiments, cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After the cells were 70-80% confluent, the medium was changed to DMEM/F12 with 0 to 2% FBS with addition of activin A at 100 ng/ml and cultured in the presence of activin A for either three, five, or seven days. After this time interval, the cells were then further treated for five to six days with combinations of retinoic acid and bFGF as shown in FIGS. 8A, 8B and 8C. Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
  • Gene expression analysis revealed that activin A or retinoic acid alone did not induce the expression of Pdx1. Similar results were observed in cultures of cells treated with retinoic acid in combination with FGF and in the presence of activin A (FIG. 8A). However, treatment of cells with retinoic acid and FGF in the absence of activin A increased the expression of Pdx1 still further (FIG. 8A). Cells treated for three days with activin A, then treated for 5 days with 1 μM retinoic acid and 50 ng/ml bFGF (also known as FGF-2) in the absence of activin A showed a level of Pdx1 expression that was approximately 3500-fold higher than that observed in samples with activin A treatment alone for 5 days (FIG. 8A). Immunocytochemistry showed that 5 to 20% of all cells expressed Pdx1 (FIGS. 9A and 9B).
  • Treatment with 1 μM retinoic acid and bFGF in the absence of activin A also caused an increase in the expression of GLUT-2 and PTF1a (FIG. 8C) that was not observed in cells treated in the presence of activin A alone. The largest increase in expression of GLUT-2 and PTF1a was observed in cells treated with 1 μM retinoic acid and 50 ng/ml bFGF. Taken together, these data suggest that the formation of pancreatic endoderm is further enhanced by removal of activin A from cell cultures after definitive endoderm has been formed.
    • Example 4 Formation of Pancreatic Endocrine Cells
  • Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 3-4 days according to the methods described in Example 1. After the cells were 50-60% confluent, the medium was changed to DMEM/F12 without FBS, containing activin A at 100 ng/ml, and the cells were cultured in this medium for one day. Following the one day culture, the medium was removed and replaced with medium containing 0.5% FBS with 100 ng/ml activin A, and the cells were cultured for one day. Following the second one-day culture, the medium was removed and replaced with medium containing 2% FBS with 100 ng/ml activin A, and the cells were cultured for one day. After this time interval, the cells were then treated for six days with combinations of retinoic acid and FGF as outlined in Example 2, then the culture medium was removed and replaced with medium comprising DMEM/F12 with 2% FBS, containing the γ-secretase inhibitors L-685,458 at 10 μM for three days. Cultures were harvested and samples of mRNA were collected for analysis. Control cultures consisting of cells treated with activin A alone for five days were also included.
  • Gene expression analysis revealed that activin A alone or in combination with retinoic acid and FGFs did not induce the expression of Ngn3 or Insulin (FIGS. 10A and 10C). A decrease in the expression of Hes-1 was also observed following treatment with L-685,458. The maximal inhibition was observed on day three post treatment (FIG. 10D). However, treatment of cells with L-685,458 induced the expression of Ngn3 to a level approximately 50-fold higher than that observed in samples treated with activin A alone or retinoic acid with FGFs in combination. A 70-fold increase of insulin expression was observed in samples treated with the γ-secretase inhibitor. NeuroD1 expression was also increased further by the L-685,458 treatment (FIG. 10A). Taken together, these data suggest that the formation of endocrine cells is further enhanced by removal of retinoic acid and FGFs from cell culture and the addition of γ-secretase inhibitors after pancreatic endoderm has been formed.
    • Example 5 Formation of Pancreatic Endocrine Cells Expressing Nkx2.2
  • Definitive endoderm cells obtained according to the methods outlined in Example 2 were treated as follows: Cells were cultured in basal medium, comprising DMEM/F12 with 2% FBS plus 50 ng/ml activin A, 50 ng/ml basic FGF and 1 μM of Retinoic Acid for 3 to 5 days. Cells were continuously cultured for another 3 to 5 days in basal medium with retinoic acid at 1 μM, alone or with bFGF. RNA samples were harvested from cells at various time points along this process to help evaluate the directed differentiation of the cells. Furthermore, culture medium and factors were regularly removed and replenished throughout the differentiation protocol. Addition of activin A showed an increase of Nkx2.2 expression about 35-fold compared to samples without activin A. Samples treated with activin A for the first three days of culture maintained Pdx1 expression at a level similar to samples containing no activin A (FIGS. 11A and 11B). Taken together, these data suggest that the expression of the pancreatic endocrine marker Nkx2.2 is further enhanced by adding Activin A to the first three days of retinoic acid and bFGF treatment.
    • Example 6 Passage and Expansion of Pancreatic Endoderm Cells in Culture
  • This example demonstrates that pancreatic endoderm cells derived from human embryonic stem cells herein can be maintained in cell culture and passaged without further differentiation. Pancreatic endoderm cells were differentiated in the presence of 100 ng/ml activin A in low serum DMEM/F12. The low serum DMEM/F12 contained 0% (v/v) fetal bovine serum (FBS) on day 1, 0.5% (v/v) FBS on day two and 2% (v/v) FBS on each day thereafter. After four days of differentiation, the cells were cultured in low serum DMEM/F12 contained 2% (v/v) FBS, 1 μM retinoic acid and 50 ng/ml bFGF for a total of six more days. After the six days of differentiation, the cells were maintained in culture in low serum DMEM/F12 contained 2% (v/v) FBS in the presence of 50 ng/ml FGF10 for a total of 6 days. During the six-day culture period, the pancreatic endoderm cells were passaged twice and cell population-doubling time is about 36 to 48 hours during this 6-day culture. On days 0, 3, and 6 of culture, Q-PCR was used to measure the expression of marker genes indicative of pancreatic endoderm. FIG. 12 shows that cells grown in the presence of 50 ng/ml FGF10 maintained expression of the pancreatic endoderm marker Pdx1 during the 6 day culture period subsequent to their derivation.
    • Example 7 Derivation of Hepatocytes from Human Embryonic Stem Cells
  • Cultures of human embryonic stem cells were maintained in undifferentiated culture conditions for 2-3 days according to the methods described in Example 1. After cells were 70-80% confluent, the medium was changed to DMEM/F12 with 2% FBS containing activin A at 100 ng/ml, and cells were cultured in the presence of activin A for seven days. After 7 days treatment with activin A, the cells were then treated for five days with the conditions shown in FIGS. 13A and 13B. After this time, the cells were harvested, and samples of mRNA were collected for analysis.
  • An increase in the expression of α-fetoprotein (AFP) and albumin was observed (FIG. 13A) for cells cultured in the absence of activin A. This was further increased by retinoic acid and FGF-4 (FIG. 13B). Taken together, these data suggest that cultures of human embryonic stem cells are capable of expressing hepatocyte markers following the treatment described above. Furthermore, human embryonic stem cells are capable of being differentiated into cells expressing markers that are characteristic of hepatocytes.
    • Example 8 Characterization of the H9 Human Embryonic Stem Cell Line
  • The quality of H9 cells was monitored over time by evaluating expression of several markers expressed by undifferentiated ES cells (Carpenter et al., 2001; Reubinoff et al., 2000; Thomson et al., 1998a). H9 cells exhibited reciprocal expression of stage-specific embryonic antigens (Table IH). H9 cells play strong immunoreactivity for SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, AP and CD9 antigens, all of which are characteristic of undifferentiated human embryonic stem cells.
  • RT-PCR was performed to assess the expression of genes characteristic of embryonic stem cells, such as, for example, OCT3/4, SOX-2, UTF-1, REX-1, Cx43, Cx45, ABCG-2 and TERT, confirming that the cells grown in this example appeared similar to previously described undifferentiated embryonic stem cells (Table IH). OCT3/4 protein expression and Alkaline Phosphatase activity (Chemicon) were confirmed by immunostaining. A majority of H9 cells were positive for OCT3/4 and AP (FIGS. 14A and 14B). Overall, these results demonstrate that the H9 cells used in this example were not significantly different in morphology, antigen immunostaining, or pluripotency marker expression when compared to reports from other laboratories.
    • Example 9 Fluorescence-Activated Cell Sorting (FACS) Analysis
  • Adhered cells were removed from culture plates by five-minute incubation with TrypLE™ Express solution (Invitrogen, CA). Released cells were resuspended in human embryonic stem cell culture medium and recovered by centrifugation, followed by washing and resuspending the cells in a staining buffer consisting of 2% BSA, 0.05% sodium azide in PBS (Sigma, MO). As appropriate, the cells were Fc-receptor blocked for 15 minutes using a 0.1% γ-globulin (Sigma) solution. Aliquots (approximately 105 cells) were incubated with either phycoerythirin (PE) or allophycocyanin (APC) conjugated monoclonal antibodies (5 μl antibody per 106 cells), as indicated in Table I, or with an unconjugated primary antibody. Controls included appropriate isotype matched antibodies, unstained cells, and cells stained only with secondary conjugated antibody. All incubations with antibodies were performed for 30 mins at 4° C. after which the cells were washed with the staining buffer. Samples that were stained with unconjugated primary antibodies were incubated for an additional 30 mins at 4° C. with secondary conjugated PE or -APC labeled antibodies. See Table I for a list of secondary antibodies used. Washed cells were pelleted and resuspended in the staining buffer, and the cell surface molecules were identified using a FACS Array (BD Biosciences) instrument, collecting at least 10,000 events.
    • Example 10 Immunocytochemistry
  • Cells seeded on 0.1% Matrigel (BD) coated dishes were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were blocked for 1 h at room temperature with PBS/0.1% BSA/10% normal chick serum/0.5% Triton X-100 and then incubated overnight with primary antibodies in PBS/0.1% BSA/10% normal chick serum at 4° C. The list of primary antibodies and their working dilutions are shown in Table IB. After three washes in PBS/0.1% BSA, fluorescent secondary antibodies at a 1:100 dilution in PBS were incubated with cells for 1 h at room temperature to allow binding. Control samples included reactions where the primary antibody was omitted or where the primary antibody was replaced with corresponding matched negative control immunoglobulins at the same concentration as the primary antibodies. Stained samples were rinsed; a drop of PROLONG® (Invitrogen, CA) containing diamidino-2-phenylindole, dihydrochloride (DAPI) was added to each sample to counter-stain the nucleus and to function as an anti-fade reagent. Images were acquired using a Nikon Confocal Eclipse C-1 inverted microscope (Nikon, Japan) and a 10-60× objective.
    • Example 11 PCR Analysis of Undifferentiated Cells
  • RNA Extraction, Purification, and cDNA Synthesis.
  • RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an ABI (ABI, CA) high capacity cDNA archive kit.
  • Real-Time PCR Amplification and Quantitative Analysis.
  • Unless otherwise stated, all reagents were purchased from Applied Biosystems. Real-time PCR reactions were performed using the ABI PRISM® 7900 Sequence Detection System. TAQMAN® UNIVERSAL PCR MASTER MIX® (ABI, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μl. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN®probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by Applied Biosystem. Primer and probe sets are listed as follows: Oct3/4 (Hs00742896), SOX-2 (Hs00602736), UTF-1 (Hs00747497), Rex-1 (Hs00399279), Connexin 43 (Hs00748445), Connexin 45 (Hs00271416), ABCG2 (Hs00184979), Otx-1 (Hs00222238), Cerberus (Hs00193796)m, Albumin (Hs00609411).
  • After an initial incubation at 50° C. for 2 min followed by 95° C. for 10 min, samples were cycled 40 times in two stages—a denaturation step at 95° C. for 15 sec followed by an annealing/extension step at 60° C. for 1 min. Data analysis was carried out using GENEAMP®7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ΔCt). The normalized amount of target was calculated as 2ΔCt, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.
    • Example 12 Karyotype Analysis
  • The karyotype of H9 cells was determined by standard G-banding karyotype analysis. A total of 100 metaphase spreads were evaluated (Applied Genetics Laboratories, Inc.). No chromosome aberrations were found in 100 cells analyzed. Cytogenetic analysis showed that the cells had a normal number of autosomes and a modal chromosome number of 46. FIG. 15 depicts a typical karyotype obtained from the human embryonic stem cell line H9.
    • Example 13 Human Embryonic Stem Cell Culture on Tissue Culture Substrate Coated with Extracellular Matrix
  • The human embryonic stem cell lines H1, H7, and H9 were obtained from WiCell Research Institute, Inc., (Madison, Wis.) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basic fibroblast growth factor (bFGF). MEF cells, derived from E13 to 13.5 mouse embryos, were purchased from Charles River. MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C (Sigma, St. Louis, Mo.) for 3 h to arrest cell division, then trypsinized and plated at 2×104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37° C. in an atmosphere of 5% CO2 within a humidified tissue culture incubator. When confluent (approximately 5 to 7 days after plating), human embryonic stem cells were treated with 1 mg/ml collagenase type IV (Invitrogen/GIBCO) for 5 to 10 min and then gently scraped off the surface using a 5 ml glass pipette. Cells were centrifuged at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells on plates coated with a 1:30 dilution of growth factor reduced MATRIGEL™ (BD Biosciences). Cells were subsequently cultured in MEF-conditioned media supplemented with 8 ng/ml bFGF and collagenase passaged on MATRIGEL coated plates for at least five passages. The cells cultured on MATRIGEL™ were routinely passaged with collagenase IV (Invitrogen/GIBCO), Dispase (BD Biosciences) or Liberase enzyme (Roche, IN).
    • Example 14 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix to Definitive Endoderm
  • Differentiation of embryonic stem cells to definitive endoderm was carried out as previously described in Nature Biotechnology 23, 1534-1541 (December 2005). Briefly, H9 cultures at approximately 60 to 70% confluency were exposed to DMEM:/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days, followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with growth factor reduced MATRIGEL at a 1:30 to 1:10 dilution or on regular MATRIGEL at a 1:30 to 1:10 dilution The plates were coated with MATRIGEL for 1 hr at room temperature.
  • At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9, and N-cadherin expression and by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceral endoderm markers, while GATA4, HNF-3 beta and SOX-17 represent definite endoderm markers, and GSC, Bry, and CXCR4 represent markers of primitive streak. FIGS. 17A, 17B, 17C, 17D, 17E and 17F depicts the expression of CXCR4 by FACS. There was a significant increase in expression of CXCR4 by cells cultured on plates coated with MATRIGEL at a 1:10 dilution as compared to lower concentrations of MATRIGEL. Furthermore, growth factor reduced MATRIGEL was not as effective in formation of definitive endoderm cells as compared to regular MATRIGEL.
  • FIG. 18 shows the real-time PCR results verifying that cells cultured on plates coated with a 1:10 dilution of MATRIGEL showed a significant upregulation of definitive endoderm markers as compared to cells cultured on a 1:30 dilution of MATRIGEL.
    • Example 15 Microarray Analysis of Changes in Gene Expression in Embryonic Stem Cells Following Formation of Definitive Endoderm
  • Total RNA was isolated from the following human embryonic stem cell cultures using an RNeasy mini kit (Qiagen): H9P83 cells cultured on MATRIGEL-coated plates and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA) for an additional three days; H9P44 cells cultured on MEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A for an additional three days. Controls for each group included cells plated on MATRIGEL-coated dishes and cultured in MEF-conditioned medium or cells plated on MEFs and cultured in ES medium.
  • Sample preparation, hybridization, and image analysis were performed according to the Affymetrix Human Genome U133 Plus 2.0 Array. Following normalization and a log transformation, data analysis was performed using OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). The variability within each treatment and among the different treatments was compared using the Pearson correlation coefficient. Variance in gene expression profiles between the different treatments along with the correlation coefficient between the lines are depicted in FIGS. 19A and 19B. Significant differences in gene expression between the samples were evaluated using analysis of variance and an F-test with adjusted P-value (Benjamini and Hochberg correction) of ≦0.05. Only genes with a present call were included in the analysis. Table IV lists the genes that are differentially expressed with a difference at least 5-fold between the various samples. The normalized intensity value of the genes that are significantly expressed along with the standard error of the mean (SEM) for each gene are listed.
    • Example 16 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm
  • Differentiation of embryonic stem cells to definitive endoderm was carried out as previously described in Nature Biotechnology 23, 1534-1541 (December 2005). Briefly, H9, H7, or H1 cells seeded on growth factor reduced MATRIGEL™ (1:30 dilution) cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A (R&D Systems, MN)) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. In all subsequent examples unless otherwise noted, this treatment regimen will be referred to as the definite endoderm (DE) protocol. In parallel, H9, H7, or H1 cells cultured on MEF feeders were also exposed to the same DE protocol outlined above.
  • At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9, CD99, and N-cadherin (CD56) expression and by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak.
  • H-lines cultured on mouse feeders and exposed to the DE protocol resulted in a robust expression of DE markers and expression of CXCR4 by FACS (FIGS. 20A, 20B and 20C). There was also a significant decrease in expression of E-cadherin following treatment with the DE protocol. Lastly, the CXCR4+population also stained positive for CD117. FIGS. 21A and 21B shows a significant upregulation of definitive endoderm markers as compared to untreated H7 (FIG. 21A) and H9 cells (FIG. 21B).
  • Unlike H-lines cultured on MEF feeders, H-lines cultured on MATRIGEL™ (1:30 dilution) and treated with the definitive endoderm protocol failed to show robust expression of definitive endoderm markers. In particular, the expression of CXCR4 by FACS and by real-time PCR was significantly lower for cells cultured on MATRIGEL™ as compared to cells cultured on mouse embryonic fibroblasts. Expression of definitive endoderm markers follows a general response pattern with H1 being greater than H9, which is greater than H7 (FIGS. 22A, 22B, 22C, 23A, 23B and 23C). From FIGS. 22A, 22B and 22C, H1 cells showed a significant increase in CXCR4 expression as compared to H7 and H9 lines. Note that in all cases, the expression of CXCR4 was lower for cells cultured on MATRIGEL™ (1:30 dilution) as compared to cells cultured on mouse embryonic fibroblasts. FIGS. 23A, 23B, and 23C shows the real-time PCR results showing that there was modest increase in upregulation of definitive endoderm markers in H7 (FIG. 23A) and H9 (FIG. 23B) lines. However, H1 (FIG. 23C) line showed a more robust upregulation of definitive endoderm markers as compared to H7 and H9 lines.
    • Example 17 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)—Role of Wnt Ligands
  • H7P44 and H9P46 embryonic stem cells were cultured on MATRIGEL™ (1:10 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. In some of the cultures 20 ng/ml Wnt-3a (Catalog#1324-WN-002, R&D Systems, MN), 20 ng/ml Wnt-5a (Catalog#654-WN-010, R&D Systems, MN), 25 ng/ml Wnt-7a (Catalog#3008-WN-025, R&D Systems, MN), or 25 ng/ml Wnt-5b (Catalog#3006-WN-025, R&D Systems, MN) was added throughout the five day treatment. FIGS. 24A and 24B depicts phase contrast images of H9P46 definitive endoderm culture in the presence of high concentration of (a) AA or (b) AA+20 ng/ml Wnt-3a. FIGS. 25A, 25B, 25C and 25D depicts the expression of CXCR4 by FACS at day 5 for H7P44, and H9P46 lines cultured on MATRIGEL™ (1:30 dilution) and exposed to the DE protocol+Wnt-3a (FIGS. 25B and 25D) and −Wnt-3a (FIGS. 25A and C). Presence of Wnt-3a in DE cultures resulted in robust expression of CXCR4 (CD184) as compared to DE cultures treated with low serum plus high concentration of AA. FIGS. 26A and 26B displays the real-time PCR data for a) H7 and b) H9 cultures treated with low serum+AA+/−Wnt ligands. For both H-lines, addition of WNT-3a resulted in significant upregulation of definitive endoderm markers. In contrast, Wnt 5a, Wnt-5b and Wnt-7a had minimal impact on expression of definitive endoderm markers.
    • Example 18 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm: Effective Dose of Wnt-3a
  • H9P46 embryonic stem cells were cultured on MATRIGEL™ coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml Activin A (AA), and 10-50 ng/ml WNt-3a (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 10-50 ng/ml Wnt-3a for an additional three days. Control cultures were not treated with Wnt-3a. FIG. 27A depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a, b) 10 ng/ml Wnt-3a, c) 20 ng/ml Wnt-3a and d) 50 ng/ml Wnt-3a. In the absence of Wnt-3a the expression of CXCR4 was very low. In contrast, addition of 10-50 ng/ml of Wnt-3a significantly increased the number of CXCR4 positive cells. Furthermore, addition of 10 ng/ml of Wnt-3a was as effective as addition of 50 ng/ml of Wnt-3a. Real-time PCR results (FIG. 28A) also confirm this finding.
  • In a separate study, H9p52 cells were plated on 1:30 low growth factor MATRIGEL™. For the first 2 days of the DE protocol a range of Wnt3A doses was used: 10 ng/ml, 5 ng/ml and 1 ng/ml. FIG. 28B shows PCR analysis of the DE markers after 5 days of treatment. The number of cells at the end of the experiment is noted in FIG. 28C. This indicates that cells are proliferating when higher doses of Wnt-3a are used. Extension to 5 days of Wnt3a treatment (5 D) had little effect on DE markers by PCR and did not significantly increase cell numbers (FIG. 28C). These data indicate that 10 ng/ml Wnt3a for 2 days is sufficient to reach optimal cell expansion and definitive endoderm differentiation.
    • Example 19 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)—Effect of GSK-3B Inhibitor
  • In order to confirm that the effect of Wnt-3a was through the Wnt pathway, a GSK-3 inhibitor was used to activate the downstream targets of Wnt, such as beta catenin. H9P46-P48 embryonic stem cells were cultured on MATRIGEL™ coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin-A (AA), and 10-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA), and 0-1000 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) for an additional three days. Control cultures were treated with low serum plus high dose of activin A+/−Wnt-3a. FIG. 29A depicts the expression of CXCR4 by FACS at day 5 in the absence of Wnt-3a or GSK-3B inhibitor, b)+20 ng/ml Wnt-3a, c)+1000 nM GSK-3B inhibitor IX, d)+500 nM GSK-3B inhibitor IX, e)+100 nM GSK-3B inhibitor IX, f)+10 nM GSK-3B inhibitor IX, g)+100 nM GSK-3B inhibitor IX for days 1-2, and h)+10 nM GSK-3B inhibitor IX for days 1-2.
  • In the absence of Wnt-3a or at 10 nm GSK-3B inhibitor the expression of CXCR4 was very low. In contrast, addition of 20 ng/ml of Wnt-3a or 100-1000 nM GSK-3B inhibitor significantly increased the number of CXCR4 positive cells. Furthermore, addition of 100 nM GSK-3B inhibitor for days 1-2 was as effective as addition of 100 nM GSK-3B inhibitor for the entire five-day period. FIGS. 30A and 30B depicts the gene expression of definitive endoderm markers for (FIG. 30A) H9P48 cells and (FIG. 30B) H9P46 cells.
  • FIG. 16 depicts the outline of a differentiation protocol in this invention, where embryonic stem cells are differentiated into definitive endoderm in a feeder free system.
    • Example 20 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)—Effective Dose of Activin-A in the Presence of GSK-3B Inhibitor or Wnt-3A
  • H9P49 and H1P46 embryonic stem cells were cultured on MATRIGEL™ coated dishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 10-100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) or 20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus 100 ng/ml of activin A. FIGS. 31A, 31B, 31C, 31D, 31E and 31F depicts the expression of CXCR4 by FACS for H9P49 and H1P46 at day 5 with a) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 10 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days, and f) 10 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days. FIGS. 31A, 31B, 31C and 31D is for H9P49 cells and FIGS. 31E and 31F is for H1P46 cells. FIGS. 32A and 32B depicts the gene expression of definitive endoderm markers for H9P49 cultures treated with 10, 50, or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a: FIG. 32A: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and FIG. 32B: SOX-17 and GATA4. This does not add up—It appears to be a problem with figure numbers or it is a cut and paste problem. It appears that robust expression of definitive endoderm markers can be obtained by using 50 ng/ml of AA+20 ng/ml of Wnt-3A or 100 nM GSK-3B inhibitor IX. Lower doses of activin A lead to formation of extraembryonic endoderm.
    • Example 21 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)—Combination of Wnt-3a and GSK-3B Inhibitor
  • H9P53 embryonic stem cells were cultured on MATRIGEL™ coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) +/−20 ng/ml Wnt-3a for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/ml activin-A (AA) for an additional three days. In parallel, H9P53 cultures were treated with 25 ng/ml BMP-4 (Catalog#314-BP-010, R&D Systems, MN) +/−20 ng/ml Wnt-3A+/−100 ng/ml activin A. Control cultures were treated with low serum plus 100 ng/ml of activin A. FIGS. 33A, 33B, 33C, 33D, 33E, and 33F depicts the expression of CXCR4 by FACS at day 5 with a) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days and 25 ng/ml BMP-4 for days 3-5, b) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX for the first two days d) 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days, e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A+100 nm GSK-3B inhibitor IX for the first two days, and f) 100 ng/ml activin A+25 ng/ml BMP-4 for all five days. FIGS. 34A and 34B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/ml of activin A plus 20 ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and panel (b): SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase over untreated cells. FIGS. 35A and 35B depicts the gene expression of definitive endoderm markers, as determined by real-time PCR for cultures of the human embryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/ml of activin A plus 10 or 100 nM GSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4. Results are expressed as fold increase over untreated cells. FIGS. 36A and 36B depicts the gene expression of definitive endoderm markers for H9P53 culture treated with combinations of activin A, Wnt-3a, GSK-3 inhibitor, and BMP-4: a) expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and b) SOX-17, HNF-3B and GATA4. Addition of BMP-4 to the DE protocol appears to induce formation of mesoderm marker BRY and combination of Wnt-3A and GSK-4B inhibitor do not lead to significant upregulation of definitive endoderm markers as compared to addition of each agent by itself in the presence of activin A.
    • Example 22 Differentiation of Human Embryonic Stem Cells Cultured on MEFs to Definitive Endoderm (DE)—Combination of Wnt-3a, Activin A, Wnt-5a, BMP-2, BMP-4, BMP-6, BMP-7, IL-4, and SDF-1 in Low Serum
  • H9P44 cells were plated onto 6 well plates previously coated with mitomycin treated mouse embryonic fibroblasts (MEF). Cells were grown until 70 to 80% confluency in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mM L-glutamine (all from Invitrogen/GIBCO) and 8 ng/ml human basic fibroblast growth factor (bFGF) (R&D Systems).
  • For DE formation, cells were treated in the presence or absence of Activin A (100 ng/ml) in addition to other growth factors detailed below. Growth factors were added to increasing concentration of FBS in a stepwise manner as indicated in the following regimen:
    • Day 0: 0% FBS in DMEM/F12 Day 1: 0.5% FBS in DMEM/F12 Day 2: 2% FBS in DMEM/F12.
  • Day 3: Cells were harvested for FACS analysis and RT-PCR.
  • All growth factors were purchased from R&D Systems, MN. A detailed description and concentration of growth factors for each treatment group is shown below.
      • 1. Control—No growth factor added
      • 2. Activin A (100 ng/ml)
      • 3. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)
      • 4. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)+BMP2 (100 ng/ml)
      • 5. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)
      • 6. Activin A (100 ng/ml)+BMP-6 (100 ng/ml)
      • 7. Activin A (100 ng/ml)+BMP-7 (100 ng/ml)
      • 8. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
      • 9. IL-4 (10 ng/ml)
      • 10. SDF1a (20 ng/ml)
      • 11. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)
      • 12. BMP2 (100 ng/ml)+BMP-4 (100 ng·ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
      • 13. Activin A (100 ng/ml) BMP-2 (100 ng/ml)+BMP-4 (100 ng·ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)
      • 14. Activin A (100 ng/ml)+IL-4 (10 ng/ml)
      • 15. Activin A (100 ng/ml)+(SDF1a (20 ng/ml)
      • 16. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt-5a (10 ng/ml)+Wnt-7a (10 ng/ml)
      • 17. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)+BMP-4 (100 ng/ml)
  • Results:
  • Cells were harvested on Day 3 of DE protocol treatment. For analysis, an aliquot of treated cells was used for RNA preparation for RT-PCR and the rest of cells used for FACS analysis. The frequency (%) of CXCR4 is shown in FIG. 37. Addition of the above growth factor(s) did not enhance expression of CXCR4 above treatment with 100 ng/ml AA in low serum.
  • For RT-PCR analysis, cells were analyzed for expression of selected panel of definitive endoderm markers. Results shown were calibrated against cells grown in the base medium but not treated with Activin A or any of the other growth factors. In agreement with the FACS data, Table V shows that there was no significant upregulation of definitive endoderm markers by addition of growth factors, such as Wnt-3a to cultures treated with a high dose of activin A in low serum. This is in contrast to the previous examples showing a significant increase in DE markers for ES cells cultured on feeder-free conditions in the presence of activin A, WNT3A, and low serum.
    • Example 23 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ or Human Fibronectin to Definitive Endoderm (DE)
  • H9P55 cells were grown and differentiated on human fibronectin or regular growth factor MATRIGEL™ (BD Biosciences). 1 ml of DMEM/F12 (Invitrogen/GIBCO) containing 1 ug/ml of human fibronectin (R&D systems, MN) was added to each well of 6 well tissue culture treated dish. Alternatively, regular growth factor MATRIGEL™ was diluted 1:10 in DMEM/F12 and 1 ml of diluted MATRIGEL™ was added to each well of 6 well tissue culture treated dish. Cells were passed with collagenase. After cells reached 80% confluency, there were treated as follows: 2 days 0.5% FBS containing 10 ng/ml mouse recombinant Wnt3a (R&D) and 100 ng/ml Activin A (R&D). This was followed by 3 days 2% FBS plus 100 ng/ml Activin A. FIGS. 38A and 38B depict the expression of CXCR4 by embryonic stem cells cultured on fibronectin and MATRIGEL, respectively. Real-time PCR results (FIG. 39) confirm that formation of definitive endoderm was equivalent on fibronectin and MATRIGEL™ coated plates.
    • Example 24 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Varying Concentrations of MATRIGEL™ to Definitive Endoderm
  • H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with regular MATRIGEL at a 1:60 to 1:10 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
  • Real time PCR results are shown in FIG. 40. Treatment of human embryonic stem cells with low serum, Activin A and Wnt3a led to the expression of CXCR4, GATA4, Goosecoid, HNF-3beta, and SOX-17 genes, suggesting that the cells were differentiating to the definitive endoderm stage. However, it does not appear that the in the presence of Wnt-3A concentration of the MATRIGEL™ coating plays an important role in differentiation.
    • Example 25 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix and Subsequently Cultured on MEFS and Differentiated to Definitive Endoderm—Role of Wnt-3a
  • Cells from the human embryonic stem cell line H9 cultured on MATRIGEL™ for at least five passages were seeded onto MEF feeders in ES media. When the cells reached 60 to 70% confluency they were exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. Additional treatment groups include Wnt-3a at 20 ng/ml for all five days+10-100 ng/ml of activin A.
  • At day 3 and 5, the cultures were analyzed by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak. hTERT and Oct-4 are markers for self renewal and pluripotency respectively. Real time-PCR results are shown in FIGS. 41A, 41B, 41C and 41D. FACS analysis was also performed at day 3 and 5. Expression levels of CXCR-4, and CD9 were analyzed and reported in FIG. 41E.
  • In the absence of Wnt3a, AFP expression levels of cells cultured in 100 ng/ml Activin A are similar to those seen in untreated controls. However, with the addition of Wnt3a to cells cultured in 100 ng/ml activin A, there is an increase in the expression of AFP that increases over time. When a lower concentration of Activin A is used, AFP expression is very high, regardless of the presence of Wnt3a (FIG. 41A). This suggests that a high concentration of Activin A is necessary to keep the cells from differentiating to extra-embryonic tissues.
  • By FACS analysis, CXCR4 positive cells ranged from 32-42% of the population in samples treated with a high concentration of Activin A but not treated with Wnt3a as compared to 23-33% of the population in samples treated with a high concentration of Activin A and Wnt3a at day 3 (FIG. 41E). By day 5 of treatment, 28-32% of cells treated with a high concentration of activin A but not treated with Wnt-3a expressed CXCR4 as compared to 43-51% of cells treated with a high concentration of Activin A and Wnt3a (FIG. 41F). In cells treated with a low concentration of Activin A, there were more CXCR4 positive cells in the treatment group without Wnt3a (11 to 20%) as compared to the Wnt-3a treated group (3 to 4%) (FIG. 41G). Overall, Wnt-3a does not appear to play a significant role in differentiation of human embryonic stem cells, cultured on MEFs, to definitive endoderm. This suggests that the feeder layer is probably secreting sufficient Wnt-3a or analogous ligand to enhance activin A induced definitive endoderm formation.
    • Example 26 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Extracellular Matrix to Definitive Endoderm Following Treatment with a Wnt Inhibitor—DKK-1
  • To determine if the addition of Wnt-3a was causing the increase in differentiation, an inhibitor of Wnt-3 signaling was added to the cultures. H9 cultures at approximately 60 to 70% confluency were exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt3a, 100 ng/ml Dikkopf-1 (DKK-1) and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. H9 cells were cultured on plates coated with Growth Factor Reduced MATRIGEL at a 1:30 dilution. The plates were coated with MATRIGEL for 1 hr at room temperature.
  • At day 5, the cultures were analyzed by real time PCR for SOX-17, SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC), HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded as visceral endoderm markers while GATA4, HNF-3beta and SOX-17 represent definite endoderm markers and GSC, Bry, and CXCR4 represent markers of primitive streak. hTERT and Oct-4 are markers for self renewal and pluripotency respectively. Results are shown in FIG. 42.
  • In the presence of Wnt3a, cells express CXCR4, GATA4, HNF-3beta and SOX17, all markers of definitive endoderm. Markers of primitive streak formation such as goosecoid were also detected at levels higher than that detected in untreated controls. With the addition of DKK1, the expression level of the aforementioned differentiation markers dramatically decrease to levels similar to that of untreated cells.
    • Example 27 Imuunofluorescence Staining of DE Markers for H9 Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated MATRIGEL and Differentiated in Low Serum Plus Activin-a and +/−Wnt-3a
  • Day 5 DE cultures of H9 cells were stained according to Example 10 for SOX-17, HNF-3B, GATA-4, N-cadherin, and E-cadherin. All nuclei were counter stained with DAPI. 20 ng/ml Wnt-3a resulted in significantly larger number of nuclei stained positive for SOX-17, HNF-3beta. and GATA-4 as compared to cultures differentiated in the absence of Wnt-3a. Furthermore, addition of Wnt-3a resulted in significant loss of expression of e-cadherin and enhanced expression of N-cadherin (FIGS. 43A and 43B).
    • Example 28 Microarray Analysis of Changes in Gene Expression in Embryonic Stem Cells Following Formation of Definitive Endoderm on MEFs Vs MATRIGEL™
  • Total RNA was isolated from the following embryonic stem cell cultures using an RNeasy mini kit (Qiagen): A) H9P33 cells cultured on MATRIGEL™-coated plates (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days; B) H9P44 cells cultured on MEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml Activin A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A for an additional three days, and C) H9P48 cells cultured on MATRIGEL™-coated plates (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A plus 20 ng/ml Wnt-3A for two days followed by treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA) for an additional three days. Controls for each group included cells plated on MATRIGEL-coated dishes and cultured in MEF-conditioned medium or cells plated on MEFs and cultured in ES medium. All groups contained three biological replicates and each biological replicate was repeated on two separate gene chips.
  • Sample preparation, hybridization, and image analysis were performed according to the Affymetrix Human Genome U133 Plus 2.0 Array. Following normalization and a log transformation, data analysis was performed using OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). Significant differences in gene expression between the samples were evaluated using analysis of variance and an F-test with adjusted P-value (Benjamini and Hochberg correction) of ≦0.05. Only genes with a present call in at least one group were included in the analysis. Table VI lists the mean normalized log-transformed signal intensity of genes showing at least 5-fold difference between group A, group B, and group C along with the adjusted P-value for each gene.
    • Example 29 Differentiation of SA002 ES Line Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)
  • SA002 P38 cells (Cellartis, Sweden) previously cultured for at least three passages on MATRIGEL-coated plates (1:30 dilution) in MEF-CM supplemented with 8 ng/ml of bFGF were exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN) +/−20 ng/ml of Wnt-3a or 100 nm GSK-3B IX inhibitor for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. Real time PCR results are shown in FIGS. 44A and 44B. Similar to H1, H7, and H9 lines, SA002 line also required addition of Wnt-3A for robust expression of DE markers. Expression of CXCR4 is depicted in FIGS. 45A, 45B and 45C: a) AA treatment b) AA+Wnt-3a c) AA+GSK-3B inhibitor.
    • Example 30 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with Human Serum to Definitive Endoderm (DE)
  • Cultures of the human embryonic stem cell line H1 at passage 55 were grown and differentiated on human serum (Sigma, #H1388, MO) coated plates. 0.5 ml of human serum was added to each well of 6 well tissue culture treated dish, incubated for 1 hr at room temperature, and aspirated before adding human embryonic stem cells. After cells reached 80% confluency, they were treated as follows: 2 days 0.5% FBS containing 10 ng/ml mouse recombinant Wnt3a (R&D) or 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA) and 100 ng/ml Activin A (R&D). This was followed by 3 days 2% FBS plus 100 ng/ml Activin A. Cultures were then analyzed by real-time PCR (FIGS. 46A and 46B). Robust expression of definitive endoderm markers were noted for cells treated with activin A+GSK-3B inhibitor or Wnt-3A as compared to cells treated with activin A only. These findings parallel our findings for human embryonic stem cells cultured on MATRIGEL™ or human fibronectin coated plates.
    • Example 31 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm (DE)—Evaluation of Various GSK-3B Inhibitor
  • The effectiveness of a number of commercially available GSK-3B inhibitors was evaluated in formation of DE from human embryonic stem cells. The following GSK-3B inhibitors were evaluated at 100 nM: GSK-3B inhibitor VIII (Catalog#361549, Calbiochem, CA), GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA), GSK-3B inhibitor XI (Catalog#361553, Calbiochem, CA), GSK-3B inhibitor XII (Catalog#361554, Calbiochem, CA). H1P54 ES cells were cultured on MATRIGEL™ coated dishes (1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin A (AA)+/−various GSK-3B inhibitors for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) for an additional three days. Control cultures were treated with low serum plus high dose of AA. FIGS. 47A and 47B depicts the gene expression of definitive endoderm markers at day 5. GSK-3B inhibitor IX and XI were both effective in inducing DE formation as compared to GSK-3B inhibitor VIII and XII.
    • Example 32 Formation of Pancreatic Endoderm by Human Embryonic Stem Cells Cultured Under Feeder-Free Conditions:—Evaluation of Retinoic Acid Analogues
  • H9P49 embryonic stem cells were cultured on MATRIGEL™ (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a (Catalog#1324-WN-002, R&D Systems, MN), and 100 ng/ml activin A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) for an additional three days. At day 5, cells were collected for evaluation by FACS and real-time PCR. As indicated in previous examples, this protocol resulted in robust upregulation of definitive endoderm markers, such as CXCR4 and SOX-17. The resulting definitive endoderm cells at day 5 were exposed to the following media conditions to induce pancreatic endoderm formation: culturing in DMEM/F12 media supplemented with 2% FBS and 1 μM all-trans retinoic acid (RA) (Catalog#R2625, Sigma, MO), or 0.1-10 μM AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid, Catalog#A8843, Sigma, MO), or 0.1-1 μM TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid, Catalog#T3757, Sigma, MO) for 3 days. AM-580 and TTNPB are retinoic acid analogs with affinity for retinoic acid receptors. RA treatment was followed by additional three day treatment in DMEM/F12 media supplemented with 2% FBS and 20-50 ng/ml bFGF (Catalog#F0291, Sigma, MO). Cultures were harvested and samples of mRNA were collected for analysis.
  • Gene expression analysis revealed that (FIGS. 48A, 48B, 48C and 48D) addition of 1 μM RA followed by exposure to bFGF significantly upregulates pancreatic endoderm markers, such as PDX-1. Furthermore, this protocol resulted in robust expression of foregut endoderm markers, such as CDX-2 and AFP. At 1 μM concentration, addition of RA analogs resulted in equivalent pancreatic endoderm and foregut markers. However, addition of 1 μM RA analogs resulted in more robust expression of AFP as compared to all-trans retinoic acid. However, addition of 10 μM AM-580 suppressed AFP and CDX-2 expression while maintaining a high expression of PDX-1.
    • Example 34 The Effect of Wnt-3a Treatment on Cytokine Expression in Human Embryonic Stem Cells
  • The effect that Wnt-3a treatment has on cytokine expression was analyzed using a protein array. Cells of the human embryonic stem cell line H9 were cultured according to the methods described in Example 15. At passage 54, cells were differentiated in the presence of 100 ng/ml Activin A+/−10 ng/ml Wnt3a for 2 days in 0.5% FBS DMEM/F12. Cells were subsequently cultured for an additional three days in 100 ng/ml Activin A and 2% FBS DMEM/F12. At the end of the 5th day, CXCR4 expression was determined by FACS for each treatment group. Cells treated with Activin A only had 1% of cells expressing CXCR4. Cells treated with Activin A and Wnt3a had 73% of cells positive for CXCR4 expression.
  • Cell lysates were prepared from cells of each treatment group, with a mammalian cell lysis kit (Sigma-Aldrich, MO). Conditioned media from each treatment group was collected and concentrated. Cytokine array analysis was completed using Cytokine Array panels provided by RayBiotech, GA (http://www.raybiotech.com/). Table VII lists cytokine, growth factor, and receptor expression following normalization of the data and background subtraction. For each panel, positive and negative controls are also included. The data shown are two independent samples per cell treatment group (1, 2).
  • Noticeable upregulation of Angiogenin, IGFBP-1 and EGF are seen in the Wnt3 treated cell conditioned media. Numerous proteins are upregulated in the Wnt3a treated cell lysates including IGFBP-1, TGFbeta-1 and TGFbeta-3. These upregulated proteins can be added back into the differentiation media to replace or enhance Wnt3a effects on definitive endoderm formation.
    • Example 35 Differentiation of Human Embryonic Stem Cells Cultured on Tissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm: Role of Wnt1
  • H1P55 ES cells were cultured on MATRIGEL™ (1:30 dilution) coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/ml activin A+/−10-20 ng/ml of WNT-1 (PeproTech, NJ, Catalogue#120-17) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/ml activin A (AA) and +/−10 or 20 ng/ml of WNT-1 for an additional three days. The following combinations of WNT1+AA were tested:
  • a) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for day three, b) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for days 3-5, c) 10 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for day three, d) 10 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for days 3-5, e) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for day three, f) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for days 3-5. FIGS. 49A and 49B displays the real-time PCR data for definitive endoderm markers following treatment of the H1 cells with low serum, AA and Wnt-1. Addition of 20 ng/ml of Wnt1 in the present of 100 ng/ml of AA resulted in significant upregulation of definitive endoderm markers (Bry, CXCR4, GSC, SOX17, HNF-3B, and GATA-4).
  • Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law.
  • TABLE IA
    LIST OF PRIMARY ANTIBODIES USED FOR FACS
    AND IMMUNOSTAINININGANALYSIS.
    Antibody Supplier Isotype Clone
    SSEA-1 Chemicon (CA) Mouse IgM MC-480
    SSEA-3 Chemicon (CA) Mouse IgG3 MC-631
    SSEA-4 Chemicon (CA) Rat IgM MC-813-70
    TRA 1-60 Chemicon (CA) Mouse IgM TRA 1-60
    TRA 1-81 Chemicon (CA) Mouse IgM TRA 1-81
    TRA 1-85 Chemicon (CA) Mouse IgG1 TRA 1-85
    AP R&D Systems Mouse IgG1 B4-78
    HNF3β R&D Systems Goat IgG
    PDX1 Santa Cruz Goat IgG A-17
    Biotechnology,
    INC
    GATA4 R&D Systems Goat IgG
    Sox
    17 R&D Systems Goat IgG
    CD 9 BD Mouse IgG1 M-L13
  • TABLE IB
    LIST OF SECONDARY CONJUGATED ANTIBODIES
    USED FOR FACS AND IMMUNOSTAINININGANALYSIS.
    Secondary Conjugated
    Antibody Supplier Dilution
    Goat Anti-Mouse IgG Jackson 1:200
    APC conjugated ImmunoResearch (PA)
    Goat Anti-Mouse IgG Jackson 1:200
    PE conjugated ImmunoResearch (PA)
    Donkey anti-rabbit PE Jackson 1:200
    or -APC conjugated ImmunoResearch (PA)
    Donkey anti-goat PE or Jackson 1:200
    -APC conjugated ImmunoResearch (PA)
    Goat anti-mouse IgM SouthernBiotech (AL) 1:200
    PE
    Goat anti-Rat IgM PE SouthernBiotech (AL) 1:200
    Goat anti-mouse IgG3 SouthernBiotech (AL) 1:200
    PE
  • TABLE IIA
    CHANGES IN PROTEIN EXPRESSION IN HUMAN
    EMBRYONIC STEM CELLS WITH TIME,
    FOLLOWING ACTIVIN A TREATMENT.
    0-DAY 2-DAY 5-DAY 8-DAY
    SSEA-3 98.67% 92.14% 42.9% 22.05% 
    CD9 92.64% 29.42% 7.27%  4.1%
    ECAM 61.23% 20.87% 14.17%  1.02%
    NCAM 7.33%  5.04% 21.1% 8.86%
    CXCR4 8.53%  20.2% 55.26%  56.92% 
  • TABLE IIB
    CHANGES IN PROTEIN EXPRESSION IN HUMAN
    EMBRYONIC STEM CELLS WITH TIME, FOLLOWING ACTIVIN A
    TREATMENT.
    1-day 3-day 5-day
    AA AA AA
    Untreated 100 ng/ml Untreated 100 ng/ml Untreated 100 ng/ml
    CXCR4+   13%   6%  7.6%   38% 3% 65.5%
    CXCR4+ 5.32% 2.97%  2.9% 31.56% 3% 55.21% 
    C-Kit+
    CXCR4+ 11.5% 14.58%  5.26% 36.67% 3% 54.5%
    EPCAM+
    CXCR4+ 12.27%  8.13% 2.72% 24.11% 3%  2.1%
    CD9+
  • TABLE IIC
    CHANGES IN PROTEIN EXPRESSION IN HUMAN
    EMBRYONIC STEM CELLS WITH TIME, FOLLOWING
    ACTIVIN A TREATMENT.
    5-day AA treatment
    CXCR4+ 92.78%
    CXCR4+/C-kit+ 92.90%
    CXCR4+/EPCAM 87.99%
    CXCR4+/CD99+ 88.78%
    CXCR4+/CD9+ 7.03%
  • TABLE III
    EXPRESSION PROFILE OF PLURIPOTENCY
    MARKERS FOR THE EMBRYONIC STEM CELL USED IN THE
    PRESENT INVENTION.
    H9
    Marker FACS RT-PCR Staining
    OCT3/4 + +
    SOX-2 +
    UTF-1 +
    REX-1 +
    hTERT +
    Cx 43 +
    Cx 45 +
    ABCG-2 +
    SSEA-1 ± (36.35%) 
    SSEA-3 + (94.38%) +
    SSEA-4 + (98.77%) +
    TRA-1-81 + (85.85%) +
    TRA-1-60 + (78.14%) +
    TRA-1-85 + (95.96%)
    CD9 + (92.02%)
    AP + (99%)   +
  • TABLE IV
    DIFFERENTIAL EXPRESSION OF GENES BETWEEN
    UNDIFFERENTIATED EMBRYONIC STEM CELLS AND
    DEFINITIVE ENDODERM STAGE CELLS CULTURED ON EITHER
    MATRIGELTM OR MOUSE EMBRYONIC FIBROBLASTS AFTER 5
    DAYS OF TREATMENT.
    H9P83 on H9P44 H9P44 on
    Gene H9P83 on Matrigel-DE on MEFs-DE
    Identifier Gene Title Gene ID Matrigel SEM stage SEM MEFs SEM stage SEM
    D87811 Homo GATA6 −2.12 0.19 2.82 2.20 −2.56 0.46 5.34 1.79
    sapiens
    mRNA for
    GATA-6,
    complete
    cds.
    /PROD = GATA-6
    /FL = gb: U66075.1
    gb: NM_005257.1
    gb: D87811.1
    AW157548 insulin-like IGFBP5 −3.28 0.17 3.31 2.11 −3.78 0.36 5.35 2.00
    growth factor
    binding
    protein
    5
    /FL = gb: M65062.1
    gb: M62782.1
    gb: NM_000599.1
    gb: AF055033.1
    NM_001898 Homo CST4 −2.15 1.26 2.54 1.95 −2.71 0.98 4.64 1.63
    sapiens
    cystatin SN
    (CST1),
    mRNA.
    /PROD = cystatin
    SN
    /FL = gb: J03870.1
    gb: NM_001898.1
    AK000680 Homo PAG1 −2.87 0.91 1.61 0.22 −4.08 0.50 1.68 0.10
    sapiens
    cDNA
    FLJ20673
    fis, clone
    KAIA4464.
    /FL = gb: AF240634.1
    gb: NM_018440.1
    NM_022642 Homo — −2.24 0.12 2.97 0.42 −3.78 0.07 2.51 0.44
    sapiens
    chorionic
    somatomammotropin
    hormone 1
    (placentallactogen)
    (CSH1),
    transcript
    variant
    4,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone
    1,
    isoform4
    /FL = gb: NM_022642.1
    NM_001317 Homo CSH1 −2.95 0.57 2.69 0.36 −4.04 0.51 1.86 0.68
    sapiens
    chorionic
    somatomammotropin
    hormone 1
    (placentallactogen)
    (CSH1),
    transcript
    variant
    1,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone
    1,
    isoform
    1precursor
    /FL = gb: NM_001317.2
    gb: J00118.1
    BC005921 Homo CSH1 −2.26 0.09 3.26 0.23 −2.96 0.37 2.58 0.45
    sapiens,
    chorionic
    somatomammotropin
    hormone 1
    (placentallactogen),
    clone
    MGC: 14518,
    mRNA,
    complete
    cds.
    /PROD = chorionic
    somatomammotropin
    hormone 1
    (placentallactogen)
    /FL = gb: BC005921.1
    AI796169 GATA- GATA3 −4.45 0.10 0.24 1.30 −4.72 0.13 0.80 2.05
    binding
    protein 3
    /FL = gb: NM_002051.1
    gb: M69106.1
    gb: BC003070.1
    NM_020991 Homo CSH1 −1.27 0.48 3.19 0.23 −2.91 0.35 2.62 0.54
    sapiens
    chorionic
    somatomammotropin
    hormone 2
    (CSH2),
    transcript
    variant
    1,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone
    2,
    isoform
    1precursor
    /FL = gb: NM_020991.2
    gb: BC002717.1
    NM_021827 Homo CCDC81 −0.37 0.35 3.16 2.05 −2.02 1.27 5.25 1.98
    sapiens
    hypothetical
    protein
    FLJ23514
    (FLJ23514),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ23514
    /FL = gb: NM_021827.1
    AB028021 Cluster Incl. FOXA2 −2.97 0.25 0.59 3.25 −3.43 0.57 4.12 2.57
    AB028021: Homo
    sapiens
    HNF-3beta
    mRNA for
    hepatocyte
    nuclear
    factor-3 beta,
    complete cds
    /cds = (196,1569)
    /gb = AB028021
    /gi = 4958949
    /ug = Hs.155651
    /len = 1944
    NM_002521 Homo NPPB 1.54 0.11 5.47 1.17 −0.15 0.38 6.24 1.23
    sapiens
    natriuretic
    peptide
    precursor B
    (NPPB),
    mRNA.
    /PROD = natriuretic
    peptide
    precursor B
    /FL = gb: NM_002521.1
    gb: M25296.1
    AA352113 ESTs ST8SIA4 −4.01 1.24 −0.99 2.04 −4.79 1.00 1.05 1.62
    BM128432 Homo IGFBP5 −2.73 1.11 2.31 2.30 −3.48 0.56 4.45 2.02
    sapiens full
    length insert
    cDNA clone
    YA81B05
    NM_002770 Homo PRSS1 −2.77 0.33 1.59 2.68 −3.13 0.48 3.88 2.95
    sapiens
    protease,
    serine, 2
    (trypsin 2)
    (PRSS2),
    mRNA.
    /PROD = protease,
    serine,
    2 (trypsin 2)
    /FL = gb: M27602.1
    gb: NM_002770.1
    NM_022579 Homo CSH1 −1.58 0.91 2.48 0.38 −3.33 0.13 1.77 0.49
    sapiens
    chorionic
    somatomammotropin
    hormone-like
    1 (CSHL1),
    transcript
    variant
    3,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone-like
    1, isoform 3
    precursor
    /FL = gb: NM_022579.1
    NM_005454 Homo CER1 2.82 0.09 5.78 1.04 1.48 0.05 6.74 1.18
    sapiens
    cerberus 1
    (Xenopus
    laevis)
    homolog
    (cysteine
    knot
    superfamily)
    (CER1),
    mRNA.
    /PROD = cerberus 1
    /FL = gb: NM_005454.1
    NM_022645 Homo CSH1 −2.30 0.33 2.95 0.31 −2.78 0.24 2.45 0.34
    sapiens
    chorionic
    somatomammotropin
    hormone 2
    (CSH2),
    transcript
    variant
    3,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone
    2,
    isoform
    3precursor
    /FL = gb: NM_022645.1
    AI821586 ESTs, LOC440981 −3.22 0.97 0.66 3.19 −2.97 0.13 4.22 2.76
    Moderately
    similar to
    JE0284 Mm-
    1 cell derived
    transplantability-
    associated
    protein 1b
    (H. sapiens)
    AL121722 Human DNA — −2.95 0.36 −0.01 2.69 −3.43 0.38 2.95 2.66
    sequence
    from clone
    RP4-788L20
    on
    chromosome
    20 Contains
    the HNF3B
    (hepatocyte
    nuclear
    factor
    3,
    beta) gene. a
    novel gene
    based on
    ESTs, ESTs,
    STSs, GSSs
    and CpG
    Islands
    NM_001311 Homo CRIP1 1.66 0.17 1.90 0.07 −2.96 0.66 1.80 0.24
    sapiens
    cysteine-rich
    protein 1
    (intestinal)
    (CRIP1),
    mRNA.
    /PROD = cysteine-
    rich
    protein 1
    (intestinal)
    /FL = gb: U58630.1
    gb: BC002738.1
    gb: NM_001311.1
    gb: U09770.1
    AY177407 Homo GSC −4.59 0.18 −1.08 2.89 −4.64 0.06 1.89 2.79
    sapiens
    homeobox
    protein
    goosecoid
    mRNA,
    complete
    cds.
    /PROD = homeobox
    protein
    goosecoid
    /FL = gb: AY177407.1
    gb: NM_173849.1
    NM_005442 Homo EOMES −0.16 0.29 2.89 1.70 0.13 0.16 4.90 1.34
    sapiens
    eomesodermin
    (Xenopus
    laevis)
    homolog
    (EOMES),
    mRNA.
    /PROD = eomesodermin
    (Xenopus
    laevis)
    homolog
    /FL = gb: AB031038.1
    gb: NM_005442.1
    L01639 Human CXCR4 0.64 0.26 3.71 1.78 −0.16 0.50 5.48 1.77
    (clone
    HSY3RR)
    neuropeptide
    Y receptor
    (NPYR)
    mRNA,
    complete
    cds.
    /PROD = neuropeptide Y
    receptor
    /FL = gb: L06797.1
    gb: NM_003467.1
    gb: AF025375.1
    gb: AF147204.1
    gb: M99293.1
    gb: L01639.1
    NM_022646 Homo — −1.57 0.60 2.67 0.26 −1.88 0.98 2.22 0.35
    sapiens
    chorionic
    somatomammotropin
    hormone 2
    (CSH2),
    transcript
    variant
    4,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone
    2,
    isoform4
    /FL = gb: NM_022646.1
    AW007532 Human IGFBP5 0.31 0.25 4.59 1.53 0.72 0.09 6.19 1.58
    insulin-like
    growth factor
    binding
    protein 5
    (IGFBP5)
    mRNA
    NM_002160 Homo TNC −0.24 0.29 2.23 0.80 −0.81 0.81 2.85 0.82
    sapiens
    hexabrachion
    (tenascin
    C, cytotactin)
    (HXB),
    mRNA.
    /PROD = hexabrachion
    (tenascin C,
    cytotactin)
    /FL = gb: M55618.1
    gb: NM_002160.1
    AA149250 ESTs, LOC645638 1.27 0.61 4.23 1.26 −0.64 0.40 2.47 1.23
    Weakly
    similar to
    WDNM RAT
    WDNM1
    PROTEIN
    PRECURSOR
    (R. norvegicus)
    AW977527 ESTs — −0.91 0.99 1.18 0.68 −2.52 1.01 1.59 0.64
    NM_022454 Homo SOX17 −1.01 0.33 2.29 2.08 −0.14 0.15 4.60 1.73
    sapiens
    hypothetical
    protein
    FLJ22252
    similar to
    SRY-box
    containing
    gene 17
    (FLJ22252),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ22252
    similar to
    SRY-
    boxcontaining
    gene
    17
    /FL = gb: NM_022454.1
    AI640307 protocadherin PCDH10 −1.89 1.37 1.53 1.32 −1.33 0.38 2.99 1.19
    10
    AJ224869 Homo — 0.98 0.18 4.22 1.88 1.34 0.17 6.36 1.49
    sapiens
    CXCR4 gene
    encoding
    receptor
    CXCR4
    AI824037 ESTs, FREM1 −1.42 0.36 1.22 1.95 −1.37 0.61 3.48 1.49
    Weakly
    similar to
    FCE2
    MOUSE
    LOW
    AFFINITY
    IMMUNOGLOBULIN
    EPSILON
    FC
    RECEPTOR
    (M. musculus)
    BE222344 splicing — 0.50 0.05 3.01 0.93 −0.94 1.18 4.27 0.94
    factor,
    arginineserine-
    rich 5
    NM_001643 Homo APOA2 −2.25 1.05 1.72 2.60 −1.20 0.44 4.47 2.42
    sapiens
    apolipoprotein
    A-II
    (APOA2),
    mRNA.
    /PROD = apolipoprotein
    A-
    II precursor
    /FL = gb: M29882.1
    gb: NM_001643.1
    gb: BC005282.1
    AI821669 ESTs — −0.94 0.79 1.71 2.19 −0.89 0.21 3.91 1.94
    NM_002608 Homo PDGFB −0.23 0.82 1.87 0.15 −2.27 0.65 1.92 0.13
    sapiens
    platelet-
    derived
    growth factor
    beta
    polypeptide
    (simian
    sarcoma
    viral (v-sis)
    oncogene
    homolog)
    (PDGFB),
    mRNA.
    /PROD = platelet-
    derived
    growth factor
    beta
    polypeptide
    (simian
    sarcoma
    viral (v-sis)
    oncogene
    homolog)
    /FL = gb: M12783.1
    gb: NM_002
    AW444761 ESTs CDKN2B −3.35 0.90 0.39 0.52 −3.11 0.88 0.80 0.25
    BF223214 ESTs — −0.48 0.08 1.69 2.04 −1.51 0.25 4.09 1.98
    AF154054 Homo GREM1 1.68 0.26 4.61 0.74 0.62 0.04 3.58 0.87
    sapiens
    DRM (DRM)
    mRNA,
    complete
    cds.
    /PROD = DRM
    /FL = gb: NM_013372.1
    gb: AF110137.2
    gb: AF045800.1
    gb: AF154054.1
    NM_021223 Homo MYL7 1.81 0.05 4.28 0.81 0.17 0.08 4.87 0.96
    sapiens
    myosin light
    chain 2a
    (LOC58498),
    mRNA.
    /PROD = myosin
    light
    chain 2a
    /FL = gb: NM_021223.1
    AI817041 G protein- CMKOR1 −0.19 0.27 2.67 1.97 0.06 0.18 5.05 1.64
    coupled
    receptor
    NM_003670 Homo BHLHB2 1.09 0.08 3.85 0.10 −0.11 0.17 3.46 0.03
    sapiens
    basic helix-
    loop-helix
    domain
    containing,
    class B, 2
    (BHLHB2),
    mRNA.
    /PROD = differentiated
    embryo
    chondrocyte
    expressed
    gene1
    /FL = gb: AB004066.1
    gb: NM_003670.1
    NM_023915 Homo GPR87 −1.70 0.61 1.64 0.18 −2.99 0.06 1.40 0.23
    sapiens G
    protein-
    coupled
    receptor 87
    (GPR87),
    mRNA.
    /PROD = G
    protein-
    coupled
    receptor 87
    /FL = gb: NM_023915.1
    gb: AF237763.1
    NM_003867 Homo FGF17 −3.05 0.39 0.03 2.07 −2.13 0.41 2.49 1.35
    sapiens
    fibroblast
    growth factor
    17 (FGF17),
    mRNA.
    /PROD = fibroblast
    growth
    factor
    17
    /FL = gb: NM_003867.1
    gb: AB009249.1
    NM_024426 Homo WT1 −3.23 0.37 −1.11 0.62 −4.20 0.56 −2.44 1.26
    sapiens
    Wilms tumor
    1 (WT1),
    transcript
    variant D,
    mRNA.
    /PROD = Wilms
    tumor
    1
    isoform D
    /FL = gb: NM_024424.1
    gb: NM_024426.1
    NM_033136 Homo FGF1 −3.10 1.42 0.09 0.83 −3.16 0.63 −0.78 1.21
    sapiens
    fibroblast
    growth factor
    1 (acidic)
    (FGF1),
    transcript
    variant
    2,
    mRNA.
    /PROD = fibroblast
    growth
    factor 1
    (acidic)
    isoform
    2precursor
    /FL = gb: NM_033137.1
    gb: NM_033136.1
    X99268 H. sapiens TWIST1 0.10 0.33 3.94 0.24 0.34 0.22 3.45 0.34
    mRNA for B-
    HLH DNA
    binding
    protein.
    /PROD = B-
    HLH DNA
    binding
    protein
    /FL = gb: NM_000474.1
    AL524520 G protein- LGR5 −2.27 1.43 0.76 1.40 −1.58 0.40 2.51 1.35
    coupled
    receptor 49
    NM_022557 Homo CSH1 −0.91 0.19 1.40 0.22 −2.12 0.08 1.47 0.14
    sapiens
    growth
    hormone 2
    (GH2),
    transcript
    variant
    2,
    mRNA.
    /PROD = growth
    hormone
    2, isoform 2
    precursor
    /FL = gb: J03756.1
    gb: NM_022557.1
    AL544576 ESTs TMEM88 −1.96 0.68 1.75 0.58 −1.45 0.86 2.08 0.70
    NM_022580 Homo CSH1 −1.20 0.86 2.30 0.39 −1.78 0.64 1.55 0.63
    sapiens
    chorionic
    somatomammotropin
    hormone-like
    1 (CSHL1),
    transcript
    variant
    4,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone-like
    1, isoform 4
    /FL = gb: NM_022580.1
    J03580 Human, PTHLH −2.72 0.33 −0.80 0.40 −4.05 0.41 −1.42 0.73
    parathyroid-
    like protein
    (associated
    with humoral
    hypercalcemia
    of
    malignancy)
    mRNA,
    complete
    cds.
    /FL = gb: J03580.1
    BC029835 Homo LOC646867 −2.66 1.12 1.01 1.85 −1.40 0.41 3.03 1.61
    sapiens,
    clone
    IMAGE: 5169759,
    mRNA.
    AI452798 ESTs MYOCD 0.98 0.13 3.31 0.66 −0.07 0.28 2.80 0.98
    NM_022559 Homo CSH1 −1.56 0.38 2.00 0.32 −2.07 0.42 1.47 0.28
    sapiens
    growth
    hormone 1
    (GH1),
    transcript
    variant
    2,
    mRNA.
    /PROD = growth
    hormone
    1, isoform 2
    precursor
    /FL = gb: NM_022559.1
    NM_001318 Homo CSH1 0.15 0.41 2.83 0.40 −1.30 0.34 2.37 0.50
    sapiens
    chorionic
    somatomammotropin
    hormone-like
    1 (CSHL1),
    transcript
    variant
    1,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone-like
    1, isoform 1
    /FL = gb: NM_001318.2
    M65062 Human IGFBP5 −2.80 1.17 2.19 2.00 −0.99 0.23 4.11 1.81
    insulin-like
    growth factor
    binding
    protein 5
    (IGFBP-5)
    mRNA,
    complete
    cds.
    /PROD = insulin-
    like growth
    factor
    binding
    protein
    5
    /FL = gb: M65062.1
    gb: M62782.1
    gb: NM_000599.1
    gb: AF055033.1
    AF207990 Homo FER1L3 0.49 0.40 3.00 0.81 −0.16 0.09 4.02 0.92
    sapiens fer-1
    like protein 3
    (FER1L3)
    mRNA,
    complete
    cds.
    /PROD = fer-1
    like protein 3
    /FL = gb: AF207990.1
    AI079944 ESTs — −0.78 0.22 0.08 0.59 −3.56 0.24 −0.13 0.73
    BC003070 Homo GATA3 1.07 0.04 3.23 0.96 0.31 0.26 4.45 0.80
    sapiens,
    GATA-
    binding
    protein 3,
    clone
    MGC: 2346,
    mRNA,
    complete
    cds.
    /PROD = GATA-
    binding
    protein 3
    /FL = gb: NM_002051.1
    gb: M69106.1
    gb: BC003070.1
    BE877796 collagen, COL8A1 −3.76 1.17 −1.28 0.73 −4.89 0.54 −1.33 1.64
    type VIII,
    alpha 1
    /FL = gb: NM_001850.1
    NM_022560 Homo CSH1 −2.23 0.74 1.95 0.08 −2.01 0.51 1.47 0.31
    sapiens
    growth
    hormone 1
    (GH1),
    transcript
    variant
    3,
    mRNA.
    /PROD = growth
    hormone
    1, isoform 3
    precursor
    /FL = gb: NM_022560.1
    BE328496 hypothetical — −0.59 0.25 1.76 0.13 −2.01 0.97 1.73 0.26
    protein
    PRO2032
    /FL = gb: AF116683.1
    gb: NM_018615.1
    NM_022469 Homo GREM2 −1.15 0.25 0.09 0.88 −3.43 0.90 0.76 0.47
    sapiens
    hypothetical
    protein
    FLJ21195
    similar to
    protein
    related to
    DAC and
    cerberus
    (FLJ21195),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ21195
    similar to
    proteinrelated
    to DAC
    and cerberus
    /FL = gb: NM_022469.1
    NM_001362 Homo DIO3 −1.73 0.70 1.99 1.92 −1.05 0.79 4.23 1.51
    sapiens
    deiodinase,
    iodothyronine,
    type III
    (DIO3),
    mRNA.
    /PROD = thyroxine
    deiodinase
    type III
    /FL = gb: NM_001362.1
    gb: S79854.1
    NM_022581 Homo CSH1 −1.56 0.67 2.08 0.42 −1.63 0.80 1.47 0.53
    sapiens
    chorionic
    somatomammotropin
    hormone-like
    1 (CSHL1),
    transcript
    variant
    5,
    mRNA.
    /PROD = chorionic
    somatomammotropin
    hormone-like
    1, isoform 5
    precursor
    /FL = gb: NM_022581.1
    NM_013372 Homo GREM1 1.61 0.07 4.15 0.64 0.91 0.15 3.20 0.81
    sapiens
    cysteine knot
    superfamily
    1, BMP
    antagonist 1
    (CKTSF1B1),
    mRNA.
    /PROD = cysteine
    knot
    superfamily
    1, BMP
    antagonist
    1
    /FL = gb: NM_013372.1
    gb: AF110137.2
    gb: AF045800.1
    gb: AF154054.1
    NM_022561 Homo CSH1 −1.32 0.60 1.91 0.25 −1.66 0.46 1.57 0.22
    sapiens
    growth
    hormone 1
    (GH1),
    transcript
    variant
    4,
    mRNA.
    /PROD = growth
    hormone
    1, isoform 4
    precursor
    /FL = gb: NM_022561.1
    NM_022659 Homo — −1.58 0.75 −1.24 1.79 −3.62 0.20 −1.81 1.25
    sapiens
    hypothetical
    protein
    FLJ11500
    similar to
    EBF2
    (FLJ11500),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ11500
    similar to
    EBF2
    /FL = gb: NM_022659.1
    AF006060 Homo CSH1 −1.21 0.06 1.13 0.05 −3.08 0.63 0.50 0.17
    sapiens
    placental
    growth
    hormone
    20 kDa
    isoform
    (hGH-V)
    mRNA,
    complete
    cds.
    /PROD = placental
    growth
    hormone
    20 kDa
    isoform
    /FL = gb: AF006060.1
    gb: NM_022556.1
    AI688418 plexin A2 PLXNA2 −0.18 0.14 0.84 1.58 −1.28 0.53 2.74 0.96
    M86849 Homo — −5.08 0.26 0.54 0.47 −1.64 0.19 −0.09 0.44
    sapiens
    connexin 26
    (GJB2)
    mRNA,
    complete
    cds.
    /PROD = connexin
    26
    /FL = gb: NM_004004.1
    gb: M86849.2
    N71923 fibronectin — 0.30 0.24 2.83 1.46 0.53 0.12 4.38 1.46
    leucine rich
    transmembrane
    protein
    3
    /FL = gb: AF169677.1
    gb: NM_013281.1
    NM_013281 Homo FLRT3 −0.20 0.33 2.14 1.61 0.00 0.15 3.96 1.39
    sapiens
    fibronectin
    leucine rich
    transmembrane
    protein 3
    (FLRT3),
    mRNA.
    /PROD = fibronectin
    leucine rich
    transmembrane
    protein3
    /FL = gb: AF169677.1
    gb: NM_013281.1
    AI601101 Homo FAM84A 0.46 0.38 3.07 0.46 −0.32 0.46 3.82 0.48
    sapiens
    cDNA:
    FLJ21410
    fis, clone
    COL03938
    NM_000325 Homo PITX2 1.37 0.17 3.51 0.47 0.89 0.16 4.02 0.47
    sapiens
    paired-like
    homeodomain
    transcription
    factor 2
    (PITX2),
    mRNA.
    /PROD = paired-
    like
    homeodomain
    transcription
    factor
    2
    /FL = gb: NM_000325.1
    gb: U69961.1
    gb: AF048720.1
    AI692659 heat shock PRDM1 0.21 0.25 1.67 0.77 −0.67 0.17 2.61 0.80
    90 kD protein
    1, alpha
    NM_000602 Homo SERPINE1 2.97 0.16 4.75 1.89 1.55 0.22 2.75 1.87
    sapiens
    serine (or
    cysteine)
    proteinase
    inhibitor,
    clade E
    (nexin,
    plasminogen
    activator
    inhibitor type
    1), member 1
    (SERPINE1),
    mRNA.
    /PROD = serine
    (or
    cysteine)
    proteinase
    inhibitor,
    cladeE
    (nexin,
    plasminogen
    activator
    inhibitor type
    1), membe
    NM_001480 Homo GALR1 −1.90 0.68 0.04 0.73 −2.42 0.72 −0.34 0.73
    sapiens
    galanin
    receptor 1
    (GALR1),
    mRNA.
    /PROD = galanin
    receptor
    1
    /FL = gb: NM_001480.2
    gb: U23854.1
    gb: L34339.1
    gb: U53511.1
    NM_000393 Homo COL5A2 4.29 0.14 5.25 0.21 2.62 0.02 5.48 0.13
    sapiens
    collagen,
    type V, alpha
    2 (COL5A2),
    mRNA.
    /PROD = collagen,
    type V,
    alpha 2
    /FL = gb: NM_000393.1
    N63706 ESTs — 0.06 0.18 1.73 1.89 −0.74 0.13 3.75 1.75
    AF132818 Homo KLF5 0.63 0.11 3.16 0.50 0.07 0.42 3.40 0.44
    sapiens
    colon
    Kruppel-like
    factor
    (CKLF)
    mRNA,
    complete
    cds.
    /PROD = colon
    Kruppel-
    like factor
    /FL = gb: AF132818.1
    gb: AF287272.1
    gb: AB030824.1
    gb: NM_001730.1
    gb: D14520.1
    X59065 H. sapiens — −1.17 1.11 −0.39 1.06 −2.61 0.26 −0.96 1.01
    FGF gene,
    exon 3
    /FL = gb: NM_000800.1
    gb: M13361.1
    R73554 Human IGFBP5 −0.12 0.15 2.63 1.41 −0.69 0.16 4.00 1.65
    insulin-like
    growth factor
    binding
    protein 5
    (IGFBP5)
    mRNA
    NM_002149 Homo HPCAL1 −0.18 0.28 1.81 0.44 −1.98 0.86 1.18 0.22
    sapiens
    hippocalcin-
    like 1
    (HPCAL1),
    mRNA.
    /PROD = hippocalcin-
    like 1
    /FL = gb: NM_002149.1
    gb: D16227.1
    AI093327 ESTs — 0.69 0.11 3.09 0.58 0.45 0.07 2.29 0.85
    NM_003240 Homo PYCR2 6.22 0.16 6.97 0.60 4.16 0.07 7.51 0.68
    sapiens
    endometrial
    bleeding
    associated
    factor (left-
    right
    determination,
    factor A;
    transforming
    growth factor
    beta
    superfamily)
    (EBAF),
    mRNA.
    /PROD = transforming
    growth
    factor, beta 4
    /FL = gb: U81523.1
    gb: NM_003240.1
    gb: AF081513.1
    AI263909 ras homolog RHOB 3.89 0.16 5.88 0.13 3.01 0.15 5.64 0.10
    gene family,
    member B
    /FL = gb: NM_004040.1
    NM_001792 Homo CDH2 3.83 0.17 6.01 0.11 2.85 0.13 5.74 0.17
    sapiens
    cadherin
    2,
    type 1, N-
    cadherin
    (neuronal)
    (CDH2),
    mRNA.
    /PROD = cadherin
    2, type
    1, N-
    cadherin
    (neuronal)
    /FL = gb: NM_001792.1
    gb: M34064.1
    NM_003897 Homo IER3 5.45 0.15 6.97 0.17 4.33 0.11 6.75 0.27
    sapiens
    immediate
    early
    response 3
    (IER3),
    mRNA.
    /PROD = immediate
    early
    response
    3
    /FL = gb: BC005080.1
    gb: BC000844.1
    gb: AF083421.1
    gb: NM_003897.1
    AF278532 Homo NTN4 −0.16 0.30 1.70 0.91 −0.67 0.48 2.66 0.74
    sapiens
    beta-netrin
    mRNA,
    complete
    cds.
    /PROD = beta-
    netrin
    /FL = gb: AF119916.1
    gb: AF297711.1
    gb: NM_021229.1
    gb: AF278532.1
    AF348491 Homo CXCR4 1.39 0.21 4.05 1.58 1.45 0.07 5.67 1.65
    sapiens
    chemokine
    receptor
    CXCR4
    mRNA,
    complete
    cds.
    /PROD = chemokine
    receptor
    CXCR4
    /FL = gb: AF348491.1
    NM_030781 Homo COLEC12 1.93 0.13 3.64 1.66 1.96 0.18 5.68 1.51
    sapiens
    scavenger
    receptor with
    C-type lectin
    (SRCL),
    mRNA.
    /PROD = scavenger
    receptor with
    C-type lectin
    /FL = gb: NM_030781.1
    NM_000599 Homo IGFBP5 −0.31 0.34 3.03 1.85 0.12 0.24 4.51 1.75
    sapiens
    insulin-like
    growth factor
    binding
    protein 5
    (IGFBP5),
    mRNA.
    /PROD = insulin-
    like growth
    factor
    binding
    protein
    5
    /FL = gb: M65062.1
    gb: M62782.1
    gb: NM_000599.1
    gb: AF055033.1
    AI348094 KIAA0882 TBC1D9 0.13 0.26 3.21 1.13 0.96 0.13 4.67 1.08
    protein
    BG287862 AHNAK AHNAK 1.47 0.20 3.72 0.51 1.37 0.17 4.34 0.58
    nucleoprotein
    (desmoyokin)
    AI676059 ESTs FOXQ1 −0.32 0.55 3.08 1.63 0.50 0.17 4.81 1.47
    AI127440 ESTs — −0.85 0.36 0.60 1.22 −0.65 0.27 2.13 1.13
    AL574210 serine (or SERPINE1 2.81 0.24 5.07 0.88 2.13 0.15 3.96 0.86
    cysteine)
    proteinase
    inhibitor,
    clade E
    (nexin,
    plasminogen
    activator
    inhibitor type
    1), member 1
    /FL = gb: NM_000602.1
    gb: M16006.1
    AB037810 Homo SIPA1L2 3.88 0.04 5.66 0.08 3.18 0.11 5.85 0.11
    sapiens
    mRNA for
    KIAA1389
    protein,
    partial cds.
    /PROD = KIAA1389
    protein
    NM_001394 Homo DUSP4 0.22 0.37 2.88 1.09 0.50 0.14 4.12 0.99
    sapiens dual
    specificity
    phosphatase
    4 (DUSP4),
    mRNA.
    /PROD = dual
    specificity
    phosphatase
    4
    /FL = gb: NM_001394.2
    gb: BC002671.1
    gb: U48807.1
    gb: U21108.1
    BC029442 Homo — 2.04 0.20 3.80 0.74 1.18 0.05 4.50 0.65
    sapiens,
    Similar to
    immunity
    associated
    protein 1,
    clone
    MGC: 32707
    IMAGE: 4618467,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to
    immunity
    associated
    protein 1
    /FL = gb: BC029442.1
    NM_000700 Homo ANXA1 5.00 0.18 6.27 1.28 3.67 0.05 4.96 1.25
    sapiens
    annexin A1
    (ANXA1),
    mRNA.
    /PROD = annexin I
    /FL = gb: BC001275.1
    gb: NM_000700.1
    BC000740 Homo CCKBR 1.08 0.36 3.93 1.84 1.93 0.03 5.86 1.82
    sapiens,
    cholecystokinin B
    receptor,
    clone
    MGC: 2199,
    mRNA,
    complete
    cds.
    /PROD = cholecystokinin B
    receptor
    /FL = gb: L07746.1
    gb: L08112.1
    gb: S70057.1
    gb: BC000740.1
    gb: L04473.1
    gb: NM_000731.1
    N36408 hypothetical FOSL2 −0.09 0.40 2.07 0.04 −0.85 0.24 1.71 0.28
    protein
    FLJ23306
    /FL = gb: NM_024530.1
    AF072242 Homo MBD2 −3.98 0.38 −0.59 0.27 −3.07 0.22 −1.70 0.68
    sapiens
    methyl-CpG
    binding
    protein
    MBD2
    (MBD2)
    mRNA,
    complete
    cds.
    /PROD = methyl-
    CpG
    binding
    protein
    MBD2
    /FL = gb: NM_003927.2
    gb: AF072242.1
    AF211891 Homo MIXL1 −0.63 0.38 0.77 0.95 −2.29 0.44 0.92 1.71
    sapiens Mix-
    like
    homeobox
    protein 1
    (MILD1)
    mRNA,
    complete
    cds.
    /PROD = Mix-
    like
    homeobox
    protein
    1
    /FL = gb: AF211891.1
    BF063186 ESTs CALD1 1.16 0.19 2.03 0.42 −1.20 1.02 1.65 0.27
    NM_000362 Homo TIMP3 0.08 0.32 1.98 0.09 −0.34 0.33 1.80 0.26
    sapiens
    tissue
    inhibitor of
    metalloproteinase 3
    (Sorsby
    fundus
    dystrophy,
    pseudoinflammatory)
    (TIMP3),
    mRNA.
    /PROD = tissue
    inhibitor of
    metalloproteinase
    3precursor
    /FL = gb: NM_000362.2
    gb: U14394.1
    gb: U67195.1
    gb: U02571.1
    AK022852 Homo SIPA1L2 2.87 0.12 4.50 0.09 1.87 0.01 4.55 0.09
    sapiens
    cDNA
    FLJ12790
    fis, clone
    NT2RP2001985,
    weakly
    similar to
    Homo
    sapiens
    high-risk
    human
    papilloma
    viruses E6
    oncoproteins
    targeted
    protein
    E6TP1 alpha
    mRNA.
    BE500942 Homo C6orf155 3.23 0.10 4.68 0.86 1.95 0.03 3.77 0.96
    sapiens
    mRNA;
    cDNA
    DKFZp761M0111
    (from
    clone
    DKFZp761M0111)
    AW665892 paternally MFAP5 −1.82 0.54 0.37 0.60 −1.54 0.39 −0.01 0.28
    expressed 3
    AK025063 Homo FAM84A −1.34 0.84 0.75 0.45 −2.35 0.85 1.01 0.53
    sapiens
    cDNA:
    FLJ21410
    fis, clone
    COL03938.
    NM_001828 Homo CLC 2.17 0.21 3.53 1.07 0.45 0.08 2.11 1.27
    sapiens
    Charot-
    Leyden
    crystal
    protein
    (CLC),
    mRNA.
    /PROD = Charot-
    Leyden
    crystal
    protein
    /FL = gb: NM_001828.3
    gb: L01664.1
    M15329 Human IL1A 0.85 0.32 2.72 0.30 −0.88 0.60 2.20 0.22
    interleukin 1-
    alpha (IL1A)
    mRNA,
    complete
    cds.
    /PROD = interleukin
    1-
    alpha
    /FL = gb: M15329.1
    BC002671 Homo DUSP4 1.79 0.03 4.30 1.39 2.10 0.10 5.60 1.22
    sapiens, dual
    specificity
    phosphatase
    4, clone
    MGC: 3713,
    mRNA,
    complete
    cds.
    /PROD = dual
    specificity
    phosphatase
    4
    /FL = gb: NM_001394.2
    gb: BC002671.1
    gb: U48807.1
    gb: U21108.1
    AA524250 deleted in DLC1 1.02 0.05 2.35 0.98 0.10 0.38 3.26 0.96
    liver cancer 1
    BC001211 Homo KIFC3 −1.53 0.72 0.81 0.38 −2.08 0.42 0.75 0.16
    sapiens,
    kinesin
    family
    member C3,
    clone
    MGC: 3226,
    mRNA,
    complete
    cds.
    /PROD = kinesin
    family
    member C3
    /FL = gb: BC001211.1
    gb: NM_005550.1
    gb: AF004426.1
    NM_004560 Homo ROR2 0.76 0.08 2.20 0.81 0.08 0.14 3.06 0.95
    sapiens
    receptor
    tyrosine
    kinase-like
    orphan
    receptor 2
    (ROR2),
    mRNA.
    /PROD = receptor
    tyrosine
    kinase-like
    orphan
    receptor
    2
    /FL = gb: M97639.1
    gb: NM_004560.1
    BC000125 Homo TGFB1 1.16 0.18 3.43 0.08 0.72 0.14 3.30 0.16
    sapiens,
    Similar to
    transforming
    growth
    factor, beta
    1, clone
    MGC: 3119,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to
    transforming
    growth
    factor, beta 1
    /FL = gb: M38449.1
    gb: BC001180.1
    gb: BC000125.1
    gb: NM_000660.1
    NM_016931 Homo NOX4 1.83 0.06 3.31 1.29 1.20 0.14 2.28 1.27
    sapiens
    NADPH
    oxidase 4
    (NOX4),
    mRNA.
    /PROD = NADPH
    oxidase
    4
    /FL = gb: AF261943.1
    gb: NM_016931.1
    gb: AF254621.1
    gb: AB041035.1
    BC001830 Homo TGFB1I1 0.95 0.17 3.59 0.73 1.52 0.09 2.74 0.69
    sapiens,
    Similar to
    transforming
    growth factor
    beta
    1
    induced
    transcript 1,
    clone
    MGC: 4078,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to
    transforming
    growth factor
    beta
    1induced
    transcript
    1
    /FL = gb: NM_015927.1
    gb: BC001830.1
    gb: AF116343.1
    NM_024576 Homo OGFRL1 3.15 0.13 4.49 0.82 1.63 0.06 3.30 0.98
    sapiens
    hypothetical
    protein
    FLJ21079
    (FLJ21079),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ21079
    /FL = gb: NM_024576.1
    NM_001963 Homo EGF 0.12 0.22 1.82 1.15 −0.62 0.36 2.68 1.23
    sapiens
    epidermal
    growth factor
    (beta-
    urogastrone)
    (EGF),
    mRNA.
    /PROD = epidermal
    growth
    factor (beta-
    urogastrone)
    /FL = gb: NM_001963.2
    BE620374 ESTs C6orf155 1.97 0.05 3.35 1.01 0.59 0.17 2.10 1.05
    AL359062 Homo COL8A1 −1.31 0.25 2.32 0.94 0.03 0.12 3.51 1.02
    sapiens
    mRNA full
    length insert
    cDNA clone
    EUROIMAGE
    1913076.
    AL117653 Homo MITF −0.12 0.61 2.32 0.10 0.21 0.13 2.56 0.06
    sapiens
    mRNA;
    cDNA
    DKFZp586C0224
    (from
    clone
    DKFZp586C0224).
    AL021977 Cluster Incl. — 1.92 0.14 4.53 0.43 2.15 0.12 4.66 0.22
    AL021977: bK447C4.1
    (novel MAFF
    (v-maf
    musculoapo
    neurotic
    fibrosarcoma
    (avian)
    oncogene
    family,
    protein F)
    LIKE protein)
    /cds = (0.494)
    /gb = AL021977
    /gi = 4914526
    /ug = Hs.51305
    /len = 2128
    NM_003564 Homo TAGLN2 5.43 0.12 6.76 0.06 3.48 0.27 6.69 0.04
    sapiens
    transgelin 2
    (TAGLN2),
    mRNA.
    /PROD = transgelin 2
    /FL = gb: D21261.1
    gb: NM_003564.1
    BC005107 Homo — 7.08 0.07 6.42 0.26 2.60 0.07 6.93 0.22
    sapiens,
    clone
    IMAGE: 3840937,
    mRNA,
    partial cds.
    /PROD = Unknown
    (protein for
    IMAGE: 3840937)
    NM_001124 Homo ADM 4.70 0.22 7.61 0.19 5.21 0.06 7.58 0.21
    sapiens
    adrenomedullin
    (ADM),
    mRNA.
    /PROD = adrenomedullin
    /FL = gb: NM_001124.1
    gb: D14874.1
    AF280545 Homo NRP2 −0.29 0.33 1.40 0.30 −1.67 0.61 1.22 0.33
    sapiens
    neuropilin-
    2b(5)
    (NRP2)
    mRNA,
    complete
    cds,
    alternatively
    spliced.
    /PROD = neuropilin-
    2b(5)
    /FL = gb: AF280544.1
    gb: AF280545.1
    NM_014624 Homo S100A6 3.08 0.38 3.10 0.18 −0.57 0.37 3.24 0.19
    sapiens
    S100
    calcium-
    binding
    protein A6
    (calcyclin)
    (S100A6),
    mRNA.
    /PROD = S100
    calcium-
    binding
    protein A6
    /FL = gb: NM_014624.2
    gb: BC001431.1
    AB030824 Homo KLF5 0.57 0.24 2.16 0.49 −0.41 0.05 2.53 0.17
    sapiens
    mRNA for
    transcription
    factor
    BTEB2,
    complete
    cds.
    /PROD = transcription
    factor
    BTEB2
    /FL = gb: AF132818.1
    gb: AF287272.1
    gb: AB030824.1
    gb: NM_001730.1
    gb: D14520.1
    NM_015675 Homo GADD45B 2.47 0.22 4.44 0.46 2.02 0.09 4.64 0.33
    sapiens
    growth arrest
    and DNA-
    damage-
    inducible,
    beta
    (GADD45B),
    mRNA.
    /PROD = DKFZP566B133
    protein
    /FL = gb: NM_015675.1
    gb: AF090950.1
    BF347089 tissue TIMP3 0.57 0.13 2.01 0.29 −0.19 0.20 2.09 0.44
    inhibitor of
    metalloproteinase 3
    (Sorsby
    fundus
    dystrophy,
    pseudoinflammatory)
    /FL = gb: NM_000362.2
    gb: U14394.1
    gb: U67195.1
    gb: U02571.1
    BF056473 ESTs — −0.64 0.87 1.79 0.10 −0.73 0.34 1.57 0.23
    AA809487 Homo — 0.52 0.22 3.68 0.43 1.39 0.22 3.89 0.48
    sapiens
    cDNA:
    FLJ21715
    fis, clone
    COL10287,
    highly similar
    to AF071569
    Homo
    sapiens
    multifunctional
    calciumcalmodulin-
    dependent
    protein
    kinase II
    delta2
    isoform
    mRNA
    AL575735 collagen, — 5.54 0.11 6.65 0.14 4.57 0.08 6.68 0.07
    type V, alpha 2
    /FL = gb: NM_000393.1
    AF003934 Homo GDF15 2.74 0.20 4.01 0.41 0.72 0.17 4.34 0.50
    sapiens
    prostate
    differentiation
    factor
    mRNA,
    complete
    cds.
    /PROD = prostate
    differentiation
    factor
    /FL = gb: U88323.1
    gb: BC000529.1
    gb: AF003934.1
    gb: NM_004864.1
    gb: AF019770.1
    gb: AB000584.1
    NM_000313 Homo PROS1 −1.54 0.10 0.98 0.83 −0.62 0.09 1.79 0.86
    sapiens
    protein S
    (alpha)
    (PROS1),
    mRNA.
    /PROD = protein
    S (alpha)
    /FL = gb: M15036.1
    gb: NM_000313.1
    NM_016651 Homo DACT1 2.37 0.26 4.79 0.67 2.46 0.14 3.92 0.78
    sapiens
    heptacellular
    carcinoma
    novel gene-3
    protein
    (LOC51339),
    mRNA.
    /PROD = heptacellular
    carcinoma
    novel gene-3
    protein
    /FL = gb: NM_016651.2
    gb: AF251079.2
    NM_020129 Homo LGALS14 1.38 0.30 3.15 0.70 0.79 0.04 2.47 0.66
    sapiens
    placental
    protein 13-
    like protein
    (LOC56891),
    mRNA.
    /PROD = placental
    protein
    13-like
    protein
    /FL = gb: NM_020129.1
    gb: AF267852.1
    NM_013451 Homo FER1L3 1.75 0.15 4.02 0.97 1.59 0.13 5.14 0.82
    sapiens fer-1
    (C. elegans)-
    like 3
    (myoferlin)
    (FER1L3),
    mRNA.
    /PROD = fer-1
    (C. elegans)-
    like 3
    (myoferlin)
    /FL = gb: NM_013451.1
    gb: AF182316.1
    R72286 microfibrillar- MFAP4 −1.15 0.60 −1.76 0.61 −2.37 0.22 −0.60 0.72
    associated
    protein 4
    AI417362 ESTs, FUT1 2.42 0.14 2.32 0.72 −0.46 0.53 1.70 0.59
    Moderately
    similar to
    ALU1_HUMAN
    ALU
    SUBFAMILY J
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    NM_001553 Homo IGFBP7 3.41 0.19 4.63 1.07 1.65 0.10 3.34 1.16
    sapiens
    insulin-like
    growth factor
    binding
    protein 7
    (IGFBP7),
    mRNA.
    /PROD = insulin-
    like growth
    factor
    binding
    protein
    7
    /FL = gb: NM_001553.1
    gb: L19182.1
    BG285011 Homo ARID5B 0.05 0.22 1.79 0.21 −0.95 0.42 1.16 0.55
    sapiens
    mRNA;
    cDNA
    DKFZp586N012
    (from
    clone
    DKFZp586N012)
    BE967311 Homo — 1.99 0.06 4.08 0.92 2.28 0.17 5.01 0.87
    sapiens
    mRNA;
    cDNA
    DKFZp762O1615
    (from
    clone
    DKFZp762O1615)
    BC005047 Homo DUSP6 2.50 0.32 4.04 0.67 1.56 0.16 2.94 0.84
    sapiens,
    clone
    MGC: 12852,
    mRNA,
    complete
    cds.
    /PROD = Unknown
    (protein for
    MGC: 12852)
    /FL = gb: NM_001946.1
    gb: AB013382.1
    gb: BC003562.1
    gb: BC003143.1
    gb: BC005047.1
    AW005572 putative 47 kDa ANKS1B −0.59 0.12 0.19 0.70 −1.59 0.07 0.86 0.92
    protein
    AW294092 ESTs RERG 0.54 0.16 −0.68 1.55 −2.78 0.92 −0.58 0.97
    NM_001899 Homo CST4 0.14 0.52 2.46 1.59 0.53 0.14 3.87 1.40
    sapiens
    cystatin S
    (CST4),
    mRNA.
    /PROD = cystatin S
    /FL = gb: NM_001899.1
    AI917371 ESTs — −1.47 0.95 0.18 1.43 −1.24 0.33 2.21 1.17
    NM_000515 Homo CSH1 −2.36 0.29 1.92 0.10 −0.73 0.57 1.37 0.21
    sapiens
    growth
    hormone 1
    (GH1),
    transcript
    variant
    1,
    mRNA.
    /PROD = growth
    hormone
    1, isoform 1
    precursor
    /FL = gb: NM_000515.2
    NM_004414 Homo DSCR1 2.14 0.03 3.85 0.26 1.74 0.16 3.34 0.36
    sapiens
    Down
    syndrome
    critical region
    gene 1
    (DSCR1),
    mRNA.
    /PROD = Down
    syndrome
    critical region
    protein
    1
    /FL = gb: U28833.2
    gb: NM_004414.2
    AI355441 sprouty — 0.24 0.48 1.72 0.50 −1.12 0.40 2.03 0.61
    (Drosophila)
    homolog 4
    AB032953 Homo ODZ2 0.00 0.14 0.65 0.35 −1.74 0.74 0.88 0.03
    sapiens
    mRNA for
    KIAA1127
    protein,
    partial cds.
    /PROD = KIAA1127
    protein
    BE048571 ESTs MGC16121 −1.66 0.78 1.44 1.00 −1.31 0.21 0.10 1.22
    AW471145 ESTs PRSS23 0.87 0.10 3.46 0.35 1.13 0.11 3.37 0.53
    BF196943 ESTs USP53 1.43 0.03 3.24 0.92 0.85 0.26 2.32 0.88
    AF498927 Homo ARHGDIB −0.03 0.19 0.04 0.96 −3.00 0.54 −0.85 0.93
    sapiens Rho
    GDP
    dissociation
    inhibitor beta
    (ARHGDIB)
    mRNA,
    complete
    cds.
    /PROD = Rho
    GDP
    dissociation
    inhibitor beta
    /FL = gb: AF498927.1
    AF329092 Homo DOC1 −2.38 0.45 1.00 0.19 −1.62 0.74 0.75 0.11
    sapiens
    GPBP-
    interacting
    protein 90
    mRNA,
    complete
    cds.
    /PROD = GPBP-
    interacting
    protein 90
    /FL = gb: AF329092.1
    BG250721 Homo — 2.48 0.04 4.54 0.95 2.63 0.14 5.31 0.83
    sapiens
    mRNA;
    cDNA
    DKFZp564C2063
    (from
    clone
    DKFZp564C2063)
    N69091 ESTs PCDH17 0.59 0.10 1.49 0.88 −1.11 0.69 2.49 0.78
    BF589359 ESTs PAG1 −1.11 0.52 −0.09 0.43 −1.54 0.12 0.50 0.13
    BF968270 ESTs SLC35F3 0.37 0.02 1.84 0.30 0.10 0.17 2.46 0.36
    NM_006183 Homo NTS 4.77 0.15 4.73 1.45 3.01 0.19 3.21 1.47
    sapiens
    neurotensin
    (NTS),
    mRNA.
    /PROD = neurotensin
    precursor
    /FL = gb: NM_006183.2
    gb: U91618.1
    D28124 Cluster Incl. NBL1 2.90 0.03 4.24 0.37 2.09 0.08 4.44 0.38
    D28124: Human
    mRNA for
    unknown
    product,
    complete cds
    /cds = (61,603)
    /gb = D28124
    /gi = 641821
    /ug = Hs.76307
    /len = 1929
    AW129593 tudor repeat TDRD7 1.19 0.19 2.40 0.87 0.92 0.04 3.27 0.89
    associator
    with
    PCTAIRE 2
    BE675435 core KLF6 0.29 0.30 2.91 1.02 0.54 0.24 3.68 0.94
    promoter
    element
    binding
    protein
    /FL = gb: AF001461.1
    gb: BC000311.1
    gb: NM_001300.2
    gb: AB017493.1
    gb: BC004301.1
    AI202327 ESTs CPEB2 1.88 0.09 3.42 0.07 0.99 0.10 3.36 0.03
    NM_002228 Homo JUN 2.12 0.16 4.09 0.56 1.62 0.13 4.53 0.41
    sapiens v-jun-
    avian
    sarcoma
    virus
    17
    oncogene
    homolog
    (JUN),
    mRNA.
    /PROD = v-jun
    avian
    sarcoma
    virus
    17
    oncogene
    homolog
    /FL = gb: NM_002228.2
    gb: BC002646.1
    AF005775 Homo CFLAR 2.88 0.18 3.70 1.56 0.35 0.17 5.22 1.69
    sapiens
    caspase-like
    apoptosis
    regulatory
    protein 2
    (clarp)
    mRNA,
    alternatively
    spliced,
    complete
    cds.
    /PROD = caspase-
    like
    apoptosis
    regulatory
    protein
    2
    /FL = gb: AF005775.1
    NM_007173 Homo PRSS23 3.02 0.10 5.11 0.21 2.90 0.05 5.22 0.23
    sapiens
    protease,
    serine, 23
    (SPUVE),
    mRNA.
    /PROD = protease,
    serine,
    23
    /FL = gb: BC001278.1
    gb: AF193611.1
    gb: AF015287.1
    gb: AL136914.1
    gb: NM_007173.1
    NM_012413 Homo QPCT 1.44 0.09 0.97 0.85 −2.46 0.65 0.19 1.10
    sapiens
    glutaminyl-
    peptide
    cyclotransferase
    (glutaminyl
    cyclase)
    (QPCT),
    mRNA.
    /PROD = glutaminyl-
    peptide
    cyclotransferase
    precursor
    /FL = gb: NM_012413.2
    BU683415 Homo KLF6 3.40 0.06 5.28 0.85 3.46 0.09 6.01 0.85
    sapiens,
    clone
    IMAGE: 4096273,
    mRNA
    AV729634 DnaJ DNAJC6 0.62 0.20 2.59 0.79 0.99 0.19 3.35 0.65
    (Hsp40)
    homolog,
    subfamily B,
    member 6
    /FL = gb: AB007942.1
    gb: NM_014787.1
    BC038556 Homo — −0.79 0.40 0.12 0.99 −2.17 0.62 −0.08 0.38
    sapiens,
    clone
    IMAGE: 3446976,
    mRNA.
    NM_014942 Homo ANKRD6 0.62 0.04 2.17 1.11 0.59 0.23 3.30 1.07
    sapiens
    KIAA0957
    protein
    (KIAA0957),
    mRNA.
    /PROD = KIAA0957
    protein
    /FL = gb: AB023174.1
    gb: NM_014942.1
    AF260333 Homo C4orf18 −1.58 0.10 0.21 1.21 −3.19 1.07 1.60 1.12
    sapiens
    AD036
    mRNA,
    complete
    cds.
    /PROD = AD036
    /FL = gb: AF260333.1
    AA448956 Homo CAMK2D 1.47 0.18 2.75 0.42 0.21 0.21 2.97 0.39
    sapiens
    cDNA:
    FLJ21715
    fis, clone
    COL10287,
    highly similar
    to AF071569
    Homo
    sapiens
    multifunctional
    calciumcalmodulin-
    dependent
    protein
    kinase II
    delta2
    isoform
    mRNA
    /FL = gb: AF071569.1
    BE349115 ESTs COL22A1 −0.02 0.52 2.04 0.25 0.24 0.06 2.38 0.22
    BF209337 Homo LOC541471 4.83 0.17 5.80 0.73 3.13 0.02 4.84 0.81
    sapiens
    cDNA
    FLJ10934
    fis, clone
    OVARC1000640
    AB019695 Homo — 2.43 0.01 4.15 0.40 1.42 0.05 3.50 0.29
    sapiens
    mRNA for
    thioredoxin
    reductase II
    beta,
    complete
    cds.
    /PROD = thioredoxin
    reductase II
    beta
    /FL = gb: AB019695.1
    AK090497 Homo LOC284576 −3.78 0.56 −2.87 1.42 −4.94 0.07 −4.74 0.53
    sapiens
    cDNA
    FLJ33178
    fis, clone
    ADRGL2002753.
    NM_006763 Homo BTG2 2.30 0.10 3.28 0.62 0.85 0.25 3.84 0.74
    sapiens BTG
    family,
    member 2
    (BTG2),
    mRNA.
    /PROD = BTG
    family,
    member 2
    /FL = gb: U72649.1
    gb: NM_006763.1
    BC002616 Homo TAGLN2 5.61 0.20 5.56 0.17 2.91 0.18 5.40 0.12
    sapiens,
    transgelin 2,
    clone
    MGC: 2989,
    mRNA,
    complete
    cds.
    /PROD = transgelin 2
    /FL = gb: BC002616.1
    AF078077 Homo GADD45B 1.30 0.17 3.41 0.31 0.61 0.12 3.05 0.29
    sapiens
    growth arrest
    and DNA-
    damage-
    inducible
    protein
    GADD45beta
    mRNA,
    complete
    cds.
    /PROD = growth
    arrest and
    DNA-
    damage-
    inducible
    proteinGADD45beta
    /FL = gb: AF087853.1
    gb: AF078077.1
    NM_001854 Homo COL11A1 2.90 0.02 4.06 1.44 1.87 0.06 2.68 1.43
    sapiens
    collagen,
    type XI,
    alpha 1
    (COL11A1),
    mRNA.
    /PROD = collagen,
    type XI,
    alpha 1
    /FL = gb: J04177.1
    gb: NM_001854.1
    AI830201 ESTs KIAA0773 −0.75 0.82 1.24 0.22 −1.87 0.51 0.40 0.22
    N95437 ESTs LMCD1 0.74 0.12 2.72 0.57 0.39 0.14 3.05 0.42
    BC002511 Homo CBR1 3.72 0.02 1.03 2.43 −4.10 0.16 −1.64 2.36
    sapiens,
    carbonyl
    reductase
    1,
    clone
    MGC: 1920,
    mRNA,
    complete
    cds.
    /PROD = carbonyl
    reductase
    1
    /FL = gb: BC002511.1
    gb: NM_001757.1
    gb: J04056.1
    AV682252 HIV-1 rev — −0.33 0.15 1.78 1.36 −0.24 0.17 0.41 1.29
    binding
    protein
    2
    AW263497 ESTs SYTL5 −1.05 0.25 1.44 0.38 −0.55 0.41 2.11 0.47
    AF130095 Homo — 5.80 0.14 6.80 0.49 4.54 0.10 7.35 0.36
    sapiens
    clone
    FLC0562
    PRO2841
    mRNA,
    complete
    cds.
    /PROD = PRO2841
    /FL = gb: AF130095.1
    H92988 tyrosine 3- C9orf19 2.00 0.06 3.54 0.91 1.93 0.14 4.61 0.81
    monooxygenasetryptophan
    5-
    monooxygenase
    activation
    protein, eta
    polypeptide
    X02761 Human FN1 5.67 0.17 6.65 0.54 4.52 0.21 7.21 0.42
    mRNA for
    fibronectin
    (FN
    precursor).
    /PROD = fibronectin
    precursor
    AI016316 ESTs — 0.24 0.18 1.19 1.17 −0.33 0.18 0.25 1.18
    NM_006622 Homo PLK2 4.64 0.14 5.88 0.45 3.50 0.06 5.19 0.47
    sapiens
    serum-
    inducible
    kinase
    (SNK),
    mRNA.
    /PROD = serum-
    inducible
    kinase
    /FL = gb: AF059617.1
    gb: NM_006622.1
    gb: AF223574.1
    NM_013238 Homo DNAJC15 −2.27 0.60 3.79 2.03 4.07 0.09 5.58 2.31
    sapiens
    DNAJ
    domain-
    containing
    (MCJ),
    mRNA.
    /PROD = DNAJ
    domain-
    containing
    /FL = gb: NM_013238.1
    gb: AF126743.1
    AK026737 Homo FN1 5.86 0.14 6.85 0.49 4.66 0.08 7.39 0.37
    sapiens
    cDNA:
    FLJ23084
    fis, clone
    LNG06602,
    highly similar
    to HSFIB1
    Human
    mRNA for
    fibronectin
    (FN
    precursor).
    NM_001458 Homo FLNC 3.28 0.17 4.17 0.70 2.11 0.17 3.39 0.64
    sapiens
    filamin C,
    gamma
    (actin-
    binding
    protein-280)
    (FLNC),
    mRNA.
    /PROD = gamma
    filamin
    /FL = gb: AF089841.1
    gb: NM_001458.1
    AK025843 Homo PALLD 1.58 0.31 3.38 0.15 1.03 0.20 3.29 0.19
    sapiens
    cDNA:
    FLJ22190
    fis, clone
    HRC01053.
    /FL = gb: AF151909.1
    gb: AF077041.1
    gb: NM_016081.1
    BC005858 Homo FN1 5.86 0.10 6.86 0.49 4.70 0.19 7.37 0.36
    sapiens,
    clone
    MGC: 3255,
    mRNA,
    complete
    cds.
    /PROD = Unknown
    (protein for
    MGC: 3255)
    /FL = gb: BC005858.1
    BG491844 v-jun avian JUN 3.53 0.08 5.45 0.47 3.42 0.11 5.91 0.34
    sarcoma
    virus
    17
    oncogene
    homolog
    /FL = gb: NM_002228.2
    gb: BC002646.1
    AA284532 tyrosine 3- C9orf19 2.19 0.11 4.12 0.84 2.43 0.08 4.93 0.90
    monooxygenasetryptophan
    5-
    monooxygenase
    activation
    protein, eta
    polypeptide
    AA192306 triadin TRDN −2.54 0.69 0.02 0.61 −1.99 0.31 0.57 0.63
    /FL = gb: U18985.1
    gb: NM_006073.1
    AF116676 Homo — 1.50 0.18 3.31 1.11 1.37 0.15 4.23 1.05
    sapiens
    PRO1957
    mRNA,
    complete
    cds.
    /PROD = PRO1957
    /FL = gb: AF116676.1
    NM_003033 Homo ST3GAL1 −0.08 0.18 1.68 1.04 0.20 0.16 2.74 0.74
    sapiens
    sialyltransferase
    4A
    (beta-
    galactosidase
    alpha-2,3-
    sialytransferase)
    (SIAT4A),
    mRNA.
    /PROD = sialyltransferase
    4A (beta-
    galactosidase
    alpha-2,3-
    sialytransferase)
    /FL = gb: L13972.1
    gb: L29555.1
    gb: NM_003033.1
    AI222435 ESTs — −4.14 1.52 −0.73 0.32 −2.33 0.62 −0.33 0.12
    NM_001924 Homo GADD45A 3.59 0.09 5.04 0.44 3.06 0.17 5.38 0.48
    sapiens
    growth arrest
    and DNA-
    damage-
    inducible,
    alpha
    (GADD45A),
    mRNA.
    /PROD = growth
    arrest and
    DNA-
    damage-
    inducible,
    alpha
    /FL = gb: M60974.1
    gb: NM_001924.2
    NM_001425 Homo EMP3 2.76 0.18 3.90 0.64 1.51 0.06 3.13 0.57
    sapiens
    epithelial
    membrane
    protein 3
    (EMP3),
    mRNA.
    /PROD = epithelial
    membrane
    protein
    3
    /FL = gb: U52101.1
    gb: NM_001425.1
    gb: U87947.1
    AB017493 Homo KLF6 1.69 0.20 4.02 1.01 1.80 0.08 4.87 0.87
    sapiens
    mRNA for
    DNA-binding
    zinc
    finger(GBF),
    complete
    cds.
    /PROD = DNA-
    binding zinc
    finger(GBF)
    /FL = gb: AF001461.1
    gb: BC000311.1
    gb: NM_001300.2
    gb: AB017493.1
    gb: BC004301.1
    X58851 Human — 0.99 0.16 2.75 1.28 1.12 0.15 4.04 1.14
    MLC1emb
    gene for
    embryonic
    myosin
    alkaline light
    chain,
    promoter
    and exon 1
    BE327172 v-jun avian — 0.86 0.15 2.94 0.51 1.14 0.08 3.57 0.45
    sarcoma
    virus
    17
    oncogene
    homolog
    U37283 Human MFAP5 0.48 0.19 1.87 0.26 0.15 0.35 1.36 0.47
    microfibril-
    associated
    glycoprotein-
    2 MAGP-2
    mRNA,
    complete
    cds.
    /PROD = microfibril-
    associated
    glycoprotein-
    2 MAGP-2
    /FL = gb: NM_003480.1
    gb: U37283.1
    AI819043 ESTs CREB5 0.92 0.26 2.63 0.73 0.65 0.15 1.80 0.79
    NM_001511 Homo CXCL1 1.01 0.13 2.95 0.35 0.31 0.08 2.39 0.27
    sapiens
    GRO1
    oncogene
    (melanoma
    growth
    stimulating
    activity,
    alpha)
    (GRO1),
    mRNA.
    /PROD = GRO1
    oncogene
    (melanoma
    growth
    stimulatingactivity,
    alpha)
    /FL = gb: NM_001511.1
    NM_006736 Homo DNAJB2 2.23 0.04 3.46 0.44 1.10 0.02 3.75 0.41
    sapiens heat
    shock
    protein,
    neuronal
    DNAJ-like 1
    (HSJ1),
    mRNA.
    /PROD = heat
    shock
    protein,
    neuronal
    DNAJ-like 1
    /FL = gb: NM_006736.1
    AA534817 ESTs, EDG3 2.32 0.06 3.28 1.04 2.08 0.04 4.58 1.04
    Weakly
    similar to
    ALU8_HUMAN
    ALU
    SUBFAMILY
    SX
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    U82164 Human CD99 4.73 0.15 5.69 0.73 3.19 0.07 6.36 0.93
    transmembrane
    protein
    CD99 type II
    mRNA,
    complete
    cds.
    /PROD = CD99
    typeII
    /FL = gb: BC002584.1
    gb: NM_002414.1
    gb: M16279.1
    gb: BC003147.1
    gb: U82164.1
    NM_000389 Homo CDKN1A 4.03 0.02 4.20 0.12 1.81 0.07 4.16 0.05
    sapiens
    cyclin-
    dependent
    kinase
    inhibitor 1A
    (p21, Cip1)
    (CDKN1A),
    mRNA.
    /PROD = cyclin-
    dependent
    kinase
    inhibitor 1A
    (p21, Cip1)
    /FL = gb: U03106.1
    gb: BC000275.1
    gb: BC001935.1
    gb: L25610.1
    gb: U09579.1
    gb: NM_000389.1
    gb: L26165.1
    NM_001299 Homo CNN1 3.75 0.15 5.75 0.50 3.47 0.11 5.14 0.43
    sapiens
    calponin
    1,
    basic,
    smooth
    muscle
    (CNN1),
    mRNA.
    /PROD = calponin
    1, basic,
    smooth
    muscle
    /FL = gb: U37019.1
    gb: NM_001299.1
    gb: D17408.1
    M36172 Human MYL4 1.55 0.21 3.30 1.05 1.21 0.13 4.11 1.06
    embryonic
    myosin alkali
    light chain
    (MLC1)
    mRNA,
    complete
    cds.
    /FL = gb: M36172.1
    gb: M24121.1
    gb: NM_002476.1
    AB033831 Homo PDGFC 0.48 0.21 0.63 0.15 −1.87 0.96 0.21 0.16
    sapiens
    hSCDGF
    mRNA for
    spinal cord-
    derived
    growth
    factor,
    complete
    cds.
    /PROD = spinal
    cord-
    derived
    growth factor
    /FL = gb: NM_016205.1
    gb: AB033831.1
    gb: AF091434.1
    gb: AF244813.1
    NM_014333 Homo IGSF4 3.11 0.09 4.12 0.27 1.85 0.06 3.79 0.24
    sapiens
    immunoglobulin
    superfamily,
    member 4
    (IGSF4),
    mRNA.
    /PROD = immunoglobulin
    superfamily,
    member 4
    /FL = gb: NM_014333.1
    gb: AF132811.1
    AF345910 Homo TTC29 0.35 0.26 1.53 0.93 −0.51 0.52 0.91 0.72
    sapiens
    NYD-SP14
    mRNA,
    complete
    cds.
    /PROD = NYD-
    SP14
    /FL = gb: AF345910.1
    NM_004297 Homo GNA14 4.59 0.06 4.52 1.59 1.95 0.11 2.95 1.50
    sapiens
    guanine
    nucleotide
    binding
    protein (G
    protein),
    alpha 14
    (GNA14),
    mRNA.
    /PROD = guanine
    nucleotide
    binding
    protein (G
    protein), alpha
    14
    /FL = gb: AF105201.1
    gb: NM_004297.1
    AK057525 Homo — 3.60 0.08 4.45 0.44 2.00 0.16 4.80 0.46
    sapiens
    cDNA
    FLJ32963
    fis, clone
    TESTI2008405.
    BC000893 Homo HIST1H2BK 2.80 0.06 3.99 0.90 2.48 0.09 4.81 0.82
    sapiens,
    H2B histone
    family,
    member A,
    clone
    MGC: 5132,
    mRNA,
    complete
    cds.
    /PROD = H2B
    histone
    family,
    member A
    /FL = gb: BC000893.1
    NM_007038 Homo ADAMTS5 0.23 0.17 1.80 0.48 0.45 0.28 1.11 0.73
    sapiens a
    disintegrin-
    like and
    metalloprotease
    (reprolysin
    type) with
    thrombospondin
    type
    1
    motif, 5
    (aggrecanase-
    2)
    (ADAMTS5),
    mRNA.
    /PROD = a
    disintegrin
    and
    metalloprotease
    withthrombospondin
    motifs-5
    preproprotein
    /FL = gb: NM_007038.1
    gb: AF14209
    AW241910 ESTs, COL22A1 −0.21 0.12 1.15 0.47 −0.57 0.18 1.90 0.51
    Weakly
    similar to
    JX0369
    collagen
    alpha 1(XIX)
    chain
    precursor
    (H. sapiens)
    AI860150 ESTs, FOSL2 −0.43 0.55 1.59 0.37 −0.34 0.10 1.08 0.20
    Weakly
    similar to
    A49134 Ig
    kappa chain
    V-I region
    (H. sapiens)
    NM_005902 Homo SMAD3 1.09 0.41 1.49 0.25 −1.10 0.89 1.73 0.10
    sapiens
    MAD
    (mothers
    against
    decapentaplegic,
    Drosophila)
    homolog 3
    (MADH3),
    mRNA.
    /PROD = MAD
    (mothers
    against
    decapentaplegic,
    Drosophila)
    homolog 3
    /FL = gb: U68019.1
    gb: U76622.1
    gb: NM_005902.1
    AA777512 Homo CAMK2D 2.27 0.15 3.67 0.47 1.67 0.08 3.99 0.51
    sapiens
    cDNA:
    FLJ21715
    fis, clone
    COL10287,
    highly similar
    to AF071569
    Homo
    sapiens
    multifunctional
    calciumcalmodulin-
    dependent
    protein
    kinase II
    delta2
    isoform
    mRNA
    AI130705 ESTs, FAM89A 0.80 0.00 1.96 0.99 0.43 0.18 2.70 0.97
    Weakly
    similar to
    A46302
    PTB-
    associated
    splicing
    factor, long
    form
    (H. sapiens)
    NM_007061 Homo CDC42EP1 1.86 0.11 2.51 0.29 0.16 0.27 2.00 0.17
    sapiens
    serum
    constituent
    protein
    (MSE55),
    mRNA.
    /PROD = serum
    constituent
    protein
    /FL = gb: M88338.1
    gb: NM_007061.1
    NM_003407 Homo ZFP36 2.55 0.14 3.53 0.55 1.49 0.28 3.98 0.47
    sapiens zinc
    finger protein
    homologous
    to Zfp-36 in
    mouse
    (ZFP36),
    mRNA.
    /PROD = zinc
    finger protein
    homologous
    to Zfp-36
    inmouse
    /FL = gb: NM_003407.1
    gb: M92843.1
    gb: M63625.1
    BC033088 Homo LMNA 2.03 0.22 2.89 0.45 0.69 0.24 1.79 0.50
    sapiens,
    Similar to
    lamin AC,
    clone
    MGC: 45654
    IMAGE: 3623265,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to lamin
    AC
    /FL = gb: BC033088.1
    U97075 Homo CFLAR 2.50 0.09 3.39 1.45 0.73 0.08 4.93 1.54
    sapiens
    FLICE-like
    inhibitory
    protein short
    form mRNA,
    complete
    cds.
    /PROD = FLICE-
    like
    inhibitory
    protein short
    form
    /FL = gb: U97075.1
    AF133207 Homo HSPB8 2.54 0.07 4.47 0.62 2.22 0.04 4.88 0.58
    sapiens
    protein
    kinase (H11)
    mRNA,
    complete
    cds.
    /PROD = protein
    kinase
    /FL = gb: AF133207.1
    NM_005979 Homo S100A13 4.10 0.11 5.67 1.17 3.92 0.02 6.81 1.20
    sapiens
    S100
    calcium-
    binding
    protein A13
    (S100A13),
    mRNA.
    /PROD = S100
    calcium-
    binding
    protein A13
    /FL = gb: BC000632.1
    gb: NM_005979.1
    AL040178 ESTs PEAR1 −0.87 0.08 0.51 0.36 −1.16 0.30 0.48 0.26
    AL117523 Homo SAMD4A 0.47 0.06 1.59 0.61 −0.24 0.30 1.28 0.53
    sapiens
    mRNA;
    cDNA
    DKFZp434H0350
    (from
    clone
    DKFZp434H0350);
    partial
    cds.
    /PROD = hypothetical
    protein
    AB051826 Homo RHOU 0.55 0.23 2.18 0.61 0.17 0.29 2.73 0.65
    sapiens
    hG28K
    mRNA for
    GTP-binding
    protein like
    1, complete
    cds.
    /PROD = GTP-
    binding
    protein like 1
    /FL = gb: AF282258.1
    gb: NM_021205.1
    gb: AB051826.1
    BC005961 Homo PTHLH −3.33 0.69 −0.88 0.81 −2.86 0.77 −1.97 1.36
    sapiens,
    parathyroid
    hormone-like
    hormone,
    clone
    MGC: 14611,
    mRNA,
    complete
    cds.
    /PROD = parathyroid
    hormone-like
    hormone
    /FL = gb: BC005961.1
    AI670948 ESTs NODAL 2.13 0.04 2.37 0.22 0.42 0.13 2.78 0.38
    AI685060 caldesmon 1 CALD1 4.34 0.33 6.42 0.73 4.45 0.09 5.63 0.82
    /FL = gb: M64110.1
    gb: NM_004342.2
    BF797381 Homo CAMK2D 3.16 0.13 4.91 0.65 3.10 0.10 5.49 0.61
    sapiens
    cDNA:
    FLJ21715
    fis, clone
    COL10287,
    highly similar
    to AF071569
    Homo
    sapiens
    multifunctional
    calciumcalmodulin-
    dependent
    protein
    kinase II
    delta2
    isoform
    mRNA
    AF026219 Homo DLC1 1.16 0.07 2.28 1.13 0.61 0.09 3.52 0.84
    sapiens HP
    protein (HP)
    mRNA,
    complete
    cds.
    /PROD = HP
    protein
    /FL = gb: AF026219.1
    gb: AF035119.1
    gb: NM_006094.2
    AK024480 Homo LOC126917 1.64 0.11 2.86 0.24 1.19 0.06 2.58 0.25
    sapiens
    mRNA for
    FLJ00074
    protein,
    partial cds.
    /PROD = FLJ00074
    protein
    N29837 ESTs LIX1 −1.43 0.15 −0.11 0.33 −1.50 0.45 −0.22 0.22
    AK001022 Homo ISL2 0.41 0.34 2.14 0.39 0.32 0.08 1.57 0.49
    sapiens
    cDNA
    FLJ10160
    fis, clone
    HEMBA1003545,
    highly
    similar to
    INSULIN
    GENE
    ENHANCER
    PROTEIN
    ISL-2.
    NM_000047 Homo ARSE 1.37 0.11 2.45 1.03 0.59 0.05 3.18 1.23
    sapiens
    arylsulfatase E
    (chondrodysplasia
    punctata 1)
    (ARSE),
    mRNA.
    /PROD = arylsulfatase E
    precursor
    /FL = gb: X83573.1
    gb: NM_000047.1
    NM_006379 Homo SEMA3C 0.24 0.25 1.08 0.00 −0.34 0.05 0.86 0.18
    sapiens
    sema
    domain,
    immunoglobulin
    domain
    (Ig), short
    basic
    domain,
    secreted,
    (semaphorin)
    3C
    (SEMA3C),
    mRNA.
    /PROD = sema
    domain,
    immunoglobulin
    domain
    (Ig),
    shortbasic
    domain,
    secreted,
    (semaphorin)
    3C
    /FL = gb: NM_006379.1
    gb: AB000220.1
    NM_007127 Homo VIL1 0.31 0.30 1.92 0.49 0.02 0.22 2.54 0.68
    sapiens villin
    1 (VIL1),
    mRNA.
    /PROD = villin 1
    /FL = gb: NM_007127.1
    U76549 Human KRT8 5.41 0.16 5.87 0.56 3.79 0.05 6.34 0.57
    cytokeratin 8
    mRNA,
    complete
    cds.
    /PROD = cytokeratin 8
    /FL = gb: BC000654.1
    gb: U76549.1
    gb: NM_002273.1
    gb: M26324.1
    gb: M34225.1
    NM_004904 Homo CREB5 0.92 0.23 1.84 0.37 0.00 0.06 1.21 0.80
    sapiens
    cAMP
    response
    element-
    binding
    protein CRE-
    BPa
    (H_GS165L15.1),
    mRNA.
    /PROD = cAMP
    response
    element-
    binding
    protein CRE-
    BPa
    /FL = gb: NM_004904.1
    gb: L05911.1
    AW082836 ESTs, WNK4 −1.17 0.26 0.98 0.38 −0.97 0.20 0.05 0.86
    Weakly
    similar to
    B34087
    hypothetical
    protein
    (H. sapiens)
    BE568134 death TNFRSF21 4.76 0.06 5.90 0.54 3.94 0.08 6.28 0.58
    receptor 6
    /FL = gb: NM_014452.1
    gb: AF068868.1
    NM_002845 Homo PTPRM 2.50 0.10 3.43 0.46 1.34 0.19 3.76 0.50
    sapiens
    protein
    tyrosine
    phosphatase,
    receptor
    type, M
    (PTPRM),
    mRNA.
    /PROD = protein
    tyrosine
    phosphatase,
    receptor
    type,
    mupolypeptide
    /FL = gb: NM_002845.1
    AI949419 ESTs — −0.11 0.21 1.72 0.64 −0.47 0.01 2.39 0.77
    AK024680 Homo NRP2 0.42 0.02 2.65 0.17 0.98 0.09 2.86 0.17
    sapiens
    cDNA:
    FLJ21027
    fis, clone
    CAE07110.
    /FL = gb: NM_018534.1
    BE542563 ESTs LOC643277 2.20 0.10 0.54 1.49 −3.73 0.40 −1.74 1.96
    AW005572 putative 47 kDa ANKS1B −1.21 0.52 −0.24 0.82 −1.19 0.55 1.05 0.64
    protein
    AW665892 paternally MFAP5 −3.87 0.82 −1.56 1.24 −2.40 0.24 −3.49 2.10
    expressed 3
    NM_006206 Homo PDGFRA 0.92 0.18 2.34 0.74 0.72 0.09 3.18 0.62
    sapiens
    platelet-
    derived
    growth factor
    receptor,
    alpha
    polypeptide
    (PDGFRA),
    mRNA.
    /PROD = platelet-
    derived
    growth factor
    receptor,
    alphapolypeptide
    /FL = gb: NM_006206.1
    gb: M21574.1
    NM_002425 Homo MMP10 −1.21 0.70 1.21 0.88 −0.37 0.28 0.52 0.45
    sapiens
    matrix
    metalloproteinase
    10
    (stromelysin
    2) (MMP10),
    mRNA.
    /PROD = matrix
    metalloproteinase
    10
    preproprotein
    /FL = gb: BC002591.1
    gb: NM_002425.1
    NM_004338 Homo C18orf1 −0.79 0.22 −0.09 0.39 −1.57 0.43 0.50 0.02
    sapiens
    chromosome
    18 open
    reading
    frame 1
    (C18ORF1),
    mRNA.
    /PROD = chromosome
    18
    open reading
    frame
    1
    /FL = gb: NM_004338.1
    gb: AF009426.1
    AF052094 Homo EPAS1 0.75 0.12 2.28 0.19 0.42 0.21 1.74 0.33
    sapiens
    clone 23698
    mRNA
    sequence.
    /FL = gb: U51626.1
    gb: U81984.1
    gb: NM_001430.1
    BF126155 ESTs S100A10 −0.32 0.25 1.24 0.28 −0.65 0.33 1.02 0.44
    AI860212 phosphoprotein PAG1 −0.17 0.22 1.37 0.28 −0.40 0.23 1.25 0.13
    associated
    with GEMs
    /FL = gb: AF240634.1
    gb: NM_018440.1
    AL110298 Homo SLC2A14 5.13 0.06 6.03 0.58 3.97 0.24 6.33 0.73
    sapiens
    mRNA;
    cDNA
    DKFZp564K1672
    (from
    clone
    DKFZp564K1672);
    partial
    cds.
    /PROD = hypothetical
    protein
    AY048775 Homo MANEA −0.47 0.40 0.71 0.90 −0.69 0.35 1.23 0.81
    sapiens
    mandaselin
    long form
    mRNA,
    complete
    cds.
    /PROD = mandaselin
    long
    form
    /FL = gb: AY048775.1
    M99436 Cluster Incl. TLE2 2.65 0.07 4.05 0.66 1.95 0.13 4.76 0.61
    M99436: Human
    transducin-
    like
    enhancer
    protein
    (TLE2)
    mRNA,
    complete cds
    /cds = (25,2256)
    /gb = M99436
    /gi = 307511
    /ug = Hs.173063
    /len = 2271
    NM_014061 Homo MAGEH1 2.04 0.23 3.84 1.17 1.08 0.24 4.86 1.12
    sapiens
    APR-1
    protein
    (APR-1),
    mRNA.
    /PROD = APR-
    1 protein
    /FL = gb: AF320912.1
    gb: AF143235.3
    gb: NM_014061.1
    AL577531 caldesmon 1 CALD1 5.79 0.06 6.06 0.75 4.07 0.06 5.13 0.77
    /FL = gb: M64110.1
    gb: NM_004342.2
    AI082237 proprotein TAGLN 1.38 0.21 3.20 0.33 1.55 0.15 2.74 0.44
    convertase
    subtilisinkexin
    type
    7
    BF055171 acyl- ACOX3 0.88 0.22 2.52 0.83 1.27 0.06 3.73 0.81
    Coenzyme A
    oxidase 3,
    pristanoyl
    /FL = gb: NM_003501.1
    AF231124 Homo SPOCK1 2.84 0.09 4.17 0.95 2.71 0.07 5.38 1.00
    sapiens
    testican-1
    mRNA,
    complete
    cds.
    /PROD = testican-1
    /FL = gb: NM_004598.1
    gb: AF231124.1
    AA588092 ESTs SLC40A1 −1.53 0.23 −0.94 0.51 −2.42 0.33 0.27 0.27
    AK094809 Homo RASGRF2 3.20 0.05 3.64 0.77 1.95 0.24 2.95 0.74
    sapiens
    cDNA
    FLJ37490
    fis, clone
    BRAWH2014934,
    highly
    similar to
    GUANINE
    NUCLEOTIDE
    RELEASING
    PROTEIN.
    NM_013959 Homo NRG1 1.50 0.20 3.22 0.68 1.34 0.13 3.78 0.75
    sapiens
    neuregulin 1
    (NRG1),
    transcript
    variant
    SMDF,
    mRNA.
    /PROD = neuregulin 1
    isoform
    SMDF
    /FL = gb: L41827.1
    gb: NM_013959.1
    NM_004887 Homo CXCL14 0.70 0.07 1.70 0.37 −0.15 0.11 2.36 0.63
    sapiens
    small
    inducible
    cytokine
    subfamily B
    (Cys-X-Cys),
    member 14
    (BRAK)
    (SCYB14),
    mRNA.
    /PROD = small
    inducible
    cytokine
    subfamily
    B(Cys-X-
    Cys),
    member 14
    (BRAK)
    /FL = gb: AF144103.1
    gb: AF106911.1
    gb: AF073957.1
    gb: BC003513.1
    gb: NM_004887.1
    T77995 Homo — −0.20 0.16 0.82 0.50 −1.70 0.24 1.17 0.06
    sapiens
    cDNA
    FLJ13392
    fis, clone
    PLACE1001280
    NM_030971 Homo SFXN3 −0.43 0.59 −0.01 0.78 −1.92 0.17 −1.25 0.43
    sapiens
    similar to rat
    tricarboxylate
    carrier-like
    protein
    (BA108L7.2),
    mRNA.
    /PROD = similar
    to rat
    tricarboxylate
    carrier-
    likeprotein
    /FL = gb: NM_030971.1
    H25097 KIAA1350 USP53 3.25 0.05 4.57 0.20 2.96 0.04 4.47 0.13
    protein
    NM_004932 Homo CDH6 −3.58 1.16 −1.12 0.51 −2.14 0.63 −0.88 0.39
    sapiens
    cadherin
    6,
    type 2, K-
    cadherin
    (fetal kidney)
    (CDH6),
    mRNA.
    /PROD = cadherin
    6, type
    2, K-
    cadherin
    (fetal kidney)
    /FL = gb: D31784.1
    gb: NM_004932.1
    N21426 hypothetical SYTL2 2.41 0.09 3.44 1.22 1.70 0.06 2.02 1.21
    protein
    FLJ20163
    AA234096 KIAA0963 MGC16121 0.38 0.16 1.74 0.63 −0.13 0.04 1.10 0.65
    protein
    AV734843 hypothetical OBFC2A 0.62 0.06 1.62 0.79 0.09 0.02 1.25 0.73
    protein
    FLJ22833
    AL519710 immunoglobulin IGSF4 3.86 0.02 4.95 0.26 3.22 0.12 4.66 0.27
    superfamily,
    member 4
    /FL = gb: NM_014333.1
    gb: AF132811.1
    N32834 HIV-1 rev — −0.28 0.36 1.58 0.97 −0.23 0.18 0.33 1.17
    binding
    protein 2
    AF132811 Homo IGSF4 2.23 0.07 3.85 0.28 2.17 0.10 3.58 0.34
    sapiens
    nectin-like
    protein 2
    (NECL2)
    mRNA,
    complete
    cds.
    /PROD = nectin-
    like protein 2
    /FL = gb: NM_014333.1
    gb: AF132811.1
    J04177 Cluster Incl. COL11A1 3.28 0.22 4.44 1.22 2.32 0.03 3.03 1.31
    J04177: Human
    alpha-1
    type XI
    collagen
    (COL11A1)
    mRNA,
    complete cds
    /cds = (161,5581)
    /gb = J04177
    /gi = 179729
    /ug = Hs.82772
    /len = 6158
    AI982754 clusterin CLU 0.17 0.14 1.86 0.94 0.39 0.13 0.97 0.83
    (complement
    lysis
    inhibitor, SP-
    40,40,
    sulfated
    glycoprotein
    2,
    testosterone-
    repressed
    prostate
    message
    2,
    apolipoprotein
    J)
    NM_003501 Homo ACOX3 0.43 0.32 2.10 0.91 0.79 0.08 3.19 0.95
    sapiens acyl-
    Coenzyme A
    oxidase 3,
    pristanoyl
    (ACOX3),
    mRNA.
    /PROD = acyl-
    Coenzyme A
    oxidase 3,
    pristanoyl
    /FL = gb: NM_003501.1
    AF144103 Homo CXCL14 1.69 0.13 2.16 0.55 0.40 0.21 2.73 0.69
    sapiens
    NJAC
    protein
    (NJAC)
    mRNA,
    complete
    cds.
    /PROD = NJAC
    protein
    /FL = gb: AF144103.1
    gb: AF106911.1
    gb: AF073957.1
    gb: BC003513.1
    gb: NM_004887.1
    NM_005451 Homo PDLIM7 2.58 0.12 3.12 0.50 1.30 0.16 2.65 0.37
    sapiens
    enigma (LIM
    domain
    protein)
    (ENIGMA),
    mRNA.
    /PROD = enigma
    protein
    /FL = gb: BC001093.1
    gb: NM_005451.2
    gb: AF265209.1
    NM_004472 Homo FOXD1 −0.85 0.80 2.04 0.55 0.71 0.23 1.70 0.74
    sapiens
    forkhead box
    D1 (FOXD1),
    mRNA.
    /PROD = fork
    head box D1
    /FL = gb: U59832.1
    gb: NM_004472.1
    AF332197 Homo SIX2 0.36 0.24 0.72 0.12 −0.44 0.35 1.05 0.15
    sapiens adult
    SIX2 (SIX2)
    mRNA,
    complete
    cds.
    /PROD = SIX2
    /FL = gb: AF332197.1
    gb: NM_016932.1
    gb: AF136940.1
    AB046817 Homo SYTL2 2.88 0.17 4.09 1.09 2.50 0.05 3.12 1.03
    sapiens
    mRNA for
    KIAA1597
    protein,
    partial cds.
    /PROD = KIAA1597
    protein
    AK093435 Homo FLJ36116 4.75 0.02 4.59 1.53 2.46 0.18 6.15 1.46
    sapiens
    cDNA
    FLJ36116
    fis, clone
    TESTI2022338.
    NM_004815 Homo ARHGAP29 2.18 0.02 3.18 0.82 1.69 0.03 4.15 0.69
    sapiens
    PTPL1-
    associated
    RhoGAP 1
    (PARG1),
    mRNA.
    /PROD = PTPL1-
    associated
    RhoGAP 1
    /FL = gb: U90920.1
    gb: NM_004815.1
    BG028597 ESTs COL11A1 0.19 0.02 1.61 1.02 −0.11 0.38 0.48 1.09
    AB019562 Homo SPP1 0.58 0.14 2.79 1.07 1.10 0.02 1.21 1.06
    sapiens
    mRNA
    expressed
    only in
    placental
    villi, clone
    SMAP41.
    NM_002346 Homo LY6E 3.54 0.06 3.92 1.16 1.81 0.23 4.93 1.39
    sapiens
    lymphocyte
    antigen
    6
    complex,
    locus E
    (LY6E),
    mRNA.
    /PROD = lymphocyte
    antigen
    6
    complex,
    locus E
    /FL = gb: U42376.1
    gb: NM_002346.1
    gb: U56145.1
    BF589515 ESTs TMEM16D 0.63 0.24 1.94 0.85 0.90 0.20 1.47 0.75
    AL037401 nuclear NR2F2 −2.00 0.30 0.58 0.67 −2.19 0.13 0.82 0.66
    receptor
    subfamily
    2,
    group F,
    member 2
    /FL = gb: M64497.1
    NM_000783 Homo CYP26A1 3.92 0.27 6.28 1.06 6.29 0.09 7.31 0.98
    sapiens
    cytochrome
    P450,
    subfamily
    XXVIA,
    polypeptide 1
    (CYP26A1),
    mRNA.
    /PROD = cytochrome
    P450,
    subfamily
    XXVIA,
    polypeptide 1
    /FL = gb: NM_000783.1
    gb: AF005418.1
    U16307 Human GLIPR1 −0.11 0.03 1.16 0.95 −0.46 0.77 0.35 0.77
    glioma
    pathogenesis-
    related
    protein
    (GliPR)
    mRNA,
    complete
    cds.
    /PROD = glioma
    pathogenesis-
    related
    protein
    /FL = gb: NM_006851.1
    gb: U16307.1
    NM_001233 Homo CAV2 4.07 0.07 4.42 0.90 2.82 0.08 3.44 0.85
    sapiens
    caveolin 2
    (CAV2),
    mRNA.
    /PROD = caveolin 2
    /FL = gb: AF035752.1
    gb: BC005256.1
    gb: NM_001233.1
    AA211909 ESTs C20orf100 0.62 0.17 1.93 1.00 −0.21 0.11 0.72 0.99
    AK057525 Homo — 2.17 0.13 2.97 0.77 0.97 0.08 3.68 0.60
    sapiens
    cDNA
    FLJ32963
    fis, clone
    TESTI2008405.
    BF344237 Homo — −2.82 0.20 −0.92 0.64 −0.73 0.67 −1.25 1.03
    sapiens
    mRNA;
    cDNA
    DKFZp564N1116
    (from
    clone
    DKFZp564N1116)
    NM_014481 Homo APEX2 2.76 0.15 3.18 0.80 1.57 0.11 3.91 0.93
    sapiens
    apurinicapyrimidinic
    endonuclease
    (APEX
    nuclease)-
    like 2 protein
    (APEXL2),
    mRNA.
    /PROD = apurinicapyrimidinic
    endonuclease
    (APEXnuclease)-
    like 2
    protein
    /FL = gb: AB049211.1
    gb: NM_014481.1
    gb: BC002959.1
    gb: AB021260.1
    gb: AF119046.1
    AI912583 HIV-1 rev KRR1 1.10 0.23 2.68 1.04 1.31 0.06 1.53 1.01
    binding
    protein 2
    BI254089 Homo ADAMTS5 −1.40 0.26 −0.01 0.31 −1.71 0.64 −0.79 0.52
    sapiens full
    length insert
    cDNA clone
    ZD50E03
    BF197655 caveolin
    2 CAV2 3.51 0.10 3.54 0.94 2.32 0.09 2.70 0.85
    /FL = gb: AF035752.1
    gb: BC005256.1
    gb: NM_001233.1
    NM_001955 Homo EDN1 2.84 0.15 2.97 0.95 1.01 0.14 1.75 1.03
    sapiens
    endothelin 1
    (EDN1),
    mRNA.
    /PROD = endothelin 1
    /FL = gb: NM_001955.1
    NM_003319 Homo TTN 1.28 0.19 0.62 0.86 −0.92 0.42 2.00 0.55
    sapiens titin
    (TTN),
    mRNA.
    /PROD = titin
    /FL = gb: NM_003319.1
    BE965029 Homo MICAL2 0.60 0.21 1.95 0.64 0.21 0.19 0.83 0.78
    sapiens
    cDNA:
    FLJ22463
    fis, clone
    HRC10126
    AI452457 ESTs C1orf168 −1.48 0.53 0.02 0.93 −1.87 0.07 1.05 0.94
    AI733465 collagen, COL9A2 0.96 0.13 1.99 1.00 1.17 0.14 2.94 0.86
    type IX,
    alpha 2
    /FL = gb: NM_001852.1
    NM_006103 Homo WFDC2 4.02 0.11 5.11 1.05 3.39 0.04 6.19 1.10
    sapiens
    epididymis-
    specific,
    whey-acidic
    protein type,
    four-disulfide
    core;
    putative
    ovarian
    carcinoma
    marker
    (HE4),
    mRNA.
    /PROD = epididymis-
    specific,
    whey-acidic
    protein
    type, four-
    disulfide
    core;
    putative
    ovarian
    carcinoma
    marker
    /FL = gb: NM_00610
    NM_017540 Homo GALNT10 1.46 0.18 2.68 0.78 0.97 0.23 3.47 0.82
    sapiens
    hypothetical
    protein
    DKFZp586H0623
    (DKFZp586H0623),
    mRNA.
    /PROD = hypothetical
    protein
    DKFZp586H0623
    /FL = gb: NM_017540.1
    W72527 phosphoserine SLC1A4 −0.65 0.09 0.38 0.80 −0.63 0.62 −0.72 0.09
    aminotransferase
    NM_003468 Homo FZD5 1.35 0.26 3.43 1.21 1.45 0.10 4.43 1.16
    sapiens
    frizzled
    (Drosophila)
    homolog 5
    (FZD5),
    mRNA.
    /PROD = frizzled 5
    /FL = gb: NM_003468.1
    gb: U43318.1
    H15920 ESTs, MRGPRF 2.34 0.18 3.62 1.02 2.11 0.11 2.87 0.84
    Weakly
    similar to
    RTA RAT
    PROBABLE
    G PROTEIN-
    COUPLED
    RECEPTOR
    RTA
    (R. norvegicus)
    U83508 Human ANGPT1 1.06 0.11 1.49 0.44 −0.32 0.27 0.32 0.79
    angiopoietin-
    1 mRNA,
    complete
    cds.
    /PROD = angiopoietin-1
    /FL = gb: NM_001146.1
    gb: D13628.1
    gb: U83508.1
    AF043179 Homo PRSS1 2.42 0.12 3.07 0.81 1.71 0.10 2.30 0.60
    sapiens T
    cell receptor
    beta chain
    (TCRBV13S1-
    TCRBJ2S1)
    mRNA,
    complete
    cds.
    /PROD = T
    cell receptor
    beta chain
    /FL = gb: AF043179.1
    AU157541 hypothetical — 1.39 0.08 2.12 0.42 0.55 0.18 1.41 0.77
    protein
    FLJ22833
    /FL = gb: NM_022837.1
    AF114264 Homo NEXN 1.37 0.21 2.86 0.51 1.72 0.16 1.96 0.76
    sapiens
    clone HH409
    unknown
    mRNA.
    /PROD = unknown
    BE965029 Homo MICAL2 1.58 0.19 2.58 0.51 1.32 0.13 1.40 0.74
    sapiens
    cDNA:
    FLJ22463
    fis, clone
    HRC10126
    AB028976 Homo SAMD4A 2.89 0.08 3.65 0.66 2.04 0.17 2.78 0.81
    sapiens
    mRNA for
    KIAA1053
    protein,
    partial cds.
    /PROD = KIAA1053
    protein
    AI670862 ESTs, FOSL2 0.17 0.21 2.05 0.57 0.45 0.16 1.18 0.65
    Weakly
    similar to
    A49134 Ig
    kappa chain
    V-I region
    (H. sapiens)
    L03203 Human PMP22 0.37 0.24 1.95 0.84 0.31 0.26 2.77 0.91
    peripheral
    myelin
    protein 22
    (GAS3)
    mRNA,
    complete
    cds.
    /PROD = peripheral
    myelin
    protein 22
    /FL = gb: L03203.1
    AI571798 Rho GDP ARHGDIA 0.89 0.06 −0.54 0.26 −3.00 0.89 −2.58 0.87
    dissociation
    inhibitor
    (GDI) alpha
    X57348 Cluster Incl. — 2.94 0.04 4.07 0.41 2.70 0.08 3.69 0.41
    X57348: H. sapiens
    mRNA
    (clone 9112)
    /cds = (165,911)
    /gb = X57348
    /gi = 23939
    /ug = Hs.184510
    /len = 1407
    AF051851 Homo SVIL 2.03 0.18 2.28 0.62 1.13 0.20 1.71 0.69
    sapiens
    supervillin
    mRNA,
    complete
    cds.
    /PROD = supervillin
    /FL = gb: AF051851.1
    gb: NM_003174.2
    gb: AF051850.1
    M95929 Human — 0.07 0.43 1.27 0.58 −0.02 0.19 0.64 0.61
    homeobox
    protein
    (PHOX1)
    mRNA, 3
    end.
    /PROD = homeobox
    protein
    BG251266 FOS-like FOSL1 1.94 0.06 2.41 0.46 1.25 0.05 1.93 0.36
    antigen-1
    /FL = gb: NM_005438.1
    AW298375 ESTs — −0.40 0.30 0.46 0.63 −0.09 0.20 −0.58 1.07
    NM_004362 Homo CLGN 0.90 0.30 1.95 0.96 0.69 0.11 3.11 1.01
    sapiens
    calmegin
    (CLGN),
    mRNA.
    /PROD = calmegin
    /FL = gb: NM_004362.1
    gb: D86322.1
    AF001540 calcineurin- — 0.10 0.15 2.84 0.70 0.42 0.44 1.64 0.39
    binding
    protein
    calsarcin-1
    NM_001191 Homo BCL2L1 1.21 0.22 0.58 0.35 −1.89 0.35 −1.09 1.03
    sapiens
    BCL2-like 1
    (BCL2L1),
    mRNA.
    /PROD = BCL2-
    like 1
    /FL = gb: NM_001191.1
    NM_003316 Homo TTC3 3.90 0.15 5.15 0.98 3.68 0.12 6.19 0.97
    sapiens
    tetratricopeptide
    repeat
    domain 3
    (TTC3),
    mRNA.
    /PROD = tetratricopeptide
    repeat
    domain
    3
    /FL = gb: D84295.1
    gb: NM_003316.1
    L16895 Human lysyl — −0.66 0.24 0.53 0.14 −0.63 0.15 0.14 0.19
    oxidase
    (LOX) gene,
    exon 7
    AI912976 ESTs RASGRF2 3.13 0.08 4.05 0.56 2.66 0.16 3.54 0.64
    NM_012242 Homo DKK1 0.47 0.29 0.73 0.04 0.49 0.51 1.09 0.23
    sapiens
    dickkopf
    (Xenopus
    laevis)
    homolog 1
    (DKK1),
    mRNA.
    /PROD = dickkopf
    (Xenopus
    laevis)
    homolog 1
    /FL = gb: AF177394.1
    gb: NM_012242.1
    gb: AF127563.1
    AL096776 Human DNA — 1.94 0.07 3.41 0.16 2.40 0.07 3.40 0.14
    sequence
    from clone
    RP4-646B12
    on
    chromosome
    1q42.11-42.3.
    Contains an
    FTH1
    (ferritin,
    heavy
    polypeptide
    1) (FTHL6)
    pseudogene,
    the gene for
    a novel Ras
    family
    protein,
    ESTs, STSs,
    GSSs and a
    putative CpG
    island
    /FL = gb: AF282258.1
    gb: NM_0212
    BC005997 Homo — 1.12 0.25 0.95 0.44 2.04 0.07 1.51 0.22
    sapiens,
    clone
    MGC: 14801,
    mRNA,
    complete
    cds.
    /PROD = Unknown
    (protein for
    MGC: 14801)
    /FL = gb: BC005997.1
    AF074979 Homo RGS20 −0.52 0.18 −0.04 0.38 −1.30 0.09 −0.34 0.13
    sapiens
    regulator of
    G protein
    signaling-Z
    (RGSZ1)
    mRNA,
    complete
    cds.
    /PROD = regulator
    of G
    protein
    signaling
    /FL = gb: AF060877.2
    gb: AF074979.1
    gb: NM_003702.2
    BF060767 ESTs ADAMTS5 −0.36 0.15 0.80 0.33 0.22 0.14 0.40 0.56
    AU151151 Homo LEPR 1.72 0.18 2.38 0.80 1.85 0.10 1.71 0.69
    sapiens
    cDNA
    FLJ13536
    fis, clone
    PLACE1006521
    L27624 Homo TFPI2 3.77 0.06 3.57 0.77 2.59 0.11 2.44 0.89
    sapiens
    tissue factor
    pathway
    inhibitor-2
    mRNA,
    complete
    cds.
    /PROD = tissue
    factor
    pathway
    inhibitor-2
    /FL = gb: D29992.1
    gb: L27624.1
    gb: NM_006528.1
    gb: BC005330.1
    NM_003174 Homo SVIL 3.15 0.09 3.68 0.44 2.91 0.01 3.27 0.46
    sapiens
    supervillin
    (SVIL),
    transcript
    variant
    1,
    mRNA.
    /PROD = supervillin,
    isoform 1
    /FL = gb: AF051851.1
    gb: NM_003174.2
    gb: AF051850.1
    AF052127 Homo RELN −3.13 1.04 −1.36 0.26 −0.88 0.17 −0.40 0.11
    sapiens
    clone 23850
    mRNA
    sequence.
    AL031290 Human DNA — 0.38 0.06 1.39 0.46 0.40 0.05 0.95 0.44
    sequence
    from clone
    774I24 on
    chromosome
    1q24.1-24.3
    Contains
    protein
    similar to
    pregnancy-
    associated
    plasma
    protein A
    precursor
    neuronal
    migration
    protein
    astrotactin,
    ESTs, STS
    and GSS
    AI129628 ESTs SAMD3 −0.30 0.18 −0.01 0.25 0.72 0.06 0.17 0.21
    NM_016206 Homo VGLL3 −0.39 0.36 0.05 1.03 −0.31 0.29 −1.05 1.01
    sapiens
    colon
    carcinoma
    related
    protein
    (LOC51159),
    mRNA.
    /PROD = colon
    carcinoma
    related
    protein
    /FL = gb: NM_016206.1
    gb: AF099505.1
    BE348291 ESTs — 1.62 0.06 −0.19 0.98 −3.65 0.10 −2.75 1.84
    AW242720 Homo LOC143381 −1.62 0.34 −1.20 0.94 −0.08 0.24 −0.08 0.77
    sapiens
    cDNA
    FLJ10561
    fis, clone
    NT2RP2002672
    NM_001146 Homo ANGPT1 1.86 0.06 2.09 0.81 1.10 0.16 0.87 1.05
    sapiens
    angiopoietin
    1 (ANGPT1),
    mRNA.
    /PROD = angiopoietin 1
    /FL = gb: NM_001146.1
    gb: D13628.1
    gb: U83508.1
    AU152579 Homo PCSK5 2.41 0.14 2.47 0.86 2.04 0.15 1.11 1.20
    sapiens
    cDNA
    FLJ13034
    fis, clone
    NT2RP3001232
    NM_006200 Homo PCSK5 3.01 0.21 2.88 0.92 2.28 0.11 1.52 1.25
    sapiens
    proprotein
    convertase
    subtilisinkexin
    type 5
    (PCSK5),
    mRNA.
    /PROD = proprotein
    convertase
    subtilisinkexin
    type
    5
    /FL = gb: U56387.2
    gb: NM_006200.1
    BF342661 KIAA0036 MAP2 −0.77 0.30 −0.19 0.44 0.12 0.17 −2.21 1.64
    gene product
    AF063824 Homo TRPC4 −3.03 0.39 0.05 0.49 1.28 0.17 0.21 0.92
    sapiens trp-
    related
    protein 4
    truncated
    variant delta
    mRNA,
    complete
    cds.
    /PROD = trp-
    related
    protein 4
    truncated
    variant delta
    /FL = gb: AF063824.1
    AA723810 cDNA for LY6K 0.07 0.20 −0.13 1.23 0.15 0.14 −0.38 1.27
    differentially
    expressed
    CO16 gene
    /FL = gb: BC001291.1
    N29877 interleukin — 1.94 0.13 0.51 0.48 0.78 0.64 −1.57 0.65
    14
    NM_007287 Homo MME 2.59 0.03 1.81 0.40 1.89 0.21 2.14 0.30
    sapiens
    membrane
    metallo-
    endopeptidase
    (neutral
    endopeptidase,
    enkephalinase,
    CALLA,
    CD10)
    (MME),
    transcript
    variant 1bis,
    mRNA.
    /PROD = membrane
    metallo-
    endopeptidase
    /FL = gb: NM_007288.1
    gb: NM_007287.1
    gb: J03779.1
    AB050856 Homo B3GALNT1 1.39 0.05 0.20 0.22 −0.13 0.21 −0.10 0.17
    sapiens
    beta3GalNAcT-
    1 mRNA
    for globoside
    synthase,
    complete
    cds,
    clone: type 2.
    /PROD = globoside
    synthase
    /FL = gb: AB050856.1
    AI827455 Homo BCL6B 0.65 0.31 0.62 0.21 1.07 0.24 0.90 0.29
    sapiens
    cDNA:
    FLJ21042
    fis, clone
    CAE11204
    AF017987 Homo SFRP1 4.15 0.22 4.37 1.24 6.02 0.06 5.74 1.01
    sapiens
    secreted
    apoptosis
    related
    protein 2
    (SARP2)
    mRNA,
    complete
    cds.
    /PROD = secreted
    apoptosis
    related
    protein 2
    /FL = gb: AF056087.1
    gb: NM_003012.2
    gb: AF017987.1
    gb: AF001900.1
    AL117451 Homo C1orf108 −0.73 0.20 0.68 0.24 1.06 0.05 0.82 0.21
    sapiens
    mRNA;
    cDNA
    DKFZp586E2317
    (from
    clone
    DKFZp586E2317).
    AW268357 ESTs, Highly USP32 0.73 0.20 0.36 0.19 0.70 0.03 0.09 0.18
    similar to
    AF155116 1
    NY-REN-60
    antigen
    (H. sapiens)
    BE465243 ESTs ARFGEF1 0.50 0.23 0.16 0.32 1.03 0.08 0.47 0.06
    AA993400 ESTs ADAL 0.89 0.22 0.48 0.28 0.83 0.10 0.29 0.43
    AI970898 Cluster Incl. ACACB 0.69 0.15 0.51 0.53 1.46 0.11 −0.87 0.83
    AI970898: wr
    21c03.x1
    Homo
    sapiens
    cDNA, 3
    end
    /clone = IMAGE-
    2488324
    /clone_end = 3
    /gb = AI970898
    /gi = 5767724
    /ug = Hs.234898
    /len = 382
    BG026457 ESTs, KIAA1909 0.60 0.07 0.23 0.59 0.98 0.03 −2.11 1.28
    Weakly
    similar to
    ALU5_HUMAN
    ALU
    SUBFAMILY
    SC
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    BC041933 Homo UBE3C 0.83 0.23 0.11 0.51 0.55 0.05 0.41 0.92
    sapiens,
    clone
    IMAGE: 5300703,
    mRNA.
    AI638611 KIAA1373 STAMBPL1 2.20 0.17 1.37 0.58 2.32 0.08 −0.39 1.24
    protein
    AI341686 ESTs, Highly MTRF1 1.36 0.14 0.45 0.48 1.43 0.09 −1.09 0.99
    similar to
    RF1M_HUMAN
    MITOCHONDRIAL
    PEPTIDE
    CHAIN
    RELEASE
    FACTOR
    1
    PRECURSOR
    (H. sapiens)
    NM_003182 Homo TAC1 1.89 0.20 1.65 0.13 2.46 0.09 1.48 0.26
    sapiens
    tachykinin,
    precursor 1
    (substance
    K, substance
    P, neurokinin
    1, neurokinin
    2,
    neuromedin
    L, neurokinin
    alpha,
    neuropeptide
    K, neuropeptide
    gamma)
    (TAC1),
    transcript
    variant beta,
    mRNA.
    /PROD = tachykinin 2
    precursor,
    isoform beta
    /FL = gb: U3
    M80634 Human FGFR2 3.63 0.06 2.25 0.39 2.79 0.19 2.07 0.84
    keratinocyte
    growth factor
    receptor
    mRNA,
    complete
    cds.
    /PROD = keratinocyte
    growth factor
    receptor
    /FL = gb: M80634.1
    gb: NM_022969.1
    gb: M97193.1
    AK021452 Homo ZNF521 −0.20 0.16 −1.15 1.12 1.31 0.05 0.36 0.18
    sapiens
    cDNA
    FLJ11390
    fis, clone
    HEMBA1000561,
    weakly
    similar to
    ZINC
    FINGER
    PROTEIN
    91.
    AA541622 ESTs SYNPO2 −0.49 0.30 −0.90 0.06 0.35 0.09 −2.58 0.93
    N50714 ESTs — −1.13 0.13 −1.34 0.52 0.10 0.12 −0.21 0.28
    NM_002674 Homo PMCH 0.62 0.24 1.10 0.48 1.64 0.03 0.09 0.80
    sapiens pro-
    melanin-
    concentrating
    hormone
    (PMCH),
    mRNA.
    /PROD = pro-
    melanin-
    concentrating
    hormone
    /FL = gb: NM_002674.1
    gb: M57703.1
    BM666010 Homo LOC200169 1.36 0.16 0.45 0.74 1.49 0.12 −1.65 1.41
    sapiens
    cDNA
    FLJ23803
    fis, clone
    HEP22811.
    AI343467 Homo — −0.44 0.08 −1.50 0.82 1.11 0.19 −0.64 0.20
    sapiens
    cDNA
    FLJ11041
    fis, clone
    PLACE1004405
    AA046424 ESTs, ACOT4 0.58 0.27 −0.94 0.66 −1.08 0.38 −1.09 0.25
    Weakly
    similar to
    YZ28_HUMAN
    HYPOTHETICAL
    PROTEIN
    ZAP128
    (H. sapiens)
    R38389 olfactomedin OLFM1 4.27 0.11 3.02 0.76 4.53 0.07 2.17 0.96
    related ER
    localized
    protein
    BF724270 ESTs — 2.03 0.22 0.72 0.42 2.36 0.06 0.01 0.97
    NM_001819 Homo CHGB 1.40 0.18 0.50 0.75 1.66 0.05 −0.74 1.03
    sapiens
    chromogranin B
    (secretogranin
    1) (CHGB),
    mRNA.
    /PROD = chromogranin B
    precursor
    /FL = gb: BC000375.1
    gb: NM_001819.1
    AK026387 Homo — 0.03 0.28 −0.05 0.52 1.23 0.26 0.52 0.67
    sapiens
    cDNA:
    FLJ22734
    fis, clone
    HUV00109.
    BF062139 polymerase — 5.23 0.06 4.31 0.74 5.80 0.07 3.37 0.86
    (RNA) III
    (DNA
    directed)
    (32 kD)
    /FL = gb: NM_006467.1
    gb: U93868.1
    BG540454 ESTs SCGB3A2 4.64 0.02 2.91 0.84 4.13 0.07 1.72 0.83
    AI659533 ArgAbl- SORBS2 0.02 0.12 1.12 0.71 2.17 0.05 −1.08 1.48
    interacting
    protein
    ArgBP2
    AA531287 ESTs — 2.94 0.27 1.32 0.72 2.57 0.04 0.22 0.90
    NM_013243 Homo SCG3 3.30 0.20 2.13 0.95 3.67 0.19 0.87 1.06
    sapiens
    secretogranin
    III (SCG3),
    mRNA.
    /PROD = secretogranin
    III
    /FL = gb: AF078851.1
    gb: NM_013243.1
    AI307586 Homo C10orf95 0.34 0.22 −1.15 0.24 −0.49 0.16 −3.61 0.99
    sapiens
    mRNA;
    cDNA
    DKFZp566H0124
    (from
    clone
    DKFZp566H0124)
    BC032004 Homo GRIA3 −0.02 0.23 −0.15 0.33 0.47 0.04 0.17 0.07
    sapiens,
    Similar to
    glutamate
    receptor,
    ionotrophic,
    AMPA 3,
    clone
    IMAGE: 4753474,
    mRNA.
    AW205739 ESTs, TYW3 0.47 0.01 0.81 1.35 3.72 0.18 2.46 1.15
    Weakly
    similar to
    ORF
    YGL050w
    (S. cerevisiae)
    NM_153262 Homo SYT14 −0.01 0.17 −0.84 0.51 0.00 0.18 −3.88 1.64
    sapiens
    hypothetical
    protein
    FLJ34198
    (FLJ34198),
    mRNA.
    /FL = gb: NM_153262.1
    AA156873 albumin PELO 0.77 0.12 −0.23 0.93 0.65 0.14 −2.33 1.01
    BC012375 Homo ARSG −0.46 0.48 −0.78 0.42 0.57 0.22 −0.41 0.55
    sapiens,
    Similar to
    KIAA1001
    protein,
    clone
    MGC: 8996
    IMAGE: 3882163,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to
    KIAA1001
    protein
    /FL = gb: AB023218.1
    gb: NM_014960.1
    gb: BC012375.1
    AA843242 ESTs BNC2 2.12 0.37 1.03 0.47 2.89 0.06 0.43 0.80
    BF792954 ESTs HDLBP −1.53 1.04 −2.75 0.91 0.30 0.16 −0.72 0.26
    AA780067 heparan HS3ST3B1 0.41 0.19 0.30 0.61 2.61 0.18 0.78 0.60
    sulfate
    (glucosamine)
    3-O-
    sulfotransferase
    3B1
    AA909330 ESTs RP1-32F7.2 0.98 0.17 −1.67 0.51 0.79 0.02 −0.38 0.12
    AF141339 Homo ZNF521 −0.41 0.12 −0.97 0.93 1.67 0.11 −0.31 0.80
    sapiens
    LYST-
    interacting
    protein LIP3
    mRNA,
    partial cds.
    /PROD = LYST-
    interacting
    protein LIP3
    BF435123 bromodomain — 2.28 0.17 1.31 0.51 2.70 0.12 1.30 1.00
    and PHD
    finger
    containing, 3
    AK056212 Homo — 1.03 0.14 0.14 0.27 1.10 0.07 −1.41 0.51
    sapiens
    cDNA
    FLJ31650
    fis, clone
    NT2RI2004079.
    NM_001446 Homo FABP7 2.07 0.15 0.74 0.29 2.20 0.04 1.08 0.16
    sapiens fatty
    acid binding
    protein
    7,
    brain
    (FABP7),
    mRNA.
    /PROD = fatty
    acid binding
    protein
    7,
    brain
    /FL = gb: U81235.1
    gb: D88648.1
    gb: U51338.1
    gb: NM_001446.1
    gb: D50373.1
    AW051591 ESTs, RNF175 1.41 0.32 0.22 0.10 2.60 0.06 0.96 0.20
    Moderately
    similar to
    unnamed
    protein
    product
    (H. sapiens)
    BC041970 Homo C9orf122 0.74 0.03 −0.35 0.49 0.76 0.06 −2.31 1.25
    sapiens,
    clone
    IMAGE: 5302687,
    mRNA.
    BC013077 Homo — 2.51 0.11 0.80 1.15 2.85 0.06 0.08 0.89
    sapiens,
    clone
    IMAGE: 3459334,
    mRNA.
    AW572379 ESTs — 1.80 0.03 0.60 0.84 1.45 0.07 1.88 0.53
    BE644917 nuclear XIST −3.88 1.26 −0.54 1.28 1.94 0.13 −0.20 1.94
    receptor
    subfamily
    1,
    group I,
    member 3
    NM_171999 Homo SALL3 2.97 0.18 2.04 0.71 3.79 0.08 1.02 0.74
    sapiens sal-
    like 3
    (Drosophila)
    (SALL3),
    mRNA.
    /PROD = sal-
    like 3
    /FL = gb: NM_171999.1
    AI654224 ESTs — 0.46 0.36 0.14 0.44 1.03 0.07 −0.81 0.85
    AA167449 nuclear XIST −3.08 0.10 −0.47 2.59 4.59 0.07 2.80 1.86
    receptor
    subfamily
    1,
    group I,
    member 3
    BF977837 KIAA0527 SUSD5 1.64 0.02 0.50 0.68 1.96 0.16 −1.52 1.66
    protein
    BC029425 Homo FILIP1 −0.83 0.45 −0.03 0.62 0.76 0.20 −0.29 0.44
    sapiens,
    Similar to
    KIAA1275
    protein,
    clone
    IMAGE: 4616553,
    mRNA.
    AI978754 ESTs — 2.76 0.14 1.54 0.57 3.57 0.06 2.31 0.69
    AA628440 nuclear XIST 0.18 0.13 1.42 1.52 4.80 0.02 3.01 1.50
    receptor
    subfamily
    1,
    group I,
    member 3
    L36861 L36861 — 2.75 0.17 2.01 0.82 3.63 0.09 3.00 0.97
    /FEATURE =
    expanded_cds
    /DEFINITION =
    HUMGCAPB
    Homo
    sapiens
    guanylate
    cyclase
    activating
    protein
    (GCAP)
    gene exons
    1-4,
    complete cds
    AW023227 ESTs MKX −0.91 0.60 −1.31 0.06 0.27 0.13 −2.90 0.52
    NM_021614 Homo KCNN2 3.45 0.13 2.52 0.83 4.40 0.14 1.54 0.80
    sapiens
    potassium
    intermediate
    small
    conductance
    calcium-
    activated
    channel,
    subfamily N,
    member 2
    (KCNN2),
    mRNA.
    /PROD = potassium
    intermediate
    small
    conductance
    calcium-
    activated
    channel,
    subfamily N,
    member 2
    /FL = gb: NM_021614.1
    gb: AF239613.1
    NM_000956 Homo PTGER2 −0.71 0.71 −0.73 0.57 0.63 0.33 −2.38 0.61
    sapiens
    prostaglandin
    E receptor
    2 (subtype
    EP2), 53 kD
    (PTGER2),
    mRNA.
    /PROD = prostaglandin E
    receptor 2
    (subtype
    EP2), 53 kD
    /FL = gb: U19487.1
    gb: NM_000956.1
    NM_013381 Homo TRHDE 1.96 0.16 0.25 0.36 1.81 0.03 0.82 0.23
    sapiens
    thyrotropin-
    releasing
    hormone
    degrading
    ectoenzyme
    (TRHDE),
    mRNA.
    /PROD = thyrotropin-
    releasing
    hormone
    degradingectoenzyme
    /FL = gb: AF126372.1
    gb: NM_013381.1
    NM_016354 Homo SLCO4A1 2.18 0.06 1.01 0.37 2.35 0.05 −0.91 1.23
    sapiens
    solute carrier
    family 21
    (organic
    anion
    transporter),
    member 12
    (SLC21A12),
    mRNA.
    /PROD = organic
    anion
    transporter
    OATP-E
    /FL = gb: AB031051.1
    gb: NM_016354.1
    gb: AF205072.1
    gb: AF187817.1
    BC028359 Homo ZNF141 −1.05 0.39 −0.59 0.65 1.25 0.17 −1.48 0.72
    sapiens,
    clone
    IMAGE: 4828836,
    mRNA.
    AI193252 ESTs, LRRN6A 4.38 0.11 2.47 0.43 4.23 0.11 1.90 0.37
    Weakly
    similar to
    AF133270 1
    SLIT2
    (H. sapiens)
    H09780 Human — 2.04 0.19 0.56 0.07 2.50 0.11 0.12 0.40
    (clone CTG-
    A4) mRNA
    sequence
    BC040605 Homo — 2.64 0.16 1.61 1.08 3.59 0.09 0.45 1.14
    sapiens,
    clone
    IMAGE: 5271039,
    mRNA.
    AW057589 ESTs — −0.96 0.26 −2.77 0.81 0.25 0.24 −1.21 0.35
    M31213 Human RET 0.42 0.10 −1.86 0.83 1.77 0.12 −0.64 0.15
    papillary
    thyroid
    carcinoma-
    encoded
    protein
    mRNA,
    complete
    cds.
    /FL = gb: M31213.1
    Z92546 Human DNA — 0.58 0.13 −1.16 0.54 1.07 0.12 −1.27 0.14
    sequence
    from clone
    CTA-65A6
    on
    chromosome
    22q11-12
    Contains the
    3 part of the
    gene for the
    ortholog of
    rat CAIN
    (KIAA0330),
    the gene for
    a novel
    Sushi
    domain
    (SCR repeat)
    containing
    protein
    similar to
    Mucins,
    ESTs, an
    STS, GSSs
    and two . . .
    AA974416 protein PPP2R2B 4.02 0.16 3.52 0.23 5.22 0.07 3.12 0.15
    phosphatase
    2 (formerly
    2A),
    regulatory
    subunit B
    (PR 52),
    beta isoform
    AW138143 ESTs SORBS2 3.44 0.16 2.89 0.88 4.47 0.10 1.88 1.15
    NM_014862 Homo ARNT2 1.29 0.08 −0.68 0.97 2.00 0.08 0.02 0.24
    sapiens
    KIAA0307
    gene product
    (KIAA0307),
    mRNA.
    /PROD = KIAA0307
    gene
    product
    /FL = gb: AB002305.1
    gb: NM_014862.1
    AI765540 ESTs — 1.28 0.22 0.07 0.39 1.50 0.07 −0.11 0.26
    NM_018013 Homo FLJ10159 1.30 0.21 0.38 0.39 2.89 0.02 0.62 0.21
    sapiens
    hypothetical
    protein
    FLJ10159
    (FLJ10159),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ10159
    /FL = gb: NM_018013.1
    BF382322 ESTs, — −0.40 0.09 −2.09 0.67 −0.12 0.08 −1.73 0.50
    Weakly
    similar to
    unnamed
    protein
    product
    (H. sapiens)
    AV699347 nuclear XIST −1.37 0.38 0.45 1.69 4.28 0.02 2.25 1.53
    receptor
    subfamily
    1,
    group I,
    member 3
    BC011549 Homo ATP5S 0.69 0.19 0.96 0.68 2.57 0.29 −0.02 0.52
    sapiens,
    clone
    MGC: 19945
    IMAGE: 4554461,
    mRNA,
    complete
    cds.
    /PROD = Unknown
    (protein for
    MGC: 19945)
    /FL = gb: BC011549.1
    NM_001889 Homo CRYZ −1.68 0.26 0.23 1.38 3.75 0.07 1.78 1.11
    sapiens
    crystallin,
    zeta
    (quinone
    reductase)
    (CRYZ),
    mRNA.
    /PROD = crystallin,
    zeta
    (quinone
    reductase)
    /FL = gb: NM_001889.1
    gb: L13278.1
    gb: S58039.1
    BC002665 Homo PLP1 4.57 0.15 2.95 0.64 4.70 0.06 2.32 0.52
    sapiens,
    proteolipid
    protein
    (Pelizaeus-
    Merzbacher
    disease,
    spastic
    paraplegia
    2,
    uncomplicated),
    clone
    MGC: 3940,
    mRNA,
    complete
    cds.
    /PROD = proteolipid
    protein
    (Pelizaeus-
    Merzbacherdisease,
    spastic
    paraplegia
    2,
    uncomplicated)
    /FL = gb: BC002665.1
    NM_001243 Homo TNFRSF8 3.75 0.03 1.45 0.35 3.24 0.11 0.89 0.46
    sapiens
    tumor
    necrosis
    factor
    receptor
    superfamily,
    member 8
    (TNFRSF8),
    mRNA.
    /PROD = CD30
    antigen
    (Ki-1
    antigen)
    /FL = gb: NM_001243.1
    gb: D86042.1
    gb: M83554.1
    NM_001195 Homo BFSP1 0.57 0.33 −1.65 1.30 0.60 0.14 −0.86 0.73
    sapiens
    beaded
    filament
    structural
    protein
    1,
    filensin
    (BFSP1),
    mRNA.
    /PROD = filensin
    /FL = gb: AF039655.1
    gb: NM_001195.2
    gb: Y16717.2
    NM_024582 Homo FAT4 2.16 0.27 −0.49 0.30 1.54 0.28 −1.35 1.01
    sapiens
    hypothetical
    protein
    FLJ23056
    (FLJ23056),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ23056
    /FL = gb: NM_024582.1
    NM_000767 Homo CYP2B6 2.60 0.18 0.87 0.96 1.47 0.12 −0.69 1.03
    sapiens
    cytochrome
    P450,
    subfamily IIB
    (phenobarbital-
    inducible),
    polypeptide
    6 (CYP2B6),
    mRNA.
    /PROD = cytochrome
    P450,
    subfamily
    IIB(phenobarbital-
    inducible),
    polypeptide 6
    /FL = gb: NM_000767.2
    gb: AF182277.1
    gb: M29874.1
    AW665509 ESTs MGC42174 −0.10 0.33 −0.87 0.72 1.20 0.08 −2.60 1.40
    NM_001104 Homo ACTN3 2.12 0.13 1.64 0.28 3.35 0.03 1.67 0.36
    sapiens
    actinin, alpha
    3 (ACTN3),
    mRNA.
    /PROD = skeletal
    muscle
    specific
    actinin, alpha 3
    /FL = gb: M86407.1
    gb: NM_001104.1
    NM_021069 Homo SORBS2 −0.49 0.43 0.30 1.36 2.03 0.07 −1.13 1.15
    sapiens
    ArgAbl-
    interacting
    protein
    ArgBP2
    (ARGBP2),
    transcript
    variant
    2,
    mRNA.
    /PROD = Arg
    Abl-
    interacting
    protein 2,
    isoform 2
    /FL = gb: AB018320.1
    gb: NM_021069.1
    T65020 ESTs — 0.51 0.16 −0.84 0.26 1.10 0.15 −1.50 0.38
    NM_152647 Homo GALK2 0.08 0.56 −1.42 0.52 0.03 0.17 −2.34 0.91
    sapiens
    hypothetical
    protein
    FLJ32800
    (FLJ32800),
    mRNA.
    /FL = gb: NM_152647.1
    BC036917 Homo C6orf141 −0.82 0.30 −1.18 0.40 0.88 0.11 −2.18 0.62
    sapiens,
    clone
    MGC: 46457
    IMAGE: 5201433,
    mRNA,
    complete
    cds.
    /PROD = Unknown
    (protein for
    MGC: 46457)
    /FL = gb: BC036917.1
    AI969112 Homo PHIP −1.07 0.13 −2.88 0.91 −0.37 0.02 −2.23 0.33
    sapiens,
    clone
    IMAGE: 5260603,
    mRNA,
    partial cds
    AW449813 KIAA0918 SLITRK5 0.32 0.26 −1.89 0.50 0.46 0.10 −0.57 0.13
    protein
    AW044658 ESTs — 0.16 0.16 −1.47 0.25 0.90 0.14 −0.89 0.16
    AI694300 ESTs — −0.52 0.02 −1.34 0.52 0.27 0.08 −1.00 0.08
    NM_017631 Homo FLJ20035 1.35 0.13 0.05 0.19 1.51 0.17 0.32 0.15
    sapiens
    hypothetical
    protein
    FLJ20035
    (FLJ20035),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ20035
    /FL = gb: NM_017631.1
    AF298547 Homo NALP2 1.54 0.08 1.22 1.61 5.04 0.05 3.23 1.36
    sapiens
    nucleotide-
    binding site
    protein
    1
    mRNA,
    complete
    cds.
    /PROD = nucleotide-
    binding site
    protein
    1
    /FL = gb: AF298547.1
    NM_030631 Homo SLC25A21 1.96 0.25 0.33 0.81 2.47 0.11 −0.28 0.63
    sapiens
    oxodicarboxylate
    carrier
    (ODC1),
    mRNA.
    /PROD = oxodicarboxylate
    carrier
    /FL = gb: NM_030631.1
    BF196255 ESTs — 2.94 0.05 1.18 0.43 3.15 0.08 0.54 0.57
    NM_003247 Homo THBS2 2.62 0.20 1.04 0.28 2.70 0.04 0.27 0.45
    sapiens
    thrombospondin 2
    (THBS2),
    mRNA.
    /PROD = thrombospondin 2
    /FL = gb: NM_003247.1
    gb: L12350.1
    NM_001446 Homo FABP7 0.95 0.09 −0.75 0.37 0.20 0.22 −1.73 0.99
    sapiens fatty
    acid binding
    protein
    7,
    brain
    (FABP7),
    mRNA.
    /PROD = fatty
    acid binding
    protein
    7,
    brain
    /FL = gb: U81235.1
    gb: D88648.1
    gb: U51338.1
    gb: NM_001446.1
    gb: D50373.1
    AW004016 ESTs ST6GAL2 1.57 0.14 0.89 0.32 3.30 0.07 1.48 0.43
    AW072790 contactin 1 CNTN1 0.50 0.20 0.18 0.98 1.84 0.10 1.01 1.39
    AL512686 Homo GNAO1 2.16 0.50 −0.28 1.03 1.67 0.18 −1.12 0.37
    sapiens
    mRNA;
    cDNA
    DKFZp761I177
    (from
    clone
    DKFZp761I177).
    AI638063 ESTs CBX5 0.49 0.16 −0.34 0.34 0.98 0.11 −2.71 1.22
    AU157049 Homo LOC153346 2.24 0.20 0.34 0.47 2.24 0.09 −0.44 0.96
    sapiens
    cDNA
    FLJ14284
    fis, clone
    PLACE1005898
    NM_002738 Homo PRKCB1 0.99 0.28 −0.15 0.45 1.42 0.08 −1.02 1.04
    sapiens
    protein
    kinase C,
    beta 1
    (PRKCB1),
    mRNA.
    /PROD = protein
    kinase C,
    beta 1
    /FL = gb: NM_002738.1
    AI185136 ESTs DYDC2 0.12 0.31 −0.80 0.08 0.72 0.08 −0.60 0.09
    AI928037 ESTs RPIB9 −0.55 0.55 −2.71 0.74 0.49 0.12 −0.89 0.27
    NM_016179 Homo TRPC4 −1.51 0.85 −1.87 1.42 1.71 0.11 −0.13 1.23
    sapiens
    transient
    receptor
    potential
    channel 4
    (TRPC4),
    mRNA.
    /PROD = transient
    receptor
    potential
    4
    /FL = gb: NM_016179.1
    gb: AF175406.1
    AW138143 ESTs SORBS2 3.29 0.13 2.35 1.05 4.28 0.10 1.32 1.07
    NM_001876 Homo CPT1A 1.63 0.18 −0.15 0.21 1.32 0.07 −0.20 0.33
    sapiens
    carnitine
    palmitoyltransferase
    I,
    liver
    (CPT1A),
    nuclear gene
    encoding
    mitochondrial
    protein,
    mRNA.
    /PROD = liver
    carnitine
    palmitoyltransferase I
    /FL = gb: L39211.1
    gb: NM_001876.1
    AA903862 ESTs C20orf54 1.61 0.15 −1.17 0.30 0.68 0.21 −1.49 0.23
    AI670947 phosphatidylinositol- — 3.91 0.05 0.55 1.54 3.33 0.10 1.17 0.68
    4-
    phosphate 5-
    kinase, type
    I, beta
    AV648405 polymerase — −0.01 0.11 0.23 0.52 1.73 0.14 −1.23 1.15
    (RNA) III
    (DNA
    directed)
    (32 kD)
    BF435123 bromodomain — 1.80 0.13 −1.35 1.01 1.14 0.03 0.37 0.24
    and PHD
    finger
    containing, 3
    NM_016582 Homo SLC15A3 1.77 0.16 −0.88 1.35 1.30 0.23 −2.04 1.23
    sapiens
    peptide
    transporter 3
    (LOC51296),
    mRNA.
    /PROD = peptide
    transporter
    3
    /FL = gb: NM_016582.1
    gb: AB020598.1
    AI830490 glycerol GK 0.61 0.01 −0.65 0.79 1.25 0.20 −0.38 0.33
    kinase
    AW134979 HSPC156 STXBP6 2.28 0.03 0.55 0.28 2.05 0.03 0.30 0.44
    protein
    AI354636 ESTs — 2.81 0.11 0.68 1.17 3.02 0.20 0.19 0.76
    BE672659 ESTs — 0.20 0.64 −0.43 0.61 1.98 0.12 −0.69 0.45
    AF284095 Homo ADRA2A 0.63 0.21 −0.74 0.98 1.85 0.03 0.55 0.31
    sapiens
    alpha-2A
    adrenergic
    receptor
    mRNA,
    complete
    cds.
    /PROD = alpha-
    2A
    adrenergic
    receptor
    /FL = gb: AF284095.1
    gb: NM_000681.1
    NM_021136 Homo RTN1 −0.30 0.03 −1.39 0.35 0.80 0.07 −1.23 0.39
    sapiens
    reticulon 1
    (RTN1),
    mRNA.
    /PROD = reticulon 1
    /FL = gb: L10333.1
    gb: L10334.1
    gb: NM_021136.1
    NM_014729 Homo TOX 0.34 0.03 −0.46 0.12 0.90 0.18 −1.86 0.40
    sapiens
    KIAA0808
    gene product
    (KIAA0808),
    mRNA.
    /PROD = KIAA0808
    gene
    product
    /FL = gb: AB018351.1
    gb: NM_014729.1
    BE674118 ESTs — 0.78 0.07 −1.32 0.73 0.28 0.19 −0.96 0.74
    BC026969 Homo WDR67 2.95 0.11 0.75 0.92 3.08 0.08 0.45 0.61
    sapiens,
    clone
    IMAGE: 5116073,
    mRNA,
    partial cds.
    AF131783 Homo PAP2D −0.42 0.26 −2.00 1.05 0.31 0.22 −1.56 0.70
    sapiens
    clone 25181
    mRNA
    sequence.
    U11058 Homo KCNMA1 1.56 0.09 −0.16 1.08 2.29 0.18 −0.79 0.86
    sapiens
    large
    conductance
    calcium- and
    voltage-
    dependent
    potassium
    channel
    alpha
    subunit
    (MaxiK)
    mRNA,
    complete
    cds.
    /PROD = large
    conductance
    calcium- and
    voltage-
    dependentpotassium
    channel
    alpha
    subunit
    /FL = gb: U23767.1
    gb: NM_002247.1
    gb: AF025999
    BF510715 fibroblast — 2.67 0.27 0.84 0.17 3.29 0.01 0.64 0.51
    growth factor
    4 (heparin
    secretory
    transforming
    protein
    1,
    Kaposi
    sarcoma
    oncogene)
    /FL = gb: M17446.1
    gb: NM_002007.1
    BE897866 ESTs ACADSB 1.41 0.23 0.08 0.34 3.10 0.10 −0.27 0.88
    AI735586 ESTs LOC152573 0.60 0.13 0.00 1.14 2.84 0.07 1.12 1.30
    AL573058 complement C1R 0.92 0.14 −1.67 0.38 0.73 0.02 −0.96 0.77
    component
    1, r
    subcomponent
    AF429305 Homo RMST −0.48 0.03 −2.49 0.51 −0.25 0.20 −1.89 0.27
    sapiens
    C23up
    NCRMS
    mRNA,
    partial
    sequence;
    alternatively
    spliced.
    BF195118 ESTs, ATP5J 0.09 0.13 −0.92 0.42 1.57 0.16 −0.53 0.08
    Weakly
    similar to
    ALU7_HUMAN
    ALU
    SUBFAMILY
    SQ
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    NM_005460 Homo SNCAIP 1.76 0.16 −0.84 0.52 1.61 0.19 −0.92 0.88
    sapiens
    synuclein,
    alpha
    interacting
    protein
    (synphilin)
    (SNCAIP),
    mRNA.
    /PROD = synuclein
    alpha
    interacting
    protein
    /FL = gb: AF076929.1
    gb: NM_005460.1
    NM_024893 Homo C20orf39 1.16 0.29 −0.56 0.42 2.36 0.15 −0.06 0.17
    sapiens
    hypothetical
    protein
    FLJ14220
    (FLJ14220),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ14220
    /FL = gb: NM_024893.1
    NM_022034 Homo CUZD1 1.31 0.08 0.80 0.28 2.78 0.07 0.24 0.58
    sapiens
    estrogen
    regulated
    gene 1
    (ERG-1),
    mRNA.
    /PROD = estrogen
    regulated
    gene 1
    /FL = gb: AF305835.1
    gb: NM_022034.1
    NM_014788 Homo TRIM14 3.12 0.09 1.22 0.59 3.54 0.08 1.02 0.45
    sapiens
    KIAA0129
    gene product
    (KIAA0129),
    mRNA.
    /PROD = KIAA0129
    gene
    product
    /FL = gb: D50919.1
    gb: NM_014788.1
    AV646597 ESTs, XIST −0.77 0.18 0.06 1.55 3.78 0.25 2.28 0.98
    Weakly
    similar to
    ALU7_HUMAN
    ALU
    SUBFAMILY
    SQ
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    BF062629 DKFZP586E1621 TMEM158 4.00 0.19 2.41 0.27 4.87 0.00 1.93 0.19
    protein
    AW440492 ATPase, ATP1A2 1.33 0.07 −2.12 1.07 1.27 0.06 −0.53 0.11
    Na+K+
    transporting,
    alpha 2 (+)
    polypeptide
    /FL = gb: NM_000702.1
    AF283777 Homo CD72 −0.08 0.45 −1.88 1.19 0.41 0.24 −1.42 1.13
    sapiens
    clone
    TCBAP0702
    mRNA
    sequence.
    NM_005375 Homo MYB 2.11 0.17 −0.52 0.18 2.24 0.03 0.10 0.08
    sapiens v-
    myb avian
    myeloblastosis
    viral
    oncogene
    homolog
    (MYB),
    mRNA.
    /PROD = v-
    myb avian
    myeloblastosis
    viral
    oncogenehomolog
    /FL = gb: NM_005375.1
    gb: AF104863.1
    gb: M15024.1
    NM_017671 Homo C20orf42 3.19 0.12 0.97 0.70 3.26 0.07 0.16 0.94
    sapiens
    hypothetical
    protein
    FLJ20116
    (FLJ20116),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ20116
    /FL = gb: NM_017671.1
    BG283790 ESTs MATR3 2.27 0.11 0.95 0.57 3.53 0.04 0.89 0.51
    NM_000702 Homo ATP1A2 2.12 0.17 0.76 0.38 2.93 0.16 0.51 0.12
    sapiens
    ATPase,
    Na+K+
    transporting,
    alpha 2 (+)
    polypeptide
    (ATP1A2),
    mRNA.
    /PROD = ATPase,
    Na+K+
    transporting,
    alpha 2
    (+)polypeptide
    /FL = gb: NM_000702.1
    NM_025135 Homo FHOD3 1.02 0.18 0.80 0.40 2.34 0.10 −0.77 0.72
    sapiens
    hypothetical
    protein
    FLJ22297
    (KIAA1695),
    mRNA.
    /PROD = hypothetical
    protein
    KIAA1695
    /FL = gb: NM_025135.1
    NM_012281 Homo KCND2 1.67 0.30 0.05 0.15 2.70 0.09 −0.26 0.38
    sapiens
    potassium
    voltage-
    gated
    channel,
    Shal-related
    subfamily,
    member 2
    (KCND2),
    mRNA.
    /PROD = potassium
    voltage-
    gated
    channel,
    Shal-
    relatedsubfamily,
    member 2
    /FL = gb: NM_012281.1
    gb: AB028967.1
    gb: AF121104.1
    BF449063 collagen, COL14A1 1.61 0.15 0.30 0.38 2.68 0.17 −0.33 0.68
    type XIV,
    alpha 1
    (undulin)
    AA280904 ESTs C9orf39 0.23 0.21 −1.58 1.26 1.13 0.14 −0.78 0.42
    NM_022467 Homo CHST8 2.11 0.20 −0.51 0.30 2.17 0.10 0.48 0.02
    sapiens N-
    acetylgalactosamine-
    4-O-
    sulfotransferase
    (GALNAC-4-
    ST1),
    mRNA.
    /PROD = N-
    acetylgalactosamine-
    4-O-
    sulfotransferase
    /FL = gb: NM_022467.1
    gb: AF300612.1
    BE468066 ESTs RMST 3.26 0.09 1.49 0.10 3.80 0.09 1.63 0.08
    AL120674 ESTs — 1.00 0.11 −1.35 1.73 1.55 0.15 −2.20 0.86
    NM_133329 Homo KCNG3 2.18 0.27 0.74 0.29 3.67 0.01 0.26 0.28
    sapiens
    potassium
    voltage-
    gated
    channel,
    subfamily G,
    member 3
    (KCNG3),
    transcript
    variant
    1,
    mRNA.
    /PROD = potassium
    voltage-
    gated
    channel,
    subfamily
    G, member 3
    isoform 1
    /FL = gb: AF454548.1
    gb: AF348982.1
    gb: AB070604.1
    gb: NM_133329.4
    AI742043 ESTs — 0.94 0.34 −0.76 0.37 1.77 0.10 −1.45 0.52
    NM_005103 Homo FEZ1 2.80 0.11 2.08 0.25 4.98 0.06 2.20 0.26
    sapiens
    fasciculation
    and
    elongation
    protein zeta
    1 (zygin I)
    (FEZ1),
    transcript
    variant
    1,
    mRNA.
    /PROD = zygin
    1, isoform 1
    /FL = gb: U60060.1
    gb: U69139.1
    gb: NM_005103.2
    NM_000277 Homo PAH 0.65 0.11 −1.08 0.08 1.46 0.18 −0.42 0.12
    sapiens
    phenylalanine
    hydroxylase
    (PAH),
    mRNA.
    /PROD = phenylalanine
    hydroxylase
    /FL = gb: U49897.1
    gb: NM_000277.1
    BF698797 ESTs — 0.93 0.10 −0.74 0.11 2.07 0.10 −0.40 0.44
    BF437747 ESTs, C20orf118 4.26 0.07 1.99 0.45 4.57 0.09 1.75 0.39
    Weakly
    similar to
    ALU7_HUMAN
    ALU
    SUBFAMILY
    SQ
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    NM_003020 Homo SCG5 4.03 0.14 2.53 0.32 5.01 0.12 3.11 0.24
    sapiens
    secretory
    granule,
    neuroendocrine
    protein 1
    (7B2 protein)
    (SGNE1),
    mRNA.
    /PROD = secretory
    granule,
    neuroendocrine
    protein 1
    (7B2protein)
    /FL = gb: BC005349.1
    gb: NM_003020.1
    NM_002800 Homo PSMB9 1.38 0.10 0.27 0.34 2.05 0.07 −1.43 1.14
    sapiens
    proteasome
    (prosome,
    macropain)
    subunit, beta
    type, 9 (large
    multifunctional
    protease
    2) (PSMB9),
    mRNA.
    /PROD = proteasome
    (prosome,
    macropain)
    subunit,
    betatype, 9
    (large
    multifunctional
    protease
    2)
    /FL = gb: U01025.1
    gb: NM_002800.1
    BE972639 ESTs LOC646326 0.00 0.15 −2.71 0.55 0.33 0.22 −2.24 0.61
    BC044830 Homo C10orf96 1.38 0.12 −0.32 0.82 2.28 0.06 −1.39 0.90
    sapiens,
    Similar to
    RIKEN
    cDNA
    1700011F14
    gene, clone
    MGC: 35062
    IMAGE: 5166167,
    mRNA,
    complete
    cds.
    /PROD = Similar
    to RIKEN
    cDNA
    1700011F14
    gene
    /FL = gb: BC044830.1
    AI961231 KIAA0808 TOX 3.28 0.13 1.23 0.06 4.00 0.01 1.13 0.37
    gene product
    /FL = gb: AB018351.1
    gb: NM_014729.1
    U17496 Human PSMB8 3.43 0.21 0.46 1.17 3.30 0.06 0.09 0.64
    proteasome
    subunit
    LMP7 (allele
    LMP7B)
    mRNA,
    complete
    cds.
    /PROD = proteasome
    subunit
    LMP7
    /FL = gb: U17497.1
    gb: U17496.1
    N23651 ESTs SDK2 −1.16 0.31 −2.65 0.42 1.27 0.09 −2.07 0.75
    NM_007015 Homo LECT1 4.83 0.12 2.27 0.84 5.36 0.09 1.83 0.69
    sapiens
    chondromodulin I
    precursor
    (CHM-I),
    mRNA.
    /PROD = chondromodulin I
    precursor
    /FL = gb: NM_007015.1
    gb: AB006000.1
    NM_015474 Homo SAMHD1 2.63 0.17 0.57 0.33 2.89 0.09 0.29 0.22
    sapiens
    DKFZP564A032
    protein
    (DKFZP564A032),
    mRNA.
    /PROD = DKFZP564A032
    protein
    /FL = gb: AF228421.1
    gb: AL050267.1
    gb: AB013847.1
    gb: NM_015474.1
    AF147427 Homo SAMHD1 1.33 0.03 −0.99 0.88 1.35 0.08 −2.30 0.97
    sapiens full
    length insert
    cDNA clone
    YP80A10.
    NM_004688 Homo NMI 3.54 0.04 0.96 0.07 3.80 0.07 1.13 0.34
    sapiens N-
    myc (and
    STAT)
    interactor
    (NMI),
    mRNA.
    /PROD = N-
    myc and
    STAT
    interactor
    /FL = gb: BC001268.1
    gb: NM_004688.1
    gb: U32849.1
    AB040812 Homo PAK7 −1.37 0.60 −3.89 1.62 −0.49 0.07 −4.18 0.12
    sapiens
    mRNA for
    protein
    kinase
    PAK5,
    complete
    cds.
    /PROD = protein
    kinase
    PAK5
    /FL = gb: AB040812.1
    AI985987 ESTs, SCNN1G −0.28 0.31 −0.99 0.35 0.12 0.19 −2.70 0.59
    Moderately
    similar to
    ALU1_HUMAN
    ALU
    SUBFAMILY J
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    NM_001877 Homo CR2 1.50 0.13 −0.43 1.05 2.35 0.14 −1.44 1.51
    sapiens
    complement
    component
    (3dEpstein
    Barr virus)
    receptor 2
    (CR2),
    mRNA.
    /PROD = complement
    component
    (3dEpstein
    Barr
    virus)receptor 2
    /FL = gb: NM_001877.1
    gb: M26004.1
    NM_001351 Homo DAZL 1.69 0.24 −0.81 0.76 1.93 0.12 −0.73 0.06
    sapiens
    deleted in
    azoospermia-
    like (DAZL),
    mRNA.
    /PROD = deleted
    in
    azoospermia-
    like
    /FL = gb: U66726.2
    gb: NM_001351.1
    gb: U65918.1
    gb: U66078.1
    NM_022168 Homo IFIH1 −2.03 1.08 −2.76 0.83 −0.10 0.15 −2.15 0.40
    sapiens
    melanoma
    differentiation
    associated
    protein-5
    (MDA5),
    mRNA.
    /PROD = melanoma
    differentiation
    associated
    protein-5
    /FL = gb: AY017378.1
    gb: NM_022168.1
    gb: AF095844.1
    AF052108 Homo LOC157627 1.49 0.33 0.22 1.02 2.26 0.40 −1.33 1.19
    sapiens
    clone 23687
    mRNA
    sequence.
    AI056877 Human DNA LOC200230 0.11 0.30 −2.37 0.94 1.15 0.18 −1.35 0.75
    sequence
    from clone
    RP4-530I15
    on
    chromosome
    20. Contains
    the 3 end of
    the PTPN1
    gene for
    protein
    tyrosine
    phosphatase,
    non-
    receptor type
    1 (EC
    3.1.3.48), the
    gene for a
    novel protein
    similar to
    placental
    protein
    DIFF40, an
    RPL36 (60S
    Ribos
    NM_002522 Homo NPTX1 2.35 0.25 −0.48 0.19 2.17 0.09 −1.19 0.83
    sapiens
    neuronal
    pentraxin I
    (NPTX1),
    mRNA.
    /PROD = neuronal
    pentraxin I
    precursor
    /FL = gb: NM_002522.1
    gb: U61849.1
    N21096 ESTs STXBP6 2.42 0.16 0.99 0.08 3.69 0.09 1.14 0.04
    AI693516 ESTs COL14A1 0.39 0.23 −1.63 0.96 0.96 0.17 −1.81 0.67
    NM_018043 Homo TMEM16A −0.90 0.37 −1.93 0.70 0.25 0.43 −3.93 0.49
    sapiens
    hypothetical
    protein
    FLJ10261
    (FLJ10261),
    mRNA.
    /PROD = hypothetical
    protein
    FLJ10261
    /FL = gb: NM_018043.1
    AF110400 Homo FGF19 2.12 0.14 −0.08 0.34 2.18 0.04 −0.91 0.18
    sapiens
    fibroblast
    growth factor
    19 (FGF19)
    mRNA,
    complete
    cds.
    /PROD = fibroblast
    growth
    factor 19
    /FL = gb: AF110400.1
    gb: NM_005117.1
    gb: AB018122.1
    AK098525 Homo HHIP 1.76 0.14 −0.57 0.63 3.32 0.06 −0.11 0.15
    sapiens
    cDNA
    FLJ25659
    fis, clone
    TST00427,
    highly similar
    to Mus
    musculus
    hedgehog-
    interacting
    protein (Hip)
    mRNA.
    AV715309 ESTs, C20orf118 4.28 0.13 1.89 0.39 4.76 0.10 1.48 0.42
    Weakly
    similar to
    ALU7_HUMAN
    ALU
    SUBFAMILY
    SQ
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    U96136 Homo CTNND2 0.78 0.11 −1.33 0.87 1.18 0.12 −1.17 0.67
    sapiens
    delta-catenin
    mRNA,
    complete
    cds.
    /PROD = delta-
    catenin
    /FL = gb: NM_001332.1
    gb: U72665.1
    gb: AB013805.1
    gb: U96136.1
    gb: AF035302.1
    NM_002590 Homo PCDH8 −0.80 0.34 −0.83 0.82 2.18 0.02 0.12 0.33
    sapiens
    protocadherin 8
    (PCDH8),
    mRNA.
    /PROD = protocadherin 8
    /FL = gb: NM_002590.2
    gb: AF061573.2
    AF107846 Homo — 0.44 0.20 −0.25 0.12 2.12 0.14 −2.56 0.81
    sapiens
    neuroendocrine-
    specific
    Golgi protein
    p55
    (XLalphas)
    gene, exon
    XL2 and
    complete cds
    BG169832 adenylate AK5 0.62 0.36 −2.79 0.50 1.03 0.36 −1.94 0.32
    kinase 5
    /FL = gb: NM_012093.1
    gb: AF062595.1
    BE968806 ESTs, ATP5S −1.15 0.14 −3.03 0.71 −0.34 0.15 −4.73 0.74
    Weakly
    similar to
    ALU4_HUMAN
    ALU
    SUBFAMILY
    SB2
    SEQUENCE
    CONTAMINATION
    WARNING
    ENTRY
    (H. sapiens)
    BU729850 hypothetical JAKMIP2 2.30 0.07 0.43 1.78 3.86 0.04 −1.56 1.92
    protein
    LOC153469
    AL832535 Homo LOC157627 2.25 0.18 −0.04 1.00 3.14 0.25 −1.50 1.19
    sapiens
    mRNA;
    cDNA
    DKFZp547J1816
    (from
    clone
    DKFZp547J1816).
    NM_000439 Homo PCSK1 −0.63 0.18 −2.68 0.53 0.69 0.30 −3.19 1.03
    sapiens
    proprotein
    convertase
    subtilisinkexin
    type 1
    (PCSK1),
    mRNA.
    /PROD = proprotein
    convertase
    subtilisinkexin
    type
    1
    /FL = gb: NM_000439.2
    gb: M90753.1
    M34455 Human INDO 4.39 0.23 1.58 0.09 5.44 0.08 1.69 0.13
    interferon-
    gamma-
    inducible
    indoleamine
    2,3-
    dioxygenase
    (IDO)
    mRNA,
    complete
    cds.
    /FL = gb: NM_002164.1
    gb: M34455.1
    AB014737 Homo SMOC2 1.25 0.12 −2.04 0.48 1.94 0.06 −2.27 1.12
    sapiens
    mRNA for
    SMAP-2b,
    complete
    cds.
    /PROD = SMAP-
    2b
    /FL = gb: AB014737.1
    BM682352 Homo HCN1 −0.41 0.14 −4.38 1.19 0.35 0.31 −3.88 0.32
    sapiens
    cDNA
    FLJ37204
    fis, clone
    BRALZ2006976.
  • TABLE V
    RELATIVE EXPRESSION OF DEFINITE ENDODERM
    MARKERS FOR CONDITIONS OUTLINED IN EXAMPLE 10. ALL
    VALUES WERE NORMALIZED TO GROUP 1 (CONTROL).
    Sox17 CXCR4 Goosecoid HNF-3B SOX-2 Oct-4
    Group 1- 1 1 1 1 1 1
    control
    Group
    2 45 19.5 2.8 0.64 0.91 1.1
    Group 3 45 30 2.9 0.74 0.70 0.76
    Group 4 8 14 2.7 1.11 0.18 0.36
    Group 5 23 16 3.1 1.76 0.16 0.41
    Group 6 41 5.8 3.0 1.87 0.61 0.57
    Group 7 25 19.5 2.7 0.62 0.34 0.48
    Group 8 6 15.9 2.9 2.0 0.13 0.43
    Group 9 1 1.4 0.9 0.89 1.2 0.85
    Group 10 22 1.5 1.4 1.20 1.36 0.68
    Group 11 54 23 2.5 0.71 0.66 0.65
    Group 12 68 0.7 0.9 1.51 0.02 0.30
    Group 13 13.9 12.7 3.0 2.1 0.11 0.30
    Group 14 52.6 20.6 2.9 0.82 0.69 0.70
    Group 15 68 27.7 2.9 0.68 0.68 0.85
    Group 16 13.9 21 2.4 0.79 0.46 0.72
    Group 17 52 14.9 3.5 2.12 0.22 0.44
  • TABLE VI
    MEAN NORMALIZED INTENSITY VALUES (IN LOG
    FORMAT) OF GENES FOR H9 EMBRYONIC STEM CELLS
    DERIVED DEFINITIVE ENDODERM STAGE CELLS CULTURED
    ON EITHER MATRIGEL ™ OR MEFS +/− WNT-3A.
    DE DE
    treatment in treatment
    DE treatment low serum + in low Benjamini
    in low serum + AA + Wnt- serum + and
    AA on 3A on AA on Hochberg-
    Gene Title MATRIGEL MATRIGEL MEFS P-value
    ESTs −4.82708 2.63968 −4.26995 8.62E−05
    microfibrillar-associated protein 4 0.063791 5.16358 −0.60091 3.48E−03
    Homo sapiens, alpha-1 (VI) collagen −3.66187 2.36459 −2.26934 1.45E−04
    ESTs −3.43712 2.14508 −2.6475 0.00E+00
    Homo sapiens cystatin SN (CST1), 0.072931 7.53908 4.63955 4.46E−03
    mRNA. /PROD = cystatin SN
    /FL = gb: J03870.1 gb: NM_001898.1
    solute carrier family 16 (monocarboxylic −0.066405 5.23572 0.279005 4.27E−04
    acid transporters), member 3
    /FL = gb: U81800.1 gb: NM_004207.1
    Homo sapiens fibroblast growth factor −0.894644 5.75417 2.4872 4.93E−03
    17 (FGF17), mRNA. /PROD = fibroblast
    growth factor
    17 /FL = gb: NM_003867.1
    gb: AB009249.1
    Human link protein mRNA, complete −1.93991 3.31348 −1.26346 1.14E−02
    cds. /PROD = link protein
    /FL = gb: NM_001884.1 gb: U43328.1
    Homo sapiens solute carrier family 16 0.710321 6.12971 1.72403 0.00E+00
    (monocarboxylic acid transporters),
    member 3 (SLC16A3), mRNA.
    /PROD = solute carrier family 16
    (monocarboxylic acidtransporters),
    member 3 /FL = gb: U81800.1
    gb: NM_004207.1
    Homo sapiens apolipoprotein A-I −1.47073 5.37558 2.46891 5.07E−03
    (APOA1), mRNA. /PROD = apolipoprotein
    A-I precursor /FL = gb: M27875.1
    gb: M11791.1 gb: NM_000039.1
    gb: BC005380.1
    Homo sapiens cytidine deaminase −3.89129 2.05822 −1.67035 2.23E−03
    (CDA), mRNA. /PROD = cytidine
    deaminase /FL = gb: L27943.1
    gb: NM_001785.1
    ESTs, Moderately similar to JE0284 −2.37712 5.75671 4.22227 2.56E−02
    Mm-1 cell derived transplantability-
    associated protein 1b (H. sapiens)
    ESTs −0.04716 5.28231 0.966974 0.00E+00
    glycophorin B (includes Ss blood group) −2.85201 3.32812 −0.12969 1.45E−04
    Homo sapiens homeobox protein −4.42042 3.55326 1.89424 2.50E−02
    goosecoid mRNA, complete cds.
    /PROD = homeobox protein goosecoid
    /FL = gb: AY177407.1 gb: NM_173849.1
    MCP-1 = monocyte chemotactic protein −2.27571 5.13499 2.95543 2.92E−02
    (human, aortic endothelial cells, mRNA,
    661 nt). /PROD = MCP-1
    Homo sapiens Mix-like homeobox −1.54648 4.47601 0.921971 2.01E−02
    protein 1 (MILD1) mRNA, complete cds.
    /PROD = Mix-like homeobox protein 1
    /FL = gb: AF211891.1
    ESTs −4.93603 2.17917 −0.23735 1.12E−04
    Homo sapiens lumican (LUM), mRNA. −4.05726 3.21064 0.948822 3.39E−02
    /PROD = lumican /FL = gb: NM_002345.1
    gb: U18728.1 gb: U21128.1
    Homo sapiens HNF-3beta mRNA for −2.71785 4.68666 2.82506 3.71E−02
    hepatocyte nuclear factor-3 beta,
    complete cds. /PROD = hepatocyte
    nuclear factor-3 beta
    /FL = gb: AB028021.1 gb: NM_021784.1
    Homo sapiens reserved (KCNK12), −0.468745 6.28184 3.77969 1.97E−02
    mRNA. /PROD = tandem pore domain
    potassium channel THIK-2
    /FL = gb: NM_022055.1 gb: AF287302.1
    Homo sapiens atrophin-1 interacting −4.30828 1.80825 −1.32021 9.63E−03
    protein 1; activin receptor interacting
    protein 1 (KIAA0705), mRNA.
    /PROD = atrophin-1 interacting protein 1;
    activinreceptor interacting protein 1
    /FL = gb: NM_012301.1 gb: AF038563.1
    ESTs −2.33636 2.25176 −2.32124 5.26E−04
    Homo sapiens glutamate decarboxylase −2.424 2.31908 −1.87965 4.07E−04
    1 (brain, 67 kD) (GAD1), transcript
    variant GAD25, mRNA.
    /PROD = glutamate decarboxylase 1,
    isoform GAD25 /FL = gb: NM_013445.1
    gb: AF178853.1 gb: BC002815.1
    Homo sapiens cardiac ventricular −0.549728 4.89072 1.4377 2.99E−03
    troponin C mRNA, complete cds.
    /PROD = cardiac ventricular troponin C
    /FL = gb: NM_003280.1 gb: AF020769.1
    ESTs −2.89554 3.42817 0.926036 2.62E−02
    Homo sapiens fibroblast growth factor 8 −4.32791 2.19561 0.015827 5.91E−03
    (androgen-induced) (FGF8), mRNA.
    /PROD = fibroblast growth factor 8
    (androgen-induced) /FL = gb: U36223.1
    gb: U46212.1 gb: NM_006119.1
    ESTs −3.09818 1.66254 −2.20564 8.62E−05
    Homo sapiens haptoglobin-related −2.6068 2.38009 −1.19632 6.95E−03
    protein (HPR), mRNA.
    /PROD = haptoglobin-related protein
    /FL = gb: NM_020995.1
    collagen, type VI, alpha 1 −1.418 3.85952 0.604245 1.21E−02
    Human (clone 8B1) Br-cadherin mRNA, −2.17941 2.59894 −1.02624 1.24E−02
    complete cds. /PROD = Br-cadherin
    /FL = gb: L34057.1 gb: L33477.1
    gb: NM_004061.1
    ESTs −1.40092 2.57297 −1.82509 1.45E−04
    Homo sapiens cystatin SA (CST2), −0.102178 5.21645 2.1671 2.40E−02
    mRNA. /PROD = cystatin SA
    /FL = gb: NM_001322.1
    Human mRNA for apolipoprotein AI (apo 0.215086 5.51109 2.49684 9.57E−03
    AI) =. /PROD = preproapolipoprotein AI
    Homo sapiens MLL septin-like fusion −3.29221 1.70902 −1.49951 2.25E−03
    (MSF), mRNA. /PROD = MLL septin-like
    fusion /FL = gb: AF123052.1
    gb: NM_006640.1
    Homo sapiens cystatin S (CST4), 0.92448 6.48842 3.87036 7.20E−03
    mRNA. /PROD = cystatin S
    /FL = gb: NM_001899.1
    Homo sapiens phorbolin-like protein −1.11883 4.73391 2.40782 7.86E−03
    MDS019 (MDS019), mRNA.
    /PROD = phorbolin-like protein MDS019
    /FL = gb: AF182420.1 gb: NM_021822.1
    Homo sapiens apolipoprotein A-II −1.03333 5.80468 4.46856 3.23E−02
    (APOA2), mRNA. /PROD = apolipoprotein
    A-II precursor /FL = gb: M29882.1
    gb: NM_001643.1 gb: BC005282.1
    ESTs −1.55475 3.48278 0.420447 2.50E−02
    Homo sapiens glutamate decarboxylase −3.86752 1.56384 −1.08675 2.52E−02
    1 (brain, 67 kD) (GAD1), transcript
    variant GAD67, mRNA.
    /PROD = glutamate decarboxylase 1,
    isoform GAD67 /FL = gb: NM_000817.1
    gb: M81883.1 gb: L16888.1
    ESTs −0.731491 5.43249 3.52168 1.60E−02
    ESTs −2.03591 3.38924 0.760984 2.34E−03
    Homo sapiens retinoid X receptor, −2.37496 2.62934 −0.32035 8.83E−04
    gamma (RXRG), mRNA.
    /PROD = retinoid X receptor, gamma
    /FL = gb: NM_006917.1 gb: U38480.1
    ESTs −0.648552 4.30576 1.43266 2.19E−03
    Homo sapiens cDNA FLJ11550 fis, −1.22228 5.37746 4.21644 1.68E−02
    clone HEMBA1002970
    ESTs −1.782 3.50391 1.0501 1.85E−02
    Homo sapiens haptoglobin (HP), mRNA. −1.10114 3.5449 0.477027 8.13E−03
    /PROD = haptoglobin /FL = gb: K00422.1
    gb: L29394.1 gb: NM_005143.1
    Homo sapiens hypothetical protein −0.431989 5.14497 3.02045 2.62E−02
    FLJ10970 (FLJ10970), mRNA.
    /PROD = hypothetical protein FLJ10970
    /FL = gb: NM_018286.1
    Homo sapiens beta-site APP cleaving −2.0354 3.70648 1.75385 8.00E−03
    enzyme (BACE) mRNA, complete cds.
    /PROD = beta-site APP cleaving enzyme
    /FL = gb: AF200343.1 gb: AF204943.1
    gb: AF190725.1 gb: AF201468.1
    gb: NM_012104.1
    Homo sapiens hypothetical protein 1.36784 6.82571 4.5979 2.10E−02
    FLJ22252 similar to SRY-box containing
    gene 17 (FLJ22252), mRNA.
    /PROD = hypothetical protein FLJ22252
    similar to SRY-boxcontaining gene 17
    /FL = gb: NM_022454.1
    Cluster Incl. AB028021: Homo sapiens −1.5339 5.12418 4.11704 4.47E−02
    HNF-3beta mRNA for hepatocyte
    nuclear factor-3 beta, complete cds
    /cds = (196, 1569) /gb = AB028021
    /gi = 4958949 /ug = Hs.155651 /len = 1944
    Homo sapiens gastrin-releasing peptide −2.74071 2.70077 0.509757 2.49E−04
    (GRP), mRNA. /PROD = gastrin-releasing
    peptide /FL = gb: NM_002091.1
    gb: K02054.1 gb: BC004488.1
    Homo sapiens sema domain, seven −1.53335 3.78503 1.48732 4.71E−02
    thrombospondin repeats (type 1 and
    type 1-like), transmembrane domain
    (TM) and short cytoplasmic domain,
    (semaphorin) 5A (SEMA5A), mRNA.
    /PROD = sema domain, seven
    thrombospondin repeats (type1 and type
    1-like), transmem
    Homo sapiens mRNA; cDNA −0.835182 5.22406 3.69882 2.08E−02
    DKFZp586J0624 (from clone
    DKFZp586J0624); complete cds.
    /PROD = hypothetical protein
    /FL = gb: AF215636.1 gb: NM_014585.1
    gb: AF231121.1 gb: AF226614.1
    gb: AL136944.1
    Human mRNA for alpha-1 type II −2.8736 2.155 −0.38021 5.70E−03
    collagen.
    Rho GDP dissociation inhibitor (GDI) −1.54385 1.7147 −2.58241 1.71E−02
    alpha
    neuropilin
    1 /FL = gb: AF016050.1 −1.62253 1.95432 −1.9667 8.83E−04
    gb: NM_003873.1 gb: AF018956.1
    Human DNA sequence from clone RP1- −3.72313 1.68755 −0.37308 1.38E−03
    181C24 on chromosome 6p11.1-12.2.
    Contains the 3 end of the BMP5 gene for
    bone morphogenetic protein 5, ESTs,
    STSs and GSSs /FL = gb: M60314.1
    gb: NM_021073.1
    myristoylated alanine-rich protein kinase −0.71724 3.51728 0.335725 4.29E−03
    C substrate (MARCKS, 80K-L)
    /FL = gb: M68956.1 gb: D10522.1
    gb: NM_002356.4
    hypothetical protein FLJ23403 −1.45618 1.81423 −2.31327 1.20E−02
    /FL = gb: NM_022068.1
    hepatocyte nuclear factor 4, alpha −4.26574 1.7879 0.445241 3.25E−02
    Homo sapiens cell adhesion molecule −0.541188 2.1751 −2.5002 1.16E−03
    with homology to L1CAM (close
    homologue of L1) (CHL1), mRNA.
    /PROD = cell adhesion molecule with
    homology to L1CAM(close homologue of
    L1) /FL = gb: AF002246.1
    gb: NM_006614.1
    matrix metalloproteinase 14 (membrane- −2.05734 2.36236 −0.5185 1.66E−02
    inserted) /FL = gb: U41078.1
    gb: NM_004995.2
    Homo sapiens glycophorin B (includes −0.947308 3.26089 0.180293 4.83E−04
    Ss blood group) (GYPB), mRNA.
    /PROD = glycophorin B precursor
    /FL = gb: J02982.1 gb: NM_002100.2
    WAS protein family, member 2 −2.18746 1.99129 −1.05968 4.00E−03
    /FL = gb: NM_006990.1 gb: AB026542.1
    Homo sapiens frizzled-related protein 0.56502 5.7261 3.67629 1.75E−02
    (FRZB), mRNA. /PROD = frizzled-related
    protein /FL = gb: U24163.1 gb: U68057.1
    gb: NM_001463.1 gb: U91903.1
    Homo sapiens glutamate decarboxylase −1.68495 2.27067 −0.96944 8.02E−03
    1 (brain, 67 kD) (GAD1), transcript
    variant GAD25, mRNA.
    /PROD = glutamate decarboxylase 1,
    isoform GAD25 /FL = gb: NM_013445.1
    gb: AF178853.1 gb: BC002815.1
    ESTs 0.812766 5.93144 3.91314 4.15E−02
    Homo sapiens clone 23736 mRNA −0.047182 5.79006 4.50744 1.74E−02
    sequence
    Homo sapiens glycophorin E (GYPE), −2.01601 1.79002 −1.50134 1.97E−02
    mRNA. /PROD = glycophorin E
    /FL = gb: NM_002102.1 gb: M29610.1
    ESTs 1.06767 5.63319 3.12487 2.00E−02
    ESTs −1.41162 2.5396 −0.57029 1.29E−02
    Human Fritz mRNA, complete cds. 0.436589 5.69814 3.91514 1.99E−02
    /PROD = Fritz /FL = gb: U24163.1
    gb: U68057.1 gb: NM_001463.1
    gb: U91903.1
    Homo sapiens , clone MGC: 4655, 2.3772 5.9184 2.47596 1.20E−02
    mRNA, complete cds. /PROD = Unknown
    (protein for MGC: 4655)
    /FL = gb: BC004908.1
    KIAA0878 protein /FL = gb: NM_014899.1 1.1189 6.41747 4.78882 2.01E−02
    gb: AB020685.1
    Homo sapiens sema domain, −0.785987 3.69668 1.27624 2.10E−02
    immunoglobulin domain (Ig), short basic
    domain, secreted, (semaphorin) 3E
    (SEMA3E), mRNA. /PROD = sema
    domain, immunoglobulin domain (Ig),
    shortbasic domain, secreted,
    (semaphorin) 3E /FL = gb: NM_012431.1
    gb: AB002329.1
    ESTs 1.48084 6.59709 4.81395 1.36E−02
    noggin /FL = gb: NM_005450.1 −1.63627 3.28161 1.32958 2.53E−02
    Homo sapiens hypothetical protein −0.904749 3.35854 0.755319 1.12E−04
    FLJ11316 (FLJ11316), mRNA.
    /PROD = hypothetical protein FLJ11316
    /FL = gb: NM_018388.1
    Homo sapiens angiopoietin 2 (ANGPT2), −2.93044 2.23779 0.59685 3.43E−02
    mRNA. /PROD = angiopoietin 2
    /FL = gb: AB009865.1 gb: AF004327.1
    gb: NM_001147.1
    Homo sapiens matrix metalloproteinase −0.723489 2.97262 −0.09689 5.44E−03
    14 (membrane-inserted) (MMP14),
    mRNA. /PROD = matrix metalloproteinase
    14 preproprotein /FL = gb: U41078.1
    gb: NM_004995.2
    G protein-coupled receptor 1.50709 6.65228 5.05327 2.00E−02
    collagen, type IX, alpha 2 1.27026 5.4659 2.93507 3.19E−03
    /FL = gb: NM_001852.1
    ESTs 0.521638 3.93176 0.620223 0.00E+00
    KIAA1462 protein −3.84563 1.65452 0.437064 3.27E−02
    Homo sapiens cartilage linking protein 1 −1.31515 2.27271 −0.80521 2.22E−02
    (CRTL1), mRNA. /PROD = cartilage
    linking protein
    1 /FL = gb: NM_001884.1
    gb: U43328.1
    Homo sapiens solute carrier family 21 0.711428 4.89808 2.43304 6.84E−03
    (prostaglandin transporter), member 2
    (SLC21A2), mRNA. /PROD = solute
    carrier family 21
    (prostaglandintransporter), member 2
    /FL = gb: U70867.1 gb: NM_005630.1
    ESTs 0.307173 4.78515 2.65653 1.72E−02
    6-phosphofructo-2-kinasefructose-2,6- −0.242865 3.97929 1.59985 5.28E−03
    biphosphatase 4
    Homo sapiens dual specificity 0.953857 5.82811 4.12159 4.00E−03
    phosphatase 4 (DUSP4), mRNA.
    /PROD = dual specificity phosphatase 4
    /FL = gb: NM_001394.2 gb: BC002671.1
    gb: U48807.1 gb: U21108.1
    ESTs, Weakly similar to T00331 −1.57372 2.70797 0.443622 6.74E−03
    hypothetical protein KIAA0555
    (H. sapiens)
    ESTs −3.57414 3.15167 3.37198 8.82E−03
    ESTs, Highly similar to IHH_HUMAN −0.653989 3.22059 0.590533 5.18E−03
    INDIAN HEDGEHOG PROTEIN
    PRECURSOR (H. sapiens)
    ESTs, Weakly similar to FCE2 MOUSE 0.494192 5.22522 3.48031 1.97E−02
    LOW AFFINITY IMMUNOGLOBULIN
    EPSILON FC RECEPTOR (M. musculus)
    homeo box HB9 /FL = gb: NM_005515.1 −1.65563 3.2238 1.63092 5.58E−03
    Homo sapiens arylsulfatase E 0.283004 4.95903 3.18424 9.22E−03
    (chondrodysplasia punctata 1) (ARSE),
    mRNA. /PROD = arylsulfatase E
    precursor /FL = gb: X83573.1
    gb: NM_000047.1
    ESTs −0.05909 3.0455 −0.29817 1.47E−02
    Homo sapiens hypothetical protein −1.48452 1.97473 −1.00431 5.72E−03
    FLJ23403 (FLJ23403), mRNA.
    /PROD = hypothetical protein FLJ23403
    /FL = gb: NM_022068.1
    ESTs −0.182403 3.01548 −0.18954 3.72E−03
    hypothetical protein FLJ23091 0.323388 5.25192 3.77987 3.57E−02
    Human dipeptidyl peptidase IV (CD26) −3.61145 0.760585 −1.25595 3.07E−02
    mRNA, complete cds. /PROD = dipeptidyl
    peptidase IV /FL = gb: M74777.1
    hypothetical protein FLJ21032 0.355672 4.67756 2.61753 4.59E−02
    Homo sapiens Kell blood group (KEL), −2.20519 1.89439 −0.38393 1.91E−02
    mRNA. /PROD = Kell blood group antigen
    /FL = gb: BC003135.1 gb: NM_000420.1
    splicing factor, arginineserine-rich 5 0.7481 5.68934 4.27169 2.97E−03
    Human prostatic secretory protein 57 −3.01313 1.46338 −0.40767 1.97E−02
    mRNA, complete cds. /PROD = PSP57
    /FL = gb: U22178.1
    Homo sapiens KIAA0878 protein 2.0265 7.00937 5.65368 2.85E−02
    (KIAA0878), mRNA. /PROD = KIAA0878
    protein /FL = gb: NM_014899.1
    gb: AB020685.1
    Homo sapiens cryptic mRNA, complete 0.104874 2.87319 −0.67353 1.61E−03
    cds. /PROD = cryptic /FL = gb: AF312769.1
    Homo sapiens cDNA FLJ13221 fis, 0.355743 3.98782 1.30963 1.38E−03
    clone NT2RP4002075
    phorbolin-like protein MDS019 −1.11756 2.83853 0.503523 1.59E−02
    Homo sapiens mRNA for KIAA1409 0.368334 2.8009 −1.03191 1.78E−02
    protein, partial cds. /PROD = KIAA1409
    protein
    Homo sapiens mRNA; cDNA −2.63427 1.64513 −0.3056 2.52E−02
    DKFZp434D0818 (from clone
    DKFZp434D0818)
    ESTs 0.35393 5.14775 3.74875 3.16E−02
    Homo sapiens TBX3-iso protein (TBX3- −2.34566 2.45238 1.07038 1.31E−02
    iso), mRNA. /PROD = TBX3-iso protein
    /FL = gb: NM_016569.1 gb: AF216750.1
    Homo sapiens chromosome 19, cosmid −0.258871 3.0636 0.223926 1.58E−03
    R31181
    Homo sapiens mRNA for GATA-6, 2.21862 6.8609 5.34263 4.29E−02
    complete cds. /PROD = GATA-6
    /FL = gb: U66075.1 gb: NM_005257.1
    gb: D87811.1
    Homo sapiens ankyrin-like with −1.10879 3.93484 2.81939 1.29E−02
    transmembrane domains 1 (ANKTM1),
    mRNA
    G protein-coupled receptor 49 0.265509 4.46257 2.50537 2.19E−02
    Homo sapiens growth differentiation 1.67253 5.34944 2.87486 8.62E−05
    factor 3 (GDF3), mRNA. /PROD = growth
    differentiation factor
    3 precursor
    /FL = gb: NM_020634.1 gb: AF263538.1
    Human (clone HSY3RR) neuropeptide Y 1.77461 6.70301 5.47995 3.00E−02
    receptor (NPYR) mRNA, complete cds.
    /PROD = neuropeptide Y receptor
    /FL = gb: L06797.1 gb: NM_003467.1
    gb: AF025375.1 gb: AF147204.1
    gb: M99293.1 gb: L01639.1
    Homo sapiens type VI collagen alpha 2 1.7011 5.33126 2.84458 5.91E−03
    chain precursor (COL6A2) mRNA,
    complete cds, alternatively spliced.
    /PROD = type VI collagen alpha 2 chain
    precursor /FL = gb: AY029208.1
    ESTs −0.349726 3.29119 0.824929 4.11E−03
    ESTs −0.903317 1.89777 −1.36429 6.84E−03
    Homo sapiens hypothetical protein 2.60483 7.10633 5.55242 1.41E−02
    FLJ10718 (FLJ10718), mRNA.
    /PROD = hypothetical protein FLJ10718
    /FL = gb: NM_018192.1
    Homo sapiens Rho GTPase activating −1.10389 1.83053 −1.28521 1.28E−02
    protein 6 (ARHGAP6), transcript variant
    2, mRNA. /PROD = Rho GTPase
    activating protein
    6 isoform 2
    /FL = gb: AF022212.2 gb: NM_001174.2
    stanniocalcin 1 /FL = gb: U25997.1 2.41135 7.29563 6.14284 2.51E−02
    gb: NM_003155.1 gb: U46768.1
    Human glycophorin HeP2 mRNA, partial −0.843493 2.71108 0.233547 2.98E−04
    cds. /PROD = glycophorin HeP2
    Homo sapiens cDNA FLJ12993 fis, 0.147259 4.12241 2.06949 6.84E−03
    clone NT2RP3000197
    Homo sapiens presenilin stabilization −0.86173 2.85614 0.56745 6.09E−03
    factor b (PSF) mRNA, complete cds;
    alternatively spliced. /PROD = presenilin
    stabilization factor b /FL = gb: AY113699.1
    Homo sapiens glycophorin Erik STA −1.19362 2.17108 −0.45439 1.45E−04
    (GPErik) gene complete cds.
    /PROD = glycophorin Erik (STA)
    /FL = gb: U00178.1
    bromodomain and PHD finger −2.8472 1.75573 0.369397 8.62E−05
    containing, 3
    ESTs 0.784344 4.74104 2.71543 7.55E−03
    ESTs −1.26251 3.4693 2.27614 3.80E−03
    ESTs −1.71713 1.23763 −1.71122 2.99E−02
    Homo sapiens microsomal glutathione 2.06233 6.81536 5.6918 3.79E−02
    S-transferase 2 (MGST2), mRNA.
    /PROD = microsomal glutathione S-
    transferase 2 /FL = gb: NM_002413.1
    gb: U77604.1
    Homo sapiens eomesodermin (Xenopus 2.65926 6.71627 4.89839 2.76E−02
    laevis) homolog (EOMES), mRNA.
    /PROD = eomesodermin (Xenopus laevis)
    homolog /FL = gb: AB031038.1
    gb: NM_005442.1
    Homo sapiens mRNA for MSX-2, 0.407211 4.11145 1.94529 1.35E−02
    complete cds. /PROD = MSX-2
    /FL = gb: D89377.1
    Homo sapiens apolipoprotein A-II −1.10237 5.10066 4.75899 1.34E−02
    (APOA2), mRNA. /PROD = apolipoprotein
    A-II precursor /FL = gb: M29882.1
    gb: NM_001643.1 gb: BC005282.1
    Homo sapiens adenylate cyclase 8 −1.3408 1.12773 −2.26149 1.07E−02
    (brain) (ADCY8), mRNA.
    /PROD = adenylate cyclase 8
    /FL = gb: NM_001115.1
    Homo sapiens glucose-6-phosphate −0.193516 3.03064 0.407738 1.38E−03
    transporter (G6PT) gene, G6PT-Dt
    allele, complete cds
    Homo sapiens glutathione S-transferase −3.10645 1.11704 −0.4221 5.37E−04
    A2 (GSTA2), mRNA. /PROD = glutathione
    S-transferase A2 /FL = gb: BC002895.1
    gb: M25627.1 gb: M16594.1 gb: M14777.1
    gb: M15872.1 gb: M21758.1
    gb: NM_000846.1
    Homo sapiens sodium dependent −0.397133 2.78341 0.231961 1.11E−02
    phosphate transporter isoform NaPi-IIb
    mRNA, complete cds. /PROD = sodium
    dependent phosphate transporter
    isoformNaPi-IIb /FL = gb: AF111856.1
    gb: NM_006424.1 gb: AF146796.1
    ESTs 2.77413 7.19192 5.906 3.17E−02
    Homo sapiens partial LHX9 gene for −2.2486 1.64952 −0.13424 7.89E−03
    LIM-homeobox 9, 3UTR.
    Homo sapiens LYST-interacting protein −0.682149 2.34271 −0.31253 9.87E−03
    LIP3 mRNA, partial cds. /PROD = LYST-
    interacting protein LIP3
    putative 47 kDa protein −0.123937 3.2914 1.05247 3.72E−03
    Homo sapiens protein S (alpha) 0.577604 4.00035 1.7883 7.89E−03
    (PROS1), mRNA. /PROD = protein S
    (alpha) /FL = gb: M15036.1
    gb: NM_000313.1
    ESTs −1.63711 0.915477 −2.15328 1.83E−02
    protocadherin 10 1.76718 5.1503 2.98569 2.52E−02
    KIAA1511 protein 0.620278 3.86886 1.57543 1.86E−03
    Homo sapiens cDNA FLJ13221 fis, 0.282785 3.44178 1.08178 1.31E−03
    clone NT2RP4002075
    Homo sapiens fibroblast growth factor −1.72385 2.26674 0.747239 6.86E−03
    receptor 4, soluble-form splice variant
    (FGFR4) mRNA, complete cds.
    /PROD = fibroblast growth factor receptor
    4, soluble-form splice variant
    /FL = gb: NM_022963.1 gb: AF202063.1
    X75208 /FEATURE = cds −1.36729 2.60456 1.08204 1.58E−02
    /DEFINITION = HSPTKR H. sapiens
    HEK2 mRNA for protein tyrosine kinase
    receptor
    Homo sapiens chemokine receptor 2.30557 6.72969 5.66933 3.31E−02
    CXCR4 mRNA, complete cds.
    /PROD = chemokine receptor CXCR4
    /FL = gb: AF348491.1
    KIAA1415 protein 0.185854 3.70346 1.74255 7.78E−04
    ESTs −0.048335 3.11786 0.836448 1.29E−02
    Homo sapiens stanniocalcin 1 (STC1), 2.39435 6.55737 5.28007 2.19E−02
    mRNA. /PROD = stanniocalcin 1
    /FL = gb: U25997.1 gb: NM_003155.1
    gb: U46768.1
    Homo sapiens high mobility group 1.04695 3.2786 0.095286 2.11E−02
    protein-R mRNA, complete cds.
    /PROD = high mobility group protein-R
    /FL = gb: AF176039.1
    ESTs, Moderately similar to −1.10358 1.66331 −0.98371 3.37E−02
    ALU4_HUMAN ALU SUBFAMILY SB2
    SEQUENCE CONTAMINATION
    WARNING ENTRY (H. sapiens)
    Homo sapiens arrestin, beta 1 (ARRB1), −0.158844 2.41791 −0.40915 1.12E−02
    transcript variant 1, mRNA.
    /PROD = arrestin beta 1, isoform A
    /FL = gb: BC003636.1 gb: AF084040.1
    gb: NM_004041.2
    ESTs −1.34399 1.80279 −0.44529 1.60E−03
    Human mRNA for pro-alpha 1 (II) 0.85982 4.1935 2.15657 1.09E−02
    collagen 3end C-term. triple helical and
    C-terminal non-helical domain.
    /PROD = pro-alpha 1 (II) collagen (313
    AA; AA 975-271c) /FL = gb: NM_001844.2
    Homo sapiens hypothetical protein 1.47773 4.42479 2.00326 1.16E−03
    DKFZp564B052 (DKFZp564B052),
    mRNA. /PROD = hypothetical protein
    DKFZp564B052 /FL = gb: NM_030820.1
    testis enhanced gene transcript (BAX −0.695228 2.99328 1.31679 1.17E−03
    inhibitor 1)
    Homo sapiens fasciculation and 1.86433 4.71354 2.20315 8.62E−05
    elongation protein zeta 1 (zygin I)
    (FEZ1), transcript variant 1, mRNA.
    /PROD = zygin 1, isoform 1
    /FL = gb: U60060.1 gb: U69139.1
    gb: NM_005103.2
    Homo sapiens matrix metalloproteinase −0.298504 3.44582 1.8336 4.80E−03
    15 (membrane-inserted) (MMP15),
    mRNA. /PROD = matrix metalloproteinase
    15 preproprotein /FL = gb: D86331.1
    gb: NM_002428.1
    heparan sulfate proteoglycan 2 −2.53327 0.924726 −0.97212 4.31E−02
    (perlecan) /FL = gb: M85289.1
    gb: NM_005529.2
    Homo sapiens dickkopf (Xenopus laevis) 0.981469 3.70716 1.09014 1.76E−03
    homolog 1 (DKK1), mRNA.
    /PROD = dickkopf (Xenopus laevis)
    homolog 1 /FL = gb: AF177394.1
    gb: NM_012242.1 gb: AF127563.1
    ESTs −0.801487 3.37342 2.21393 1.22E−02
    ESTs −2.57626 1.40231 0.050493 6.60E−03
    ESTs 0.499579 4.10541 2.39934 4.29E−03
    Homo sapiens bone morphogenetic 2.04506 6.19114 5.02927 4.98E−02
    protein 2 (BMP2), mRNA. /PROD = bone
    morphogenetic protein 2 precursor
    /FL = gb: NM_001200.1
    Human extracellular matrix protein 1 0.634778 3.65829 1.37564 6.11E−04
    (ECM1) mRNA, complete cds.
    /PROD = extracellular matrix protein 1
    /FL = gb: NM_004425.2 gb: U65932.1
    gb: U68186.1
    Homo sapiens nuclear receptor −1.92783 2.32456 1.27907 2.99E−02
    subfamily 0, group B, member 1
    (NR0B1), mRNA. /PROD = adrenal
    hypoplasia protein /FL = gb: NM_000475.2
    Homo sapiens cDNA: FLJ23067 fis, −2.25626 1.95987 0.882498 6.62E−03
    clone LNG04993.
    Homo sapiens ADP-ribosylation factor 4- 1.63466 5.70351 4.48037 1.90E−02
    like (ARF4L), mRNA. /PROD = ADP-
    ribosylation factor 4-like
    /FL = gb: U25771.1 gb: L38490.1
    gb: NM_001661.1 gb: BC000043.1
    Homo sapiens HT016 mRNA, complete 0.218762 3.97514 2.45222 3.37E−02
    cds. /PROD = HT016 /FL = gb: AF225426.1
    Homo sapiens, tropomodulin, clone 0.348796 3.54199 1.46427 1.29E−03
    MGC: 3643, mRNA, complete cds.
    /PROD = tropomodulin
    /FL = gb: NM_003275.1 gb: M77016.1
    gb: BC002660.1
    Homo sapiens hypothetical protein 0.903825 4.65366 3.1349 2.48E−02
    FLJ22471 (FLJ22471), mRNA.
    /PROD = hypothetical protein FLJ22471
    /FL = gb: NM_025140.1
    ESTs −0.120583 2.94229 0.738304 7.20E−03
    ESTs, Moderately similar to JC4969 pig- −2.39713 0.906454 −1.04495 1.69E−02
    c protein (H. sapiens)
    Homo sapiens LIM homeobox protein 1 −0.77166 2.27746 0.085559 4.26E−02
    (LHX1), mRNA. /PROD = LIM homeobox
    protein
    1 /FL = gb: NM_005568.1
    gb: U14755.1
    Homo sapiens, hypothetical protein −2.43105 0.646883 −1.50617 8.27E−03
    MGC2865, clone MGC: 20246
    IMAGE: 4635389, mRNA, complete cds.
    /FL = gb: BC016043.1
    ESTs −2.01523 2.16773 1.12403 1.99E−02
    Homo sapiens oxoglutarate −0.032595 2.3997 −0.38533 1.45E−02
    dehydrogenase (lipoamide) (OGDH),
    mRNA. /PROD = oxoglutarate
    dehydrogenase (lipoamide)
    /FL = gb: D10523.1 gb: BC004964.1
    gb: NM_002541.1
    ESTs, Weakly similar to T32252 2.20966 5.35974 3.33255 1.12E−04
    hypothetical protein T15B7.2-
    Caenorhabditis elegans (C. elegans)
    Homo sapiens, cleft lip and palate 1.31578 3.52357 0.556735 2.92E−03
    associated transmembrane protein 1,
    clone MGC: 10593, mRNA, complete
    cds. /PROD = cleft lip and palate
    associated transmembraneprotein 1
    /FL = gb: BC004865.1
    Homo sapiens clone TUA8 Cri-du-chat 0.09218 3.37126 1.49746 6.46E−03
    region mRNA
    Homo sapiens transcriptional activator of 0.666391 4.50481 3.22532 5.80E−03
    the c-fos promoter (CROC4), mRNA.
    /PROD = transcriptional activator of the c-
    fos promoter /FL = gb: NM_006365.1
    gb: U49857.1
    Homo sapiens hypothetical protein −1.38519 2.23626 0.763963 7.36E−03
    FLJ21195 similar to protein related to
    DAC and cerberus (FLJ21195), mRNA.
    /PROD = hypothetical protein FLJ21195
    similar to proteinrelated to DAC and
    cerberus /FL = gb: NM_022469.1
    ras homolog gene family, member B −0.123101 2.52093 0.071885 1.86E−02
    /FL = gb: AF498971.1 gb: NM_004040.1
    cartilage linking protein 1 0.879449 3.47789 0.987243 1.03E−03
    Homo sapiens BCL2adenovirus E1B 3.30506 6.91803 5.44746 1.79E−03
    19 kD-interacting protein 3 (BNIP3)
    mRNA, complete cds.
    /PROD = BCL2adenovirus E1B 19 kD-
    interacting protein 3 /FL = gb: AF002697.1
    gb: NM_004052.2 gb: U15174.1
    Homo sapiens polycythemia rubra vera −1.19629 2.40987 0.93741 9.40E−03
    1; cell surface receptor (PRV1), mRNA.
    /PROD = polycythemia rubra vera 1; cell
    surfacereceptor /FL = gb: NM_020406.1
    gb: AF146747.1
    Homo sapiens hypothetical protein −0.044112 3.42017 1.80688 3.77E−03
    FLJ11560 (FLJ11560), mRNA.
    /PROD = hypothetical protein FLJ11560
    /FL = gb: NM_025182.1
    plexin A2 0.930527 4.36203 2.74167 1.52E−02
    Homo sapiens mRNA; cDNA 1.37193 4.24502 2.08129 5.37E−04
    DKFZp547H236 (from clone
    DKFZp547H236). /PROD = hypothetical
    protein
    Homo sapiens cystatin C (amyloid 2.08601 5.30525 3.51965 2.20E−03
    angiopathy and cerebral hemorrhage)
    (CST3), mRNA. /PROD = cystatin C
    (amyloid angiopathy and
    cerebralhemorrhage)
    /FL = gb: NM_000099.1
    ESTs, Moderately similar to NFY-C −0.749407 2.00148 −0.24268 2.34E−03
    (H. sapiens)
    ESTs 2.54243 5.75201 3.97307 2.60E−02
    Homo sapiens sialyltransferase (STHM), −0.68709 2.89586 1.50348 3.70E−02
    mRNA. /PROD = sialyltransferase
    /FL = gb: U14550.1 gb: NM_006456.1
    Homo sapiens SG2NA beta isoform −1.15566 1.13989 −1.53439 1.55E−02
    mRNA, partial cds. /PROD = SG2NA beta
    isoform
    Homo sapiens hypothetical protein 1.49091 4.80138 3.15205 2.63E−02
    FLJ12838 (FLJ12838), mRNA.
    /PROD = hypothetical protein FLJ12838
    /FL = gb: NM_024641.1
    Homo sapiens solute carrier 0.403706 2.12385 −1.10043 1.78E−02
    (SLC25A18) mRNA, complete cds;
    nuclear gene for mitochondrial product.
    /PROD = solute carrier
    /FL = gb: AY008285.1
    KIAA0346 protein −1.34736 2.49605 1.41497 3.90E−03
    Human clone 23826 mRNA sequence 1.80782 4.07112 1.44315 2.28E−04
    Homo sapiens receptor tyrosine kinase- 1.53333 4.73839 3.05612 1.63E−02
    like orphan receptor 2 (ROR2), mRNA.
    /PROD = receptor tyrosine kinase-like
    orphan receptor
    2 /FL = gb: M97639.1
    gb: NM_004560.1
    Homo sapiens MYC-associated zinc −0.055569 2.67075 0.511292 2.63E−02
    finger protein (purine-binding
    transcription factor) (MAZ), mRNA.
    /PROD = MYC-associated zinc finger
    protein(purine-binding transcription
    factor) /FL = gb: D85131.1
    gb: NM_002383.1
    Homo sapiens cDNA FLJ30081 fis, 0.677905 3.33728 1.12499 1.12E−04
    clone BGGI12000693, weakly similar to
    POLYHOMEOTIC-PROXIMAL
    CHROMATIN PROTEIN.
    Homo sapiens elongation of very long 0.099979 3.23372 1.50802 2.59E−03
    chain fatty acids (FEN1Elo2, SUR4Elo3,
    yeast)-like 2 (ELOVL2), mRNA.
    /PROD = elongation of very long chain
    fatty acids(FEN1Elo2, SUR4Elo3,
    yeast)-like 2 /FL = gb: NM_017770.1
    Homo sapiens thyrotropin-releasing −1.65672 1.46994 −0.25839 3.02E−02
    hormone (TRH), mRNA.
    /PROD = thyrotropin-releasing hormone
    /FL = gb: NM_007117.1
    ESTs, Weakly similar to A46302 PTB- 0.894051 4.21835 2.69535 1.57E−02
    associated splicing factor, long form
    (H. sapiens)
    Human MLC1emb gene for embryonic 1.3489 5.11354 4.03961 1.61E−02
    myosin alkaline light chain, promoter and
    exon 1
    Homo sapiens microseminoprotein, −2.37904 1.3101 0.169915 2.46E−02
    beta-(MSMB), mRNA.
    /PROD = microseminoprotein, beta-
    /FL = gb: NM_002443.1
    Homo sapiens cDNA FLJ11390 fis, −1.12944 2.02889 0.364373 0.00E+00
    clone HEMBA1000561, weakly similar to
    ZINC FINGER PROTEIN 91.
    ESTs, Weakly similar to ALU8_HUMAN 2.76608 6.07949 4.57766 1.93E−02
    ALU SUBFAMILY SX SEQUENCE
    CONTAMINATION WARNING ENTRY
    (H. sapiens)
    Homo sapiens cDNA FLJ13810 fis, −2.92389 0.51764 −0.85322 8.08E−03
    clone THYRO1000279
    Homo sapiens, aminolevulinate, delta-, 1.3099 4.44525 2.76918 7.69E−03
    dehydratase, clone MGC: 5057, mRNA,
    complete cds. /PROD = aminolevulinate,
    delta-, dehydratase /FL = gb: BC000977.1
    gb: M13928.1 gb: NM_000031.1
    Homo sapiens kidney-specific 1.91929 5.08917 3.44968 6.91E−03
    membrane protein NX-17 mRNA,
    complete cds. /PROD = kidney-specific
    membrane protein NX-17
    /FL = gb: AF229179.1
    Homo sapiens alpha 2,8- −1.10548 2.36359 1.05469 4.63E−03
    sialyltransferase mRNA, complete cds.
    /PROD = alpha 2,8-sialyltransferase
    /FL = gb: L43494.1 gb: D26360.1
    gb: L32867.1 gb: NM_003034.1
    tyrosine 3-monooxygenasetryptophan 5- 2.98486 6.17918 4.60758 8.01E−03
    monooxygenase activation protein, eta
    polypeptide
    Homo sapiens cardiac ankyrin repeat −0.002291 3.35645 1.98688 3.44E−02
    protein (CARP), mRNA. /PROD = cardiac
    ankyrin repeat protein
    /FL = gb: NM_014391.1
    Homo sapiens porcupine (MG61), 1.51754 4.96311 3.68103 9.86E−03
    mRNA. /PROD = porcupine
    /FL = gb: AF317059.1 gb: AF317058.1
    gb: NM_022825.1
    collagen, type V, alpha 1 1.5875 3.98844 1.66569 1.17E−03
    /FL = gb: D90279.1 gb: NM_000093.1
    gb: M76729.1
    Homo sapiens forkhead box F2 −1.54469 1.02305 −1.11517 6.95E−03
    (FOXF2), mRNA. /PROD = forkhead box
    F2 /FL = gb: U13220.1 gb: NM_001452.1
    PTPRF interacting protein, binding −1.92021 3.74378 2.77277 2.10E−02
    protein 2 (liprin beta 2)
    Homo sapiens PRO1957 mRNA, 1.68941 5.30311 4.22705 1.43E−02
    complete cds. /PROD = PRO1957
    /FL = gb: AF116676.1
    Homo sapiens hypothetical protein 0.564613 5.98527 5.24692 2.85E−02
    FLJ23514 (FLJ23514), mRNA.
    /PROD = hypothetical protein FLJ23514
    /FL = gb: NM_021827.1
    Homo sapiens mRNA; cDNA −1.80897 1.69722 0.523009 2.47E−02
    DKFZp586G2120 (from clone
    DKFZp586G2120); complete cds.
    /PROD = hypothetical protein
    /FL = gb: AL136924.1
    Cluster Incl. L37033: Human FK-506 0.615619 3.478 1.66132 1.54E−02
    binding protein homologue (FKBP38)
    mRNA, complete cds /cds = (140, 1207)
    /gb = L37033 /gi = 965469 /ug = Hs.173464
    /len = 1613
    ESTs −0.004009 3.63657 2.61352 4.29E−02
    Homo sapiens apolipoprotein C-I 3.24954 6.71073 5.50997 2.16E−02
    (APOC1), mRNA. /PROD = apolipoprotein
    C-I precursor /FL = gb: NM_001645.2
    ESTs 1.38635 4.93112 3.83641 1.39E−02
    Homo sapiens cDNA FLJ34035 fis, −3.58002 −0.48718 −2.00751 1.46E−02
    clone FCBBF2004788.
    Homo sapiens, clone IMAGE: 3509274, 0.493686 2.94923 0.794513 1.82E−02
    mRNA, partial cds
    Homo sapiens putative sterol reductase 1.66857 4.3767 2.47833 5.37E−04
    SR-1 (TM7SF2) mRNA, complete cds.
    /PROD = putative sterol reductase SR-1
    /FL = gb: AF096304.1
    nuclear factor of kappa light polypeptide 2.91463 6.48938 5.47812 1.97E−02
    gene enhancer in B-cells inhibitor, alpha
    /FL = gb: NM_020529.1 gb: BC002601.1
    gb: BC004983.1 gb: M69043.1
    hypothetical protein FLJ12666 0.215767 3.10249 1.40459 1.91E−02
    Homo sapiens ectodermal dysplasia 1, 0.796627 4.22895 3.07833 1.01E−02
    anhidrotic (ED1), mRNA.
    /PROD = ectodermal dysplasia 1,
    anhidrotic /FL = gb: AF060999.1
    gb: NM_001399.1 gb: AF040628.1
    gb: AF061189.1
    glycoprotein M6A /FL = gb: D49958.1 0.274378 3.59045 2.3278 2.64E−02
    ESTs 1.04714 3.87608 2.12752 3.75E−02
    ESTs −1.48489 1.371 −0.35043 1.88E−03
    ESTs, Weakly similar to KIAA1330 −0.925232 2.41149 1.18187 2.65E−03
    protein (H. sapiens)
    putative 47 kDa protein 0.156484 2.78771 0.861726 2.60E−02
    Homo sapiens hypothetical protein −0.595622 4.84785 3.94683 3.34E−02
    FLJ32835 (FLJ32835), mRNA.
    /FL = gb: NM_152506.1
    Homo sapiens GS1999full mRNA, 0.137029 2.75189 0.849942 0.00E+00
    complete cds. /FL = gb: AB048286.1
    Homo sapiens hexabrachion (tenascin 3.96862 5.66572 2.85391 1.39E−02
    C, cytotactin) (HXB), mRNA.
    /PROD = hexabrachion (tenascin C,
    cytotactin) /FL = gb: M55618.1
    gb: NM_002160.1
    Homo sapiens Pig10 (PIG10) mRNA, 0.129454 2.86013 1.09765 1.16E−03
    complete cds. /PROD = Pig10
    /FL = gb: AF059611.1 gb: AF010314.1
    gb: NM_003633.1 gb: BC000418.1
    gb: AF005381.1
    Homo sapiens annexin A6 (ANXA6), 3.89026 6.43752 4.49981 8.81E−04
    transcript variant 1, mRNA.
    /PROD = annexin VI isoform 1
    /FL = gb: D00510.1 gb: J03578.1
    gb: NM_001155.2
    Homo sapiens c-mer proto-oncogene 1.78994 4.92901 3.61665 1.84E−02
    tyrosine kinase (MERTK), mRNA.
    /PROD = c-mer proto-oncogene tyrosine
    kinase /FL = gb: NM_006343.1
    gb: U08023.1
    Homo sapiens caspase-like apoptosis 0.434199 5.04863 5.2167 2.46E−02
    regulatory protein 2 (clarp) mRNA,
    alternatively spliced, complete cds.
    /PROD = caspase-like apoptosis
    regulatory protein 2 /FL = gb: AF005775.1
    Human embryonic myosin alkali light 1.94171 5.24959 4.11475 2.11E−02
    chain (MLC1) mRNA, complete cds.
    /FL = gb: M36172.1 gb: M24121.1
    gb: NM_002476.1
    Homo sapiens phosphatidylinositol-4- 1.34876 4.12788 2.46657 3.66E−03
    phosphate 5-kinase, type I, beta
    (PIP5K1B), mRNA.
    /PROD = phosphatidylinositol-4-
    phosphate 5-kinase, typeI, beta
    /FL = gb: NM_003558.1
    Homo sapiens mRNA; cDNA 1.36443 4.34866 2.94283 2.92E−03
    DKFZp434E082 (from clone
    DKFZp434E082)
    Homo sapiens dystrophin (muscular 1.40427 4.41881 3.04441 1.83E−02
    dystrophy, Duchenne and Becker types),
    includes DXS142, DXS164, DXS206,
    DXS230, DXS239, DXS268, DXS269,
    DXS270, DXS272 (DMD), transcript
    variant Dp427p2, mRNA.
    /PROD = dystrophin Dp427p2 isoform
    /FL = gb: NM_004010.1
    Homo sapiens, clone MGC: 14801, 2.03928 3.96694 1.51059 5.65E−04
    mRNA, complete cds. /PROD = Unknown
    (protein for MGC: 14801)
    /FL = gb: BC005997.1
    Homo sapiens hypothetical protein −0.290244 2.2418 0.40157 6.04E−03
    BC017868 (LOC159091), mRNA.
    /FL = gb: BC017868.1 gb: NM_138819.1
    ESTs, Highly similar to AF229172 1 −0.475471 1.94826 0.002417 5.28E−03
    class III myosin (H. sapiens)
    Homo sapiens solute carrier family 9 0.047487 2.80601 1.19645 2.80E−03
    (sodiumhydrogen exchanger), isoform 5
    (SLC9A5), mRNA. /PROD = solute carrier
    family 9 (sodiumhydrogenexchanger),
    isoform 5 /FL = gb: AF111173.1
    gb: NM_004594.1
    Homo sapiens F37Esophageal cancer- −0.128814 3.17069 2.10422 1.54E−02
    related gene-coding leucine-zipper motif
    (FEZ1), mRNA. /PROD = F37Esophageal
    cancer-related gene-codingleucine-
    zipper motif /FL = gb: AF123659.1
    gb: NM_021020.1
    Homo sapiens deiodinase, −0.136937 5.08366 4.22897 1.41E−02
    iodothyronine, type III (DIO3), mRNA.
    /PROD = thyroxine deiodinase type III
    /FL = gb: NM_001362.1 gb: S79854.1
    Homo sapiens insulin-like growth factor 2.6126 5.13945 3.30402 9.72E−04
    binding protein 6 (IGFBP6), mRNA.
    /PROD = insulin-like growth factor binding
    protein
    6 /FL = gb: BC005007.1
    gb: M62402.1 gb: BC003507.1
    gb: NM_002178.1
    Homo sapiens U2 small nuclear −0.821533 1.81533 0.096512 4.72E−02
    ribonucleoprotein auxiliary factor (65 kD)
    (U2AF65), mRNA. /PROD = U2 small
    nuclear ribonucleoprotein auxiliaryfactor
    (65 kD) /FL = gb: NM_007279.1
    Homo sapiens hypothetical protein −1.11464 1.81375 0.394686 9.09E−03
    DKFZp434F0318 (DKFZP434F0318),
    mRNA. /PROD = hypothetical protein
    DKFZp434F0318 /FL = gb: NM_030817.1
    Homo sapiens BACE mRNA for beta- 0.48553 3.13284 1.44588 4.99E−04
    site APP cleaving enzyme I-476,
    complete cds. /PROD = beta-site APP
    cleaving enzyme I-476
    /FL = gb: AB050436.1
    ESTs, Weakly similar to ALUC_HUMAN 1.07951 4.06134 2.7193 2.11E−02
    !!!! ALU CLASS C WARNING ENTRY !!!
    (H. sapiens)
    Homo sapiens mRNA for KIAA0876 −0.432037 1.97736 0.073246 3.37E−02
    protein, partial cds. /PROD = KIAA0876
    protein
    Homo sapiens mRNA for protein- 0.160986 2.75713 1.04172 2.11E−02
    tyrosine kinase, complete cds.
    /PROD = protein-tyrosine kinase
    /FL = gb: U05682.1 gb: D17517.1
    gb: D50479.1
    KIAA0418 gene product −0.677643 1.83134 0.034385 1.45E−04
    Homo sapiens phosphofructokinase, 2.039 4.51747 2.70388 1.28E−03
    liver (PFKL), mRNA.
    /PROD = phosphofructokinase, liver
    /FL = gb: NM_002626.1 gb: BC004920.1
    gb: X15573.1
    Homo sapiens enolase 2, (gamma, 2.60624 5.37458 3.92621 3.17E−03
    neuronal) (ENO2), mRNA.
    /PROD = enolase 2, (gamma, neuronal)
    /FL = gb: NM_001975.1 gb: BC002745.1
    gb: M22349.1
    Homo sapiens AD036 mRNA, complete −0.503334 2.65787 1.60278 3.61E−02
    cds. /PROD = AD036 /FL = gb: AF260333.1
    ESTs −2.07933 1.00157 −0.1307 2.39E−02
    Homo sapiens keratin 19 (KRT19), 3.80582 6.96535 5.91937 2.11E−02
    mRNA. /PROD = keratin 19
    /FL = gb: NM_002276.1 gb: BC002539.1
    pleiomorphic adenoma gene-like 1 −0.646793 1.88692 0.221481 4.21E−03
    /FL = gb: U72621.3
    Homo sapiens, Similar to lipase protein, −1.16073 1.9994 0.961706 1.89E−02
    clone MGC: 2843, mRNA, complete cds.
    /PROD = Similar to lipase protein
    /FL = gb: NM_020676.1 gb: BC001698.1
    Cluster Incl. AB002344: Human mRNA 2.15679 4.91332 3.47797 7.39E−03
    for KIAA0346 gene, partial cds
    /cds = (0, 4852) /gb = AB002344
    /gi = 2280479 /ug = Hs.103915 /len = 6121
    Cluster Incl. N80935: zb07g06.s1 Homo 2.12419 4.48236 2.66555 1.50E−02
    sapiens cDNA, 3 end /clone = IMAGE-
    301402 /clone_end = 3 /gb = N80935
    /gi = 1243636 /ug = Hs.22483 /len = 527
    tryptase, alpha 2.99108 5.46095 3.76442 1.31E−03
    ESTs, Weakly similar to Z132_HUMAN 1.04865 3.65906 2.12166 2.43E−02
    ZINC FINGER PROTEIN 13 (H. sapiens)
    Homo sapiens core histone 1.67293 4.73906 3.66212 3.62E−02
    macroH2A2.2 (MACROH2A2), mRNA.
    /PROD = core histone macroH2A2.2
    /FL = gb: AF151534.1 gb: NM_018649.1
    ESTs −0.146413 2.69334 1.39451 1.02E−02
    Human rho GDI mRNA, complete cds. 0.019358 1.75075 −0.65155 1.85E−02
    /PROD = human rho GDI
    /FL = gb: M97579.1 gb: D13989.1
    gb: NM_004309.1
    heat shock 90 kD protein 1, alpha 1.69225 4.20576 2.60887 2.10E−02
    Homo sapiens BTB (POZ) domain 1.10085 3.7628 2.32464 1.85E−02
    containing 2 (BTBD2), mRNA.
    /PROD = BTB (POZ) domain containing 2
    /FL = gb: NM_017797.1
    Homo sapiens mandaselin long form −0.148053 2.58361 1.2296 1.57E−02
    mRNA, complete cds.
    /PROD = mandaselin long form
    /FL = gb: AY048775.1
    signal transducer and activator of −0.837261 1.90254 0.560225 8.83E−04
    transcription 3 (acute-phase response
    factor)
    Homo sapiens lymphocyte antigen 6 0.866364 4.91832 4.92642 2.78E−02
    complex, locus E (LY6E), mRNA.
    /PROD = lymphocyte antigen 6 complex,
    locus E /FL = gb: U42376.1
    gb: NM_002346.1 gb: U56145.1
    ESTs −1.24632 1.09042 −0.61316 4.05E−02
    Homo sapiens, clone IMAGE: 4047715, −2.50816 1.81 1.52693 2.63E−02
    mRNA.
    Cluster Incl. AB002344: Human mRNA 1.89578 4.61333 3.31717 1.12E−02
    for KIAA0346 gene, partial cds
    /cds = (0, 4852) /gb = AB002344
    /gi = 2280479 /ug = Hs.103915 /len = 6121
    Homo sapiens singed (Drosophila)-like 4.23317 6.74774 5.25403 5.12E−03
    (sea urchin fascin homolog like) (SNL),
    mRNA. /PROD = singed (Drosophila)-like
    (sea urchin fascinhomolog like)
    /FL = gb: BC000521.1 gb: NM_003088.1
    gb: U03057.1 gb: U09873.1
    Homo sapiens mRNA; cDNA −2.72593 1.58998 1.27994 4.54E−02
    DKFZp586L0120 (from clone
    DKFZp586L0120).
    Homo sapiens mitogen-activated protein 0.647994 3.46459 2.28644 6.65E−03
    kinase 10 (MAPK10), mRNA.
    /PROD = mitogen-activated protein kinase
    10 /FL = gb: U07620.1 gb: U34819.1
    gb: U34820.1 gb: NM_002753.1
    Homo sapiens transmembrane tyrosine −0.006089 2.96035 1.93237 7.69E−03
    kinase mRNA, complete cds.
    /PROD = tyrosine kinase
    /FL = gb: L08961.1
    Homo sapiens mRNA; cDNA 2.83663 5.30784 3.79206 6.22E−03
    DKFZp762H185 (from clone
    DKFZp762H185)
    tyrosine 3-monooxygenasetryptophan 5- 3.48271 6.19368 4.92556 2.11E−02
    monooxygenase activation protein, eta
    polypeptide
    Homo sapiens mRNA; cDNA 0.581419 3.38088 2.21428 3.89E−02
    DKFZp434K0621 (from clone
    DKFZp434K0621); partial cds
    ESTs, Weakly similar to ALUF_HUMAN −1.03247 1.75349 0.587598 2.11E−02
    !!!! ALU CLASS F WARNING ENTRY !!!
    (H. sapiens)
    tudor repeat associator with PCTAIRE 2 2.15771 4.69127 3.27451 2.63E−02
    Homo sapiens mRNA; cDNA 4.49405 7.38448 6.33474 1.39E−02
    DKFZp564K1672 (from clone
    DKFZp564K1672); partial cds.
    /PROD = hypothetical protein
    ESTs −0.184212 2.15367 0.554696 1.15E−02
    Homo sapiens bHLH factor Hes4 0.47816 3.40725 2.40001 3.16E−02
    (LOC57801), mRNA. /PROD = bHLH
    factor Hes4 /FL = gb: NM_021170.1
    gb: AB048791.1
    Homo sapiens guanylate cyclase −0.714216 1.95339 0.684983 3.38E−03
    activator 1B (retina) (GUCA1B), mRNA.
    /PROD = guanylate cyclase activator 1B
    (retina) /FL = gb: M95174.1
    gb: NM_002098.1 gb: M97496.1
    KIAA0918 protein −1.39708 0.961939 −0.57499 1.40E−02
    Homo sapiens mRNA for KIAA1161 0.781796 3.32846 1.98143 6.60E−03
    protein, partial cds. /PROD = KIAA1161
    protein
    Homo sapiens, Similar to B9 protein, 1.43881 3.80457 2.29205 8.62E−05
    clone MGC: 11339, mRNA, complete
    cds. /PROD = Similar to B9 protein
    /FL = gb: BC002944.1
    hypothetical protein FLJ12666 2.34087 5.11201 4.02837 1.97E−02
    Homo sapiens FLICE-like inhibitory 0.135783 4.45631 4.92758 1.96E−02
    protein short form mRNA, complete cds.
    /PROD = FLICE-like inhibitory protein
    short form /FL = gb: U97075.1
    Homo sapiens olfactory receptor, family −0.309574 2.08723 0.636487 4.36E−03
    52, subfamily A, member 1 (OR52A1),
    mRNA. /PROD = olfactory receptor, family
    52, subfamily A, member 1
    /FL = gb: NM_012375.1
    Human DNA sequence from clone RP4- 2.82768 5.54517 4.43909 2.04E−03
    781L3 on chromosome 1p34.3-36.11
    Contains a pseudogene similar to
    IFITM3 (interferon
    inducedntransmembrane protein 3 (1-
    8U)), STSs and GSSs
    Homo sapiens KIAA0127 gene product 2.05236 4.43794 3.00216 3.12E−02
    (KIAA0127), mRNA. /PROD = KIAA0127
    gene product /FL = gb: D50917.1
    gb: NM_014755.1
    Homo sapiens clone HB-2 mRNA −0.503949 2.27896 1.25171 3.01E−02
    sequence
    hypothetical protein FLJ23091 −0.76092 3.70916 3.04154 2.45E−02
    ESTs −0.403665 2.01868 0.643615 5.11E−04
    ESTs 2.14401 4.70353 3.47114 3.15E−03
    Homo sapiens regulator of G protein 3.0091 5.37511 3.95132 1.29E−03
    signalling 5 (RGS5) mRNA, complete
    cds. /PROD = regulator of G protein
    signalling 5 /FL = gb: AF493929.1
    endothelin receptor type A −1.83423 2.54948 1.95432 1.51E−02
    /FL = gb: NM_001957.1 gb: L06622.1
    H. sapiens skeletal embryonic myosin 0.690058 3.3896 2.36018 3.27E−02
    light chain 1 (MLC1) mRNA.
    /PROD = myosin light chain 1
    Homo sapiens Kruppel-like factor 8 0.326119 2.825 1.63929 3.92E−02
    (KLF8), mRNA. /PROD = Kruppel-like
    factor
    8 /FL = gb: U28282.1
    gb: NM_007250.1
    hypothetical protein DKFZp434F2322 0.070576 2.58185 1.42121 5.81E−03
    Homo sapiens transcription factor 2, −1.12537 3.45355 2.54347 1.15E−02
    hepatic; LF-B3; variant hepatic nuclear
    factor (TCF2), transcript variant a,
    mRNA. /PROD = transcription factor 2,
    isoform a /FL = gb: NM_000458.1
    H2A histone family, member X 1.37085 2.48515 −0.04057 1.34E−02
    Homo sapiens H1 histone family, 3.21701 5.68105 4.50969 4.29E−03
    member X (H1FX), mRNA. /PROD = H1
    histone family, member X
    /FL = gb: D64142.1 gb: BC000426.1
    gb: NM_006026.1
    ESTs, Weakly similar to KIAA1399 −0.831241 2.82364 2.79997 4.18E−02
    protein (H. sapiens)
    Homo sapiens PNAS-145 mRNA, 2.84962 5.38084 4.2868 8.61E−03
    complete cds. /PROD = PNAS-145
    /FL = gb: U03105.1 gb: NM_006813.1
    gb: AF279899.1
    Homo sapiens erythrocyte membrane −0.306603 2.11391 0.923806 4.81E−03
    protein band 4.9 (dematin) (EPB49),
    mRNA. /PROD = erythrocyte membrane
    protein band 4.9 (dematin)
    /FL = gb: NM_001978.1 gb: U28389.1
    ESTs 0.783545 3.23146 2.07738 2.13E−02
    ESTs 1.17515 3.61815 2.46265 1.56E−03
    nudix (nucleoside diphosphate linked 2.09472 5.82355 5.68859 3.63E−02
    moiety X)-type motif 4
    Novel human mRNA from chromosome −0.533665 3.44679 3.05803 3.99E−02
    22. /PROD = hypothetical protein
    Homo sapiens partial mRNA for putative 1.55224 3.89366 2.67866 1.53E−03
    nuclear factor. /PROD = putative nuclear
    factor /FL = gb: NM_017688.1
    Homo sapiens BCL2adenovirus E1B 4.51142 6.99506 5.92755 2.25E−03
    19 kD-interacting protein 3 (BNIP3),
    nuclear gene encoding mitochondrial
    protein, mRNA.
    /PROD = BCL2adenovirus E1B 19 kD-
    interacting protein 3 /FL = gb: AF002697.1
    gb: NM_004052.2 gb: U15174.1
    Homo sapiens bone morphogenetic −0.6099 1.76351 0.614791 6.46E−03
    protein 5 (BMP5), mRNA. /PROD = bone
    morphogenetic protein 5
    /FL = gb: M60314.1 gb: NM_021073.1
    Homo sapiens nuclear receptor −0.213822 4.14059 3.29782 3.42E−02
    subfamily 0, group B, member 1
    (NR0B1), mRNA. /PROD = adrenal
    hypoplasia protein /FL = gb: NM_000475.2
    Homo sapiens mRNA; cDNA 1.76846 4.196 3.11284 1.23E−02
    DKFZp434P228 (from clone
    DKFZp434P228)
    Homo sapiens pilin-like transcription 3.14467 5.51066 4.38212 3.80E−03
    factor (PILB), mRNA. /PROD = pilin-like
    transcription factor /FL = gb: AF122004.1
    gb: NM_012228.1
    Homo sapiens complement component −1.89639 1.65637 1.59643 1.74E−02
    5 (C5), mRNA. /PROD = complement
    component
    5 /FL = gb: M57729.1
    gb: NM_001735.1
    Homo sapiens adaptor-related protein −0.208283 2.17404 1.07398 1.39E−02
    complex 3, beta 2 subunit (AP3B2),
    mRNA. /PROD = adaptor-related protein
    complex
    3, beta 2subunit
    /FL = gb: AF022152.1 gb: NM_004644.1
    gb: U37673.1
    Homo sapiens dynein, axonemal, light 0.458271 2.88755 1.8494 3.66E−03
    polypeptide 4 (DNAL4), mRNA.
    /PROD = dynein, axonemal, light
    polypeptide
    4 /FL = gb: BC002968.1
    gb: NM_005740.1
    Homo sapiens, Similar to RIKEN cDNA 0.087468 2.45867 1.42701 3.35E−03
    C330013D18 gene, clone MGC: 11226,
    mRNA, complete cds
    Homo sapiens cDNA: FLJ22731 fis, −2.30948 1.62817 2.24143 4.15E−02
    clone HSI15841.
    ESTs 0.532996 4.6172 3.79228 2.76E−02
    Homo sapiens CXCR4 gene encoding 3.11086 7.24917 6.36084 3.13E−02
    receptor CXCR4
    stanniocalcin
    1 2.48686 6.20245 5.70081 4.94E−02
    KIAA0761 protein −2.09591 1.57935 1.05907 1.86E−02
    ESTs −1.16374 2.24964 2.52108 3.83E−02
    Homo sapiens cDNA FLJ36116 fis, 3.02587 6.7146 6.15456 3.88E−02
    clone TESTI2022338.
    Homo sapiens, clone MGC: 24252 −1.53346 2.53854 1.59251 3.91E−03
    IMAGE: 3932604, mRNA, complete cds.
    /PROD = Unknown (protein for
    MGC: 24252) /FL = gb: BC014364.1
    Homo sapiens cDNA FLJ13392 fis, −1.90184 1.76129 1.16856 1.47E−02
    clone PLACE1001280
    Homo sapiens hypothetical protein 1.70617 5.11419 4.73391 2.71E−02
    FLJ12538 similar to ras-related protein
    RAB17 (FLJ12538), mRNA.
    /PROD = hypothetical protein FLJ12538
    similar toras-related protein RAB17
    /FL = gb: AL136645.1 gb: NM_022449.1
    Human DNA sequence from clone −0.889422 2.90076 2.11955 3.52E−02
    RP11-446H13 on chromosome 10.
    Contains the 3 end of the gene for a
    novel protein similar to KIAA1059
    (ortholog of mouse VPS10 domain
    receptor protein SORCS), an RPL23A
    (60S ribosmal protein 23A) pseudogene,
    ESTs, STSs an
    Homo sapiens calmegin (CLGN), −0.104278 2.97817 3.11092 1.89E−02
    mRNA. /PROD = calmegin
    /FL = gb: NM_004362.1 gb: D86322.1
    Homo sapiens testican 3 (HSAJ1454), −2.81226 0.7939 1.55068 3.99E−02
    mRNA. /PROD = testican 3
    /FL = gb: NM_016950.1 gb: BC000460.1
    gb: BC003017.1
    Homo sapiens gelsolin (amyloidosis, 1.6198 4.54047 4.45008 1.07E−02
    Finnish type) (GSN), mRNA.
    /PROD = gelsolin (amyloidosis, Finnish
    type) /FL = gb: NM_000177.1
    Homo sapiens endothelin receptor type 0.763007 4.05983 3.55203 3.99E−02
    A (EDNRA), mRNA. /PROD = endothelin
    receptor type A /FL = gb: NM_001957.1
    gb: L06622.1
    Homo sapiens cDNA: FLJ22808 fis, −0.367449 2.7584 2.41394 4.31E−02
    clone KAIA2925.
    Homo sapiens hypothetical protein 1.59498 4.6335 4.89157 4.85E−02
    FLJ10312 (FLJ10312), mRNA.
    /PROD = hypothetical protein FLJ10312
    /FL = gb: NM_030672.1
    Homo sapiens lipase mRNA, complete −2.2131 1.0208 0.55849 3.57E−02
    cds. /PROD = lipase /FL = gb: AF225418.1
    Homo sapiens clone FLC1492 PRO3121 −0.300037 −0.46894 −3.39203 1.80E−02
    mRNA, complete cds. /PROD = PRO3121
    /FL = gb: AF130082.1
    protease, serine, 4 (trypsin 4, brain) −0.345569 2.86256 3.35886 4.65E−02
    Homo sapiens ret finger protein-like 2 −2.15511 1.1797 0.503917 1.94E−02
    (RFPL2), mRNA. /PROD = ret finger
    protein-like 2 /FL = gb: NM_006605.1
    Homo sapiens beta3GalNAcT-1 mRNA −2.73257 0.21269 −0.0984 2.48E−02
    for globoside synthase, complete cds,
    clone: type 2. /PROD = globoside
    synthase /FL = gb: AB050856.1
    Homo sapiens hypothetical protein −3.34565 −0.42738 −0.71255 3.07E−02
    FLJ11155 (FLJ11155), mRNA.
    /PROD = hypothetical protein FLJ11155
    /FL = gb: NM_018342.1
    Homo sapiens, dual specificity 2.11487 5.16681 5.60057 3.11E−02
    phosphatase 4, clone MGC: 3713,
    mRNA, complete cds. /PROD = dual
    specificity phosphatase
    4
    /FL = gb: NM_001394.2 gb: BC002671.1
    gb: U48807.1 gb: U21108.1
    Homo sapiens MAD (mothers against 0.917516 4.29847 3.5309 2.44E−02
    decapentaplegic, Drosophila) homolog 6
    (MADH6), mRNA. /PROD = MAD
    (mothers against
    decapentaplegic, Drosophila) homolog 6
    /FL = gb: U59914.1 gb: NM_005585.1
    secreted frizzled-related protein 1 1.64933 4.41286 4.21799 4.12E−02
    /FL = gb: AF056087.1 gb: NM_003012.2
    gb: AF017987.1 gb: AF001900.1
    Homo sapiens secreted apoptosis 3.18446 5.9316 5.74251 3.31E−02
    related protein 2 (SARP2) mRNA,
    complete cds. /PROD = secreted
    apoptosis related protein 2
    /FL = gb: AF056087.1 gb: NM_003012.2
    gb: AF017987.1 gb: AF001900.1
    KIAA0882 protein 2.05587 4.63577 4.67451 4.89E−02
    Homo sapiens nudix (nucleoside 3.91519 6.54632 6.42767 4.63E−02
    diphosphate linked moiety X)-type motif
    4 (NUDT4), mRNA. /PROD = nudix
    (nucleoside diphosphate linked
    moietyX)-type motif 4
    /FL = gb: NM_019094.1 gb: AF191653.1
    gb: AF191649.1 gb: AF191650.1
    Homo sapiens cDNA FLJ31061 fis, −1.30452 2.06218 1.20749 1.72E−02
    clone HSYRA2000927.
    Homo sapiens diphosphoinositol 2.62612 5.53515 5.12935 3.96E−02
    polyphosphate phosphohydrolase type 2
    beta (NUDT4) mRNA, complete cds.
    /PROD = diphosphoinositol
    polyphosphate phosphohydrolasetype 2
    beta /FL = gb: NM_019094.1
    gb: AF191653.1 gb: AF191649.1
    gb: AF191650.1
    Homo sapiens titin (TTN), mRNA. −0.466154 2.46856 2.00309 4.18E−03
    /PROD = titin /FL = gb: NM_003319.1
    carboxypeptidase E 2.72316 5.80072 5.15668 2.99E−02
    /FL = gb: NM_001873.1
    ESTs −1.80443 0.692933 0.613229 1.46E−03
    Homo sapiens hypothetical protein −1.60435 1.6149 2.4204 2.10E−02
    FLJ39502 (FLJ39502), mRNA.
    /FL = gb: NM_173648.1
    Homo sapiens renal tumor antigen −0.305406 2.4556 2.09136 3.30E−02
    (RAGE), mRNA. /PROD = renal tumor
    antigen /FL = gb: NM_014226.1
    gb: AB022694.1
    T-box 3 (ulnar mammary syndrome) −1.70952 1.31576 0.679584 1.29E−02
    Homo sapiens mitogen-activated protein −1.14241 1.82177 1.23928 2.48E−02
    kinase kinase kinase 8 (MAP3K8),
    mRNA. /PROD = mitogen-activated
    protein kinase kinase kinase8
    /FL = gb: NM_005204.1 gb: D14497.1
    Homo sapiens enoyl-Coenzyme A, 0.226733 3.18231 2.59938 6.22E−03
    hydratase3-hydroxyacyl Coenzyme A
    dehydrogenase (EHHADH), nuclear
    gene encoding mitochondrial protein,
    mRNA. /PROD = enoyl-Coenzyme A,
    hydratase3-hydroxyacylCoenzyme A
    dehydrogenase /FL = gb: NM_001966.1
    gb: L07077.1
    ESTs 1.65525 3.9984 3.98375 1.97E−02
    Homo sapiens secreted frizzled-related 3.91678 7.18429 6.24313 2.35E−02
    protein 1 (SFRP1), mRNA.
    /PROD = secreted frizzled-related protein
    1 /FL = gb: AF056087.1 gb: NM_003012.2
    gb: AF017987.1 gb: AF001900.1
    ESTs 2.28659 5.21675 4.59884 3.11E−02
    Homo sapiens selenoprotein P, plasma, 2.73332 5.13625 5.04138 1.75E−02
    1 (SEPP1), mRNA.
    /PROD = selenoprotein P precursor
    /FL = gb: NM_005410.1
    hypothetical protein FLJ12838 2.21522 5.4785 4.50835 4.55E−02
    /FL = gb: NM_024641.1
    Homo sapiens mRNA; cDNA −2.59761 0.092924 −0.31569 4.19E−02
    DKFZp761M1216 (from clone
    DKFZp761M1216)
    Homo sapiens KPL1 (KPL1) mRNA, −0.08886 2.51294 2.15967 1.97E−02
    complete cds. /PROD = KPL1
    /FL = gb: AF081583.1 gb: U89715.1
    gb: NM_021200.1
    Homo sapiens, synovial sarcoma, X −1.57209 1.39869 2.12662 6.84E−03
    breakpoint 1, clone MGC: 5162, mRNA,
    complete cds. /PROD = synovial
    sarcoma, X breakpoint 1
    /FL = gb: BC001003.2 gb: NM_005635.1
    Homo sapiens, Similar to testican 3, −2.778 0.563149 1.66935 1.87E−02
    clone MGC: 8506, mRNA, complete cds.
    /PROD = Similar to testican 3
    /FL = gb: NM_016950.1 gb: BC000460.1
    gb: BC003017.1
    ESTs 0.502949 3.0682 2.72874 2.02E−02
    Homo sapiens cDNA FLJ32963 fis, 1.49644 3.87336 3.68478 6.89E−03
    clone TESTI2008405.
    triadin /FL = gb: U18985.1 −1.61408 0.713083 0.566175 1.87E−02
    gb: NM_006073.1
    Homo sapiens adlican mRNA, complete 0.872786 3.20404 3.02406 4.21E−02
    cds. /PROD = adlican
    /FL = gb: AF245505.1
    Human midkine mRNA, complete cds. 3.55275 5.93819 5.68363 1.89E−02
    /PROD = midkine /FL = gb: NM_002391.1
    gb: M69148.1
    Homo sapiens, synovial sarcoma, X −0.806785 1.90972 2.5215 4.17E−02
    breakpoint 4, clone MGC: 12411, mRNA,
    complete cds. /PROD = synovial
    sarcoma, X breakpoint 4
    /FL = gb: BC005325.1
    ESTs −3.61193 −0.9408 −1.53439 2.43E−03
    ESTs −0.619714 1.93617 1.44843 4.14E−02
    ESTs 0.446983 3.14315 2.48676 1.98E−02
    Homo sapiens flavin containing −0.476203 1.99948 1.55491 3.73E−02
    monooxygenase 5 (FMO5), mRNA.
    /PROD = flavin containing
    monooxygenase
    5 /FL = gb: L37080.1
    gb: NM_001461.1
    Homo sapiens aminopeptidase A −0.758987 2.07596 1.20831 3.00E−02
    mRNA, complete cds.
    /PROD = aminopeptidase A
    /FL = gb: L12468.1 gb: NM_001977.1
    gb: L14721.1
    Homo sapiens hypothetical protein −0.713164 2.03145 1.24823 2.17E−03
    DKFZp761H1710 (DKFZP761H1710),
    mRNA. /PROD = hypothetical protein
    DKFZp761H1710 /FL = gb: NM_031297.1
    ESTs −1.94194 0.608912 0.013238 1.72E−02
    Homo sapiens sialyltransferase 8 (alpha- −1.07243 1.83889 0.855205 2.16E−02
    2,8-polysialytransferase) D (SIAT8D),
    mRNA. /PROD = sialyltransferase 8
    (alpha-2,8-polysialytransferase) D
    /FL = gb: NM_005668.1 gb: L41680.1
    Homo sapiens, clone IMAGE: 5194204, −2.13256 0.383746 1.02639 4.55E−02
    mRNA.
    hypothetical protein MGC4342 −0.403018 2.01722 1.41263 1.97E−02
    /FL = gb: NM_024329.1 gb: BC003033.1
    Homo sapiens PHD finger protein 1 0.620171 2.95191 2.42296 2.32E−02
    (PHF1), transcript variant 2, mRNA.
    /PROD = PHD finger protein 1, isoform b
    /FL = gb: NM_024165.1 gb: AF052205.1
    actinin, alpha 4 0.436893 3.11051 2.23625 5.44E−03
    Homo sapiens PCTAIRE protein kinase 2.38171 4.87071 4.16957 2.90E−03
    1 (PCTK1), mRNA. /PROD = PCTAIRE
    protein kinase
    1 /FL = gb: NM_006201.1
    Human DNA sequence from clone 0.167347 2.56439 1.93736 3.93E−02
    RP11-165F24 on chromosome 9.
    Contains the 3 end of the gene for a
    novel protein (similar to Drosophila
    CG6630 and CG11376, KIAA1058, rat
    TRG), an RPL12 (60S ribosomal protein
    L12) pseudogene, ESTs, STSs, GSSs
    and a C . . .
    insulin-like growth factor binding protein 0.921068 3.60279 2.67905 4.80E−02
    3 /FL = gb: NM_000598.1
    Homo sapiens, clone IMAGE: 3840937, 3.59778 6.12936 6.92649 8.62E−05
    mRNA, partial cds. /PROD = Unknown
    (protein for IMAGE: 3840937)
    Homo sapiens phosphoglucomutase 1 4.74364 7.09524 6.43056 2.21E−02
    (PGM1), mRNA.
    /PROD = phosphoglucomutase 1
    /FL = gb: NM_002633.1 gb: BC001756.1
    gb: M83088.1
    apolipoprotein C-I 2.77701 5.23054 4.44049 3.39E−02
    Homo sapiens insulin induced gene 1 2.11462 4.44128 3.77765 3.21E−02
    (INSIG1), mRNA. /PROD = insulin
    induced gene 1 /FL = gb: NM_005542.1
    Human a6(IV) collagen (COL4A6) 0.798849 3.23623 2.41372 1.38E−03
    mRNA, complete cds. /PROD = A type IV
    collagen /FL = gb: U04845.1
    Homo sapiens mRNA for alpha 1,6- 1.31479 3.6699 2.90742 3.48E−02
    fucosyltransferase, complete cds.
    /PROD = alpha 1,6-fucosyltransferase
    /FL = gb: AB049740.2
    ESTs, Moderately similar to Six5 0.434259 2.81512 1.9556 1.23E−02
    (M. musculus)
    solute carrier family 2 (facilitated glucose 5.23156 7.63734 6.74444 1.99E−02
    transporter), member 3
    /FL = gb: NM_006931.1 gb: M20681.1
    Human acid sphingomyelinase (ASM) 1.03965 3.45689 2.52125 2.62E−02
    mRNA, complete cds. /PROD = acid
    sphingomyelinase /FL = gb: NM_000543.1
    gb: M59916.1
    ESTs, Weakly similar to unnamed 3.0131 5.41021 4.46614 4.79E−03
    protein product (H. sapiens)
    Homo sapiens cDNA FLJ33178 fis, 0.137476 −1.9718 −4.73502 2.23E−03
    clone ADRGL2002753.
    Homo sapiens, parathyroid hormone-like 2.46289 −0.34647 −1.96791 3.05E−02
    hormone, clone MGC: 14611, mRNA,
    complete cds. /PROD = parathyroid
    hormone-like hormone
    /FL = gb: BC005961.1
    Homo sapiens colon carcinoma related 2.50614 −0.01889 −1.04926 2.52E−02
    protein (LOC51159), mRNA.
    /PROD = colon carcinoma related protein
    /FL = gb: NM_016206.1 gb: AF099505.1
    KIAA0036 gene product 3.05174 −0.66523 −2.2068 2.62E−02
    Homo sapiens cDNA FLJ13536 fis, 4.27968 1.86625 1.70965 1.11E−02
    clone PLACE1006521
    Homo sapiens cDNA: FLJ22463 fis, 3.68063 1.24806 1.39728 1.42E−02
    clone HRC10126
    Homo sapiens UDP-N- 2.71164 0.270906 0.433883 1.98E−02
    acetylglucosamine: a-1,3-D-mannoside
    beta-1,4-N-
    acetylglucosaminyltransferase IV-
    homolog (HGNT-IV-H), mRNA.
    /PROD = UDP-N-acetylglucosamine: a-
    1,3-D-mannosidebeta-1,4-N-
    acetylglucosaminyltransferase IV-
    homolog /FL = gb: AB024729.1 gb: NM_0
    tissue factor pathway inhibitor 2 4.19529 1.63281 1.58393 7.53E−03
    /FL = gb: D29992.1 gb: L27624.1
    gb: NM_006528.1 gb: BC005330.1
    ESTs 2.8924 0.521234 0.762594 2.82E−02
    putative gene product −0.518738 −0.66615 1.82038 4.91E−02
    Homo sapiens mRNA for KIAA1758 2.42082 −0.03923 0.213473 1.25E−02
    protein, partial cds. /PROD = KIAA1758
    protein
    prostaglandin E receptor 4 (subtype 2.58497 0.196846 0.527172 1.42E−02
    EP4) /FL = gb: L25124.1 gb: D28472.1
    gb: NM_000958.1 gb: L28175.1
    Homo sapiens cDNA: FLJ22463 fis, 2.85068 0.473875 0.830447 1.97E−02
    clone HRC10126
    Homo sapiens heparan sulfate 3.18462 0.571052 0.709206 4.54E−02
    (glucosamine) 3-O-sulfotransferase 2
    (HS3ST2), mRNA. /PROD = heparan
    sulfate D-glucosaminyl3-O-
    sulfotransferase 2 /FL = gb: AF105375.1
    gb: AF105374.1 gb: NM_006043.1
    Homo sapiens schwannomin interacting 5.5957 3.21054 3.57915 2.11E−02
    protein 1 (SCHIP-1), mRNA.
    /PROD = schwannomin interacting protein
    1 /FL = gb: AF145713.1 gb: NM_014575.1
    Human lysyl oxidase (LOX) gene, exon 7 2.11886 −0.24936 0.138069 2.55E−03
    Human nephropontin mRNA, complete 7.82555 5.27856 5.06542 4.61E−02
    cds. /PROD = nephropontin
    /FL = gb: M83248.1
    Human mRNA for KIAA0386 gene, 3.9213 1.56305 1.13663 1.93E−02
    complete cds. /FL = gb: AB002384.1
    ESTs 3.22146 0.851355 0.404487 3.41E−03
    Human glioma pathogenesis-related 3.20187 0.502872 0.348681 1.34E−02
    protein (GliPR) mRNA, complete cds.
    /PROD = glioma pathogenesis-related
    protein /FL = gb: NM_006851.1
    gb: U16307.1
    ESTs 2.96697 0.440894 0.773495 1.97E−02
    Homo sapiens cDNA FLJ13384 fis, 5.9221 3.48419 3.9531 3.38E−02
    clone PLACE1001062, highly similar to
    Homo sapiens mRNA for lysine-
    ketoglutarate reductasesaccharopine
    dehydrogenase.
    Homo sapiens mRNA; cDNA 6.06894 3.31919 3.4798 4.97E−02
    DKFZp761I1912 (from clone
    DKFZp761I1912)
    ESTs 4.12419 1.53997 1.88239 5.68E−03
    Homo sapiens fibroblast growth factor 2 5.49297 2.64534 2.76054 4.71E−02
    (basic) (FGF2), mRNA.
    /PROD = fibroblast growth factor 2 (basic)
    /FL = gb: NM_002006.1 gb: M27968.1
    ribosomal protein L34 pseudogene 1 3.45116 2.78236 5.10812 2.42E−02
    HIV-1 rev binding protein 2 4.4245 1.47599 1.52824 2.10E−02
    H. sapiens FGF gene, exon 3 2.04849 −0.34703 −0.95745 3.33E−02
    /FL = gb: NM_000800.1 gb: M13361.1
    ESTs 2.03304 −0.9164 −0.83676 4.19E−02
    ESTs 2.87393 0.428858 1.02011 9.72E−04
    Homo sapiens clone 23700 mRNA 4.60148 1.64243 1.72052 4.74E−02
    sequence
    ESTs, Highly similar to S21424 nestin 3.08579 0.725853 1.40345 1.99E−02
    (H. sapiens)
    Homo sapiens actin, gamma 2, smooth 7.17155 4.15208 4.18551 4.72E−02
    muscle, enteric (ACTG2), mRNA.
    /PROD = actin, gamma 2 propeptide
    /FL = gb: NM_001615.2
    hypothetical protein FLJ22833 2.80785 0.480636 1.25154 2.43E−02
    Homo sapiens LIM homeobox protein 6 2.52571 −0.06036 0.459727 4.37E−02
    (LHX6), mRNA. /PROD = LIM homeobox
    protein
    6 /FL = gb: AB031041.1
    gb: NM_014368.1 gb: AL136570.1
    Homo sapiens, clone IMAGE: 5271039, 3.05576 0.189935 0.452681 4.05E−02
    mRNA.
    Homo sapiens cAMP response element- 2.90971 0.487264 1.21334 4.24E−02
    binding protein CRE-BPa
    (H_GS165L15.1), mRNA. /PROD = cAMP
    response element-binding protein CRE-
    BPa /FL = gb: NM_004904.1 gb: L05911.1
    G protein-coupled receptor 1 1.15804 −1.24345 −0.49352 2.28E−03
    /FL = gb: NM_005279.1
    Homo sapiens neuropilin (NRP) and 3.25525 0.20233 0.301354 2.83E−02
    tolloid (TLL)-like 1 (NETO1), transcript
    variant
    3, mRNA. /PROD = neuropilin-
    and tolloid-like protein 1 isoform
    3precursor /FL = gb: AF448838.1
    gb: NM_138966.2
    Homo sapiens mRNA for KIAA0559 2.37088 −0.29358 0.199028 2.81E−02
    protein, partial cds. /PROD = KIAA0559
    protein
    Human 65-kilodalton phosphoprotein 3.25522 0.620942 1.15317 2.88E−02
    (p65) mRNA, complete cds.
    /PROD = phosphoprotein p65
    /FL = gb: M22300.1 gb: NM_002298.2
    gb: J02923.1
    Human DNA sequence from clone 4.77522 2.18971 2.77459 4.04E−02
    RP11-31K16 on chromosome 9.
    Contains a snoRNA binding domain
    pseudogene, the ELAVL2 gene for
    ELAV (embryonic lethal, abnormal
    vision, Drosophila)-like 2, ESTs, STSs,
    GSSs and a CpG island
    hypothetical protein FLJ11006 1.75389 −0.66646 0.100591 7.78E−04
    Homo sapiens mRNA; cDNA 3.97344 1.32598 1.8683 3.19E−02
    DKFZp566A1046 (from clone
    DKFZp566A1046)
    ESTs 4.13356 1.60769 2.27415 1.85E−02
    H. sapiens gene from PAC 106H8. 3.05906 −0.08711 −0.13845 4.34E−02
    Human T-cell receptor rearranged beta- 3.43862 0.94127 1.64514 5.31E−03
    chain V-region (V-D-J) mRNA, complete
    cds. /FL = gb: M15564.1
    peptidylprolyl isomerase A (cyclophilin 1.76352 0.908832 3.26124 2.19E−02
    A)
    Homo sapiens mRNA for KIAA1597 6.20675 3.06104 3.12403 1.78E−02
    protein, partial cds. /PROD = KIAA1597
    protein
    HIV-1 rev binding protein 2 3.54001 0.617783 0.325316 3.52E−02
    Homo sapiens nidogen 2 (NID2), mRNA. 3.39549 0.915898 1.65422 4.70E−03
    /PROD = nidogen 2 /FL = gb: NM_007361.1
    gb: D86425.1
    Homo sapiens mRNA; cDNA 3.49826 0.859233 1.46022 3.01E−02
    DKFZp566A1046 (from clone
    DKFZp566A1046)
    Homo sapiens muscleblind (Drosophila)- 3.53962 0.508166 0.298438 3.90E−02
    like (MBNL), mRNA.
    /PROD = muscleblind (Drosophila)-like
    /FL = gb: NM_021038.1 gb: AB007888.1
    hypothetical protein FLJ20163 5.26708 2.17148 2.01643 3.33E−02
    KIAA0455 gene product 3.25374 0.057742 −0.01612 2.66E−02
    hypothetical protein FLJ22833 2.93229 0.533105 1.40606 3.19E−02
    /FL = gb: NM_022837.1
    ESTs 2.19313 −0.32822 0.427128 1.87E−02
    Homo sapiens NADPH oxidase 4 5.57794 2.61602 2.28462 4.04E−02
    (NOX4), mRNA. /PROD = NADPH
    oxidase
    4 /FL = gb: AF261943.1
    gb: NM_016931.1 gb: AF254621.1
    gb: AB041035.1
    ESTs 3.3266 0.840887 1.69605 0.00E+00
    ESTs 1.99635 −0.56359 0.236236 1.39E−02
    Homo sapiens serumglucocorticoid 4.81433 2.46478 3.47797 2.02E−02
    regulated kinase (SGK), mRNA.
    /PROD = serumglucocorticoid regulated
    kinase /FL = gb: BC001263.1
    gb: NM_005627.1 gb: AF153609.1
    A kinase (PRKA) anchor protein 2 6.03149 3.65135 4.63469 1.17E−02
    ESTs 3.8522 1.17894 0.483367 3.08E−02
    Homo sapiens similar to rat 1.74077 −1.45032 −1.24979 5.69E−04
    tricarboxylate carrier-like protein
    (BA108L7.2), mRNA. /PROD = similar to
    rat tricarboxylate carrier-likeprotein
    /FL = gb: NM_030971.1
    Homo sapiens, lectin, galactoside- 4.53668 1.85893 2.59596 3.84E−03
    binding, soluble, 3 (galectin 3), clone
    MGC: 2058, mRNA, complete cds.
    /PROD = lectin, galactoside-binding,
    soluble, 3(galectin 3)
    /FL = gb: NM_002306.1 gb: M35368.1
    gb: BC001120.1 gb: M36682.1
    gb: M57710.1 gb: AB006780.1
    ESTs 1.06738 −1.51321 −0.67814 7.68E−03
    Homo sapiens heptacellular carcinoma 5.55437 3.02649 3.91576 2.22E−02
    novel gene-3 protein (LOC51339),
    mRNA. /PROD = heptacellular carcinoma
    novel gene-3 protein
    /FL = gb: NM_016651.2 gb: AF251079.2
    Homo sapiens, Similar to transforming 4.82642 2.07249 2.74019 1.03E−02
    growth factor beta 1 induced transcript 1,
    clone MGC: 4078, mRNA, complete cds.
    /PROD = Similar to transforming growth
    factor beta
    1 induced transcript 1
    /FL = gb: NM_015927.1 gb: BC001830.1
    gb: AF116343.1
    ESTs, Highly similar to FXD3_HUMAN 3.81523 1.18624 1.98102 4.17E−02
    FORKHEAD BOX PROTEIN D3
    (H. sapiens)
    ESTs 4.52094 1.70664 2.3196 2.78E−02
    Homo sapiens potassium 3.68048 0.897369 1.5433 1.32E−02
    intermediatesmall conductance calcium-
    activated channel, subfamily N, member
    2 (KCNN2), mRNA. /PROD = potassium
    intermediatesmall conductancecalcium-
    activated channel, subfamily N, member
    2 /FL = gb: NM_021614.1 gb: AF239613.1
    Homo sapiens transporter similar to 5.68324 3.35483 4.46553 3.00E−03
    yeast MRS2 (MRS2L), mRNA.
    /PROD = transporter similar to yeast
    MRS2 /FL = gb: AF288288.1
    gb: NM_020662.1
    Homo sapiens a disintegrin-like and 4.54713 2.07152 1.10726 5.91E−03
    metalloprotease (reprolysin type) with
    thrombospondin type 1 motif, 5
    (aggrecanase-2) (ADAMTS5), mRNA.
    /PROD = a disintegrin and
    metalloprotease withthrombospondin
    motifs-5 preproprotein
    /FL = gb: NM_007038.1 gb: AF14209
    HIV-1 rev binding protein 2 3.85003 0.823769 0.407316 3.76E−02
    Human, parathyroid-like protein 1.73564 −1.5631 −1.41799 4.04E−02
    (associated with humoral hypercalcemia
    of malignancy) mRNA, complete cds.
    /FL = gb: J03580.1
    ESTs 5.27633 2.76577 3.71542 1.39E−02
    ESTs 1.7022 −1.29085 −0.81236 1.72E−02
    pyruvate dehydrogenase phosphatase 6.62966 4.26189 5.37384 1.04E−03
    /FL = gb: NM_018444.1 gb: AF155661.1
    ESTs 3.90566 1.11378 1.80307 1.57E−02
    Homo sapiens DRM (DRM) mRNA, 6.16645 3.12369 3.57831 1.59E−02
    complete cds. /PROD = DRM
    /FL = gb: NM_013372.1 gb: AF110137.2
    gb: AF045800.1 gb: AF154054.1
    Homo sapiens cDNA: FLJ22769 fis, 2.36483 −0.5543 0.025213 1.45E−04
    clone KAIA1316
    ESTs 4.31647 1.44801 2.11226 2.19E−02
    ESTs 1.24891 −1.42983 −0.57145 1.29E−02
    Homo sapiens mRNA; cDNA 2.85031 0.231102 1.16439 1.08E−02
    DKFZp586N012 (from clone
    DKFZp586N012)
    jagged 1 (Alagille syndrome) 2.54951 0.151382 1.31733 4.46E−03
    ESTs, Weakly similar to I38588 reverse 1.16209 −1.26828 −0.1187 3.40E−02
    transcriptase homolog (H. sapiens)
    KIAA1339 protein 4.41803 2.09451 3.35786 3.90E−02
    ESTs 3.83407 0.892884 1.56759 8.98E−03
    DKFZP434D156 protein 4.42983 2.05154 3.28988 1.60E−02
    Homo sapiens mRNA expressed only in 3.84408 0.705031 1.20856 2.25E−02
    placental villi, clone SMAP41.
    RAS guanyl releasing protein 2 (calcium 2.7724 0.235409 1.34452 1.96E−02
    and DAG-regulated)
    KIAA0164 gene product 4.99867 2.5327 3.72535 2.85E−02
    /FL = gb: NM_014739.1 gb: D79986.1
    Homo sapiens mRNA; cDNA 5.14752 2.61217 3.76652 3.78E−02
    DKFZp761M0111 (from clone
    DKFZp761M0111)
    Homo sapiens NPD009 mRNA, 2.15383 −0.27664 0.990295 2.09E−03
    complete cds. /PROD = NPD009
    /FL = gb: NM_020686.1 gb: AF237813.1
    muscleblind (Drosophila)-like 3.23823 0.467215 1.39628 7.60E−03
    /FL = gb: NM_021038.1 gb: AB007888.1
    ESTs 4.49318 1.35004 1.91395 3.79E−02
    muscleblind (Drosophila)-like 1.84877 −0.57315 0.732468 8.06E−03
    /FL = gb: NM_021038.1 gb: AB007888.1
    ESTs 6.068 3.42334 4.51189 1.89E−02
    ESTs, Weakly similar to unnamed 3.24528 0.740266 1.98306 3.89E−04
    protein product (H. sapiens)
    Human complement cytolysis inhibitor 6.50766 4.03647 5.33533 1.77E−02
    (CLI) mRNA, complete cds.
    /FL = gb: J02908.1 gb: NM_001831.1
    gb: M64722.1 gb: M25915.1
    ESTs 5.36759 2.42249 3.24765 3.23E−03
    ESTs 0.957239 −1.43055 −0.04796 5.55E−04
    transporter similar to yeast MRS2 3.77395 1.18311 2.36844 1.83E−02
    Homo sapiens, Similar to regulator for 5.09957 2.72179 4.14116 2.72E−02
    ribosome resistance homolog (S. cerevisiae),
    clone MGC: 2755, mRNA,
    complete cds. /PROD = Similar to
    regulator for ribosome
    resistancehomolog (S. cerevisiae)
    /FL = gb: BC001811.1
    SWISNF related, matrix associated, 2.54229 −0.29226 0.695255 1.89E−02
    actin dependent regulator of chromatin,
    subfamily a, member 2
    /FL = gb: NM_003070.1 gb: D26155.1
    Homo sapiens mRNA; cDNA 3.34446 0.795373 2.07147 1.03E−02
    DKFZp586L2424 (from clone
    DKFZp586L2424)
    ESTs 4.63117 2.08252 3.36946 2.25E−03
    Homo sapiens bicarbonate transporter- 3.17407 0.745852 2.17046 1.71E−03
    related protein BTR1 mRNA, complete
    cds. /PROD = bicarbonate transporter-
    related protein BTR1
    /FL = gb: AF336127.1
    ESTs 1.08377 −1.27197 0.228305 4.76E−02
    ESTs 2.94657 0.127188 1.20032 1.43E−02
    Homo sapiens mRNA; cDNA 2.47798 −0.26439 0.894731 4.51E−03
    DKFZp434B2016 (from clone
    DKFZp434B2016).
    ESTs 2.39983 −1.04089 −0.57976 3.37E−02
    Homo sapiens synaptotagmin interacting 2.11009 −0.57109 0.653181 1.78E−02
    protein STIP1 mRNA, partial cds.
    /PROD = synaptotagmin interacting
    protein STIP1
    Homo sapiens connexin 26 (GJB2) 2.86049 −0.57377 −0.09328 4.27E−04
    mRNA, complete cds. /PROD = connexin
    26 /FL = gb: NM_004004.1 gb: M86849.2
    Homo sapiens brain-derived 1.71599 −0.79412 0.616733 3.09E−02
    neurotrophic factor (BDNF), mRNA.
    /PROD = brain-derived neurotrophic
    factor /FL = gb: NM_001709.1
    ESTs, Weakly similar to unknown 2.33677 −0.04066 1.5217 1.87E−03
    (H. sapiens)
    ESTs 2.57265 0.177794 1.74876 2.74E−04
    ESTs 2.6809 0.209095 1.70624 1.21E−02
    Homo sapiens DKC1 gene, exons 1 to 4.18307 1.70283 3.20863 2.00E−02
    11
    Homo sapiens T cell receptor beta chain 4.45228 1.38318 2.30031 3.27E−03
    (TCRBV13S1-TCRBJ2S1) mRNA,
    complete cds. /PROD = T cell receptor
    beta chain /FL = gb: AF043179.1
    Homo sapiens PRO0066 mRNA, 0.466715 −1.87679 −0.22665 4.25E−03
    complete cds. /PROD = PRO0066
    /FL = gb: AF113007.1
    axonal transport of synaptic vesicles 5.20385 2.74124 4.27648 3.33E−03
    Homo sapiens, Similar to cyclin M2, 0.139313 −2.31405 −0.76333 1.46E−02
    clone MGC: 12933 IMAGE: 4308662,
    mRNA, complete cds. /PROD = Similar to
    cyclin M2 /FL = gb: BC021222.1
    Homo sapiens clone CDABP0095 3.87201 2.7936 5.73958 9.63E−03
    mRNA sequence
    H. sapiens mRNA for ribosomal protein 2.00401 0.354674 2.73013 4.70E−04
    L18a homologue. /PROD = ribosomal
    protein L18a homologue
    ESTs, Weakly similar to ALU7_HUMAN 3.43639 0.843826 2.28468 3.37E−02
    ALU SUBFAMILY SQ SEQUENCE
    CONTAMINATION WARNING ENTRY
    (H. sapiens)
    Homo sapiens, clone IMAGE: 5242616, −0.525567 −2.07832 0.406964 2.23E−02
    mRNA.
    Homo sapiens C1orf24 mRNA, complete 5.17968 2.58724 4.04213 1.98E−04
    cds. /PROD = C1orf24
    /FL = gb: AF288391.1 gb: AB050477.1
    gb: NM_022083.1
    Homo sapiens clone IMAGE: 451939, 5.15776 2.22786 3.40438 2.60E−02
    mRNA sequence
    Homo sapiens cDNA FLJ14942 fis, 0.682724 −2.08716 −0.74074 1.45E−02
    clone PLACE1011185, highly similar to
    INSERTION ELEMENT IS1 PROTEIN
    INSB.
    hypothetical protein FLJ20425 4.23689 1.90847 3.69988 1.15E−02
    /FL = gb: NM_017816.1 gb: AL136750.1
    Homo sapiens matrix metalloproteinase 2.28308 −0.65903 0.521586 8.87E−03
    10 (stromelysin 2) (MMP10), mRNA.
    /PROD = matrix metalloproteinase 10
    preproprotein /FL = gb: BC002591.1
    gb: NM_002425.1
    ESTs 3.67167 2.20619 4.86553 3.16E−03
    Homo sapiens prominin (mouse)-like 1 5.46002 2.79737 4.29571 1.38E−03
    (PROML1), mRNA. /PROD = prominin
    (mouse)-like 1 /FL = gb: NM_006017.1
    gb: AF027208.1
    Homo sapiens mRNA; cDNA 2.40278 −0.68409 0.399469 1.48E−02
    DKFZp434M2415 (from clone
    DKFZp434M2415).
    Homo sapiens hypothetical protein 2.1471 0.312211 2.65409 2.28E−04
    FLJ20701 (FLJ20701), mRNA.
    /PROD = hypothetical protein FLJ20701
    /FL = gb: NM_017933.1
    Homo sapiens cDNA FLJ35259 fis, 6.79033 3.86627 5.12737 1.23E−03
    clone PROST2004251.
    Homo sapiens hypothetical protein 4.0369 1.34318 2.84336 7.69E−03
    FLJ39553 (FLJ39553), mRNA.
    /FL = gb: NM_173549.1
    Human microfibril-associated 3.89326 0.527618 1.35764 1.81E−03
    glycoprotein-2 MAGP-2 mRNA,
    complete cds. /PROD = microfibril-
    associated glycoprotein-2 MAGP-2
    /FL = gb: NM_003480.1 gb: U37283.1
    Homo sapiens proprotein convertase 3.76897 0.368734 1.17396 1.38E−02
    subtilisinkexin type 5 (PCSK5), mRNA.
    /PROD = proprotein convertase
    subtilisinkexin type
    5 /FL = gb: U56387.2
    gb: NM_006200.1
    Cluster Incl. AI735391: at10e09.x1 Homo 2.59941 0.203564 2.03637 1.12E−04
    sapiens cDNA, 3 end /clone = IMAGE-
    2354728 /clone_end = 3 /gb = AI735391
    /gi = 5056915 /ug = Hs.20137 /len = 567
    ESTs 2.30031 −1.01596 −0.08964 1.46E−02
    Kruppel-like factor 4 (gut) 3.50041 1.09954 2.95915 1.22E−02
    Homo sapiens full length insert cDNA −1.40946 −3.04877 −0.39378 3.32E−02
    YI25A03
    ESTs 0.806666 −2.01081 −0.53076 1.91E−02
    Homo sapiens mRNA; cDNA 3.1892 0.012768 1.13562 1.35E−03
    DKFZp761G02121 (from clone
    DKFZp761G02121); partial cds
    Homo sapiens TMEFF2 mRNA, 2.28638 −0.89508 0.225546 1.21E−03
    complete cds. /FL = gb: AB017269.1
    gb: NM_016192.2 gb: AF179274.2
    gb: AF242222.1
    Homo sapiens, clone MGC: 3328, 3.54625 0.772486 2.30168 4.16E−02
    mRNA, complete cds. /PROD = Unknown
    (protein for MGC: 3328)
    /FL = gb: BC001745.1 gb: NM_014392.1
    ESTs 2.18149 −0.58303 0.984522 2.68E−02
    synaptojanin 2 4.166 1.83143 3.84199 0.00E+00
    KIAA0367 protein 1.5999 −1.50555 −0.26286 1.58E−02
    ESTs, Moderately similar to 0.868185 −1.46804 0.573078 1.69E−02
    ALU8_HUMAN ALU SUBFAMILY SX
    SEQUENCE CONTAMINATION
    WARNING ENTRY (H. sapiens)
    bromodomain adjacent to zinc finger 1.60872 −1.12544 0.518361 2.11E−02
    domain, 1B
    Human complement cytolysis inhibitor 6.61592 3.91665 5.596 1.66E−02
    (CLI) mRNA, complete cds.
    /FL = gb: J02908.1 gb: NM_001831.1
    gb: M64722.1 gb: M25915.1
    forkhead box O1A (rhabdomyosarcoma) 5.04975 2.59802 4.53545 1.60E−03
    /FL = gb: NM_002015.2 gb: AF032885.1
    gb: U02310.1
    Homo sapiens cDNA: FLJ22727 fis, 4.88391 2.09093 3.68875 1.91E−02
    clone HSI15054
    cadherin
    6, type 2, K-cadherin (fetal 3.27371 0.243335 1.61199 2.74E−04
    kidney) /FL = gb: D31784.1
    gb: NM_004932.1
    ESTs 3.97749 0.786845 1.99803 1.73E−02
    endonuclease G-like 1 1.11519 −2.09018 −0.88324 1.69E−02
    /FL = gb: AB020523.1 gb: NM_005107.1
    Homo sapiens cDNA FLJ13034 fis, 4.59641 0.64328 1.1121 1.66E−02
    clone NT2RP3001232
    Human DNA sequence from clone RP4- 2.91607 0.209555 1.92644 1.93E−02
    614O4 on chromosome 20q11.1-12
    Contains the 3 part of the MMP24
    (matrix metalloproteinase 24
    (membrane-inserted)) gene, the
    ITGB4BP (integrin beta 4 binding
    protein) gene, the 3 end of a novel gene,
    the 3 end o . . .
    Homo sapiens, collapsin response 0.672753 −1.75265 0.250803 8.65E−03
    mediator protein-5; CRMP3-associated
    molecule, clone MGC: 11247, mRNA,
    complete cds. /PROD = collapsin
    response mediator protein-5; CRMP3-
    associated molecule
    /FL = gb: BC002874.1
    ESTs, Moderately similar to 2.91576 0.289656 2.10334 2.57E−03
    ALU7_HUMAN ALU SUBFAMILY SQ
    SEQUENCE CONTAMINATION
    WARNING ENTRY (H. sapiens)
    ESTs, Moderately similar to CA1C RAT 6.2681 1.94518 1.82747 4.12E−02
    COLLAGEN ALPHA 1(XII) CHAIN
    (R. norvegicus)
    Homo sapiens alpha-aminoadipate 5.2604 2.72549 4.6685 4.33E−03
    semialdehyde synthase mRNA,
    complete cds. /PROD = alpha-
    aminoadipate semialdehyde synthase
    /FL = gb: AF229180.1
    Homo sapiens Wnt inhibitory factor-1 1.03265 −1.51207 0.433159 8.62E−05
    (WIF-1), mRNA. /PROD = Wnt inhibitory
    factor-1 /FL = gb: AF122922.1
    gb: NM_007191.1
    ribosomal protein S11 4.95358 2.25158 4.04683 2.98E−04
    Homo sapiens tumor necrosis factor, 2.42729 −0.73843 0.613222 1.93E−02
    alpha-induced protein 6 (TNFAIP6),
    mRNA. /PROD = tumor necrosis factor,
    alpha-induced protein 6
    /FL = gb: NM_007115.1
    Homo sapiens hairyenhancer-of-split 2.3848 −0.44154 1.27956 3.16E−03
    related with YRPW motif 2 (HEY2),
    mRNA. /PROD = hairyenhancer-of-split
    related with YRPW motif2
    /FL = gb: NM_012259.1 gb: AF311884.1
    gb: AB044755.1 gb: AF232238.1
    gb: AF237949.1 gb: AF173901.1
    Homo sapiens semenogelin I (SEMG1), 0.585816 −2.46893 −0.95223 3.05E−02
    mRNA. /PROD = semenogelin I
    /FL = gb: J04440.1 gb: NM_003007.1
    Homo sapiens, Similar to cadherin 6, 2.38648 −0.02212 2.14359 2.59E−03
    type 2, K-cadherin (fetal kidney), clone
    MGC: 1470, mRNA, complete cds.
    /PROD = Similar to cadherin 6, type 2, K-
    cadherin (fetalkidney)
    /FL = gb: BC000019.1
    Homo sapiens mRNA; cDNA 2.16899 −0.56794 1.27546 3.16E−03
    DKFZp434H0350 (from clone
    DKFZp434H0350); partial cds.
    /PROD = hypothetical protein
    Homo sapiens growth factor receptor- 4.2346 1.17569 2.73613 4.80E−03
    bound protein 14 (GRB14), mRNA.
    /PROD = growth factor receptor-bound
    protein 14 /FL = gb: L76687.1
    gb: NM_004490.1
    Homo sapiens, Similar to TAF5-like RNA 2.10954 −0.1313 2.24853 8.24E−04
    polymerase II, p300CBP-associated
    factor (PCAF)-associated factor, 65 kDa,
    clone MGC: 46101 IMAGE: 5551246,
    mRNA, complete cds. /PROD = Similar to
    TAF5-like RNA polymerase II, p300CBP-
    associated factor (PCAF)-associat
    Homo sapiens mRNA; cDNA 1.42404 −1.99222 −0.77126 3.21E−02
    DKFZp761J1324 (from clone
    DKFZp761J1324)
    solute carrier family 30 (zinc 2.82535 0.288493 2.38889 0.00E+00
    transporter), member 1
    antizyme inhibitor 3.65887 0.37346 1.74912 1.86E−02
    Homo sapiens GRO1 oncogene 2.51473 0.107263 2.38874 7.16E−04
    (melanoma growth stimulating activity,
    alpha) (GRO1), mRNA. /PROD = GRO1
    oncogene (melanoma growth
    stimulatingactivity, alpha)
    /FL = gb: NM_001511.1
    Homo sapiens proprotein convertase 4.90999 0.871309 1.52441 1.71E−02
    subtilisinkexin type 5 (PCSK5), mRNA.
    /PROD = proprotein convertase
    subtilisinkexin type
    5 /FL = gb: U56387.2
    gb: NM_006200.1
    ESTs 0.254736 −1.96122 0.51777 4.00E−02
    G-protein gamma-12 subunit 3.93653 1.06178 2.88889 5.11E−04
    /FL = gb: NM_018841.1 gb: AF119663.1
    platelet-derived growth factor alpha 3.50343 0.651246 2.50207 3.99E−03
    polypeptide
    clusterin (complement lysis inhibitor, SP- 2.88196 −0.42677 0.9712 8.48E−03
    40,40, sulfated glycoprotein 2,
    testosterone-repressed prostate
    message
    2, apolipoprotein J)
    ESTs 2.82081 −1.69816 −1.89985 4.10E−02
    Homo sapiens insulinoma-associated 1 2.94347 0.120494 2.02248 1.43E−03
    (INSM1), mRNA. /PROD = insulinoma-
    associated 1 /FL = gb: NM_002196.1
    gb: M93119.1
    LBP protein 32 /FL = gb: NM_014552.1 2.68546 −0.16678 1.70626 1.87E−03
    gb: AF198489.1
    ESTs, Highly similar to T42654 2.84504 0.515703 2.95379 7.92E−04
    hypothetical protein DKFZp434G1115.1
    (H. sapiens)
    Homo sapiens Friend leukemia virus 1.7744 −2.28064 −1.56765 3.14E−02
    integration 1 (FLI1), mRNA.
    /PROD = Friend leukemia virus
    integration
    1 /FL = gb: BC001670.1
    gb: NM_002017.2 gb: M98833.3
    ESTs 0.846429 −2.42046 −0.91891 1.57E−02
    Homo sapiens oxidative 3 alpha 1.30153 −1.64227 0.196394 3.20E−03
    hydroxysteroid dehydrogenase; retinol
    dehydrogenase; 3-hydroxysteroid
    epimerase (RODH), mRNA.
    /PROD = oxidative 3 alpha hydroxysteroid
    dehydrogenase; retinol dehydrogenase;
    3-hydroxysteroid epimerase
    /FL = gb: AF016509.1 gb: A
    minor histocompatibility antigen HA-1 1.89498 −0.37136 2.15157 4.61E−02
    UDP-glucose pyrophosphorylase 2 4.26327 0.968939 2.4776 6.20E−03
    Homo sapiens regulator of G-protein 5.42952 3.05504 5.49711 1.21E−03
    signalling 2, 24 kD (RGS2), mRNA.
    /PROD = regulator of G-protein signalling
    2, 24 kD /FL = gb: L13463.1
    gb: NM_002923.1
    ESTs 4.49987 1.01623 2.35659 1.86E−03
    ESTs 0.623422 −1.94907 0.317138 8.95E−04
    ESTs 0.326084 −1.69499 1.13586 1.10E−02
    ESTs 3.82952 0.673296 2.37112 3.92E−02
    Homo sapiens cDNA FLJ10160 fis, 2.80876 −0.24405 1.56853 1.39E−02
    clone HEMBA1003545, highly similar to
    INSULIN GENE ENHANCER PROTEIN
    ISL-2.
    thyroid hormone receptor-associated 4.66717 2.01883 4.2391 4.63E−03
    protein, 150 kDa subunit
    /FL = gb: NM_005119.1 gb: AF117756.1
    ESTs 2.4548 −0.3811 1.65212 4.08E−03
    Homo sapiens mRNA; cDNA 3.13835 0.605922 2.95511 1.53E−03
    DKFZp667D095 (from clone
    DKFZp667D095)
    Homo sapiens synaptojanin 2 mRNA, 2.94452 0.23595 2.4148 3.63E−04
    complete cds. /PROD = synaptojanin 2
    /FL = gb: AF318616.1
    Homo sapiens procollagen C- 3.37999 0.343624 2.21149 1.46E−02
    endopeptidase enhancer (PCOLCE),
    mRNA. /PROD = procollagen C-
    endopeptidase enhancer
    /FL = gb: BC000574.1 gb: NM_002593.2
    gb: AB008549.1 gb: L33799.1
    ESTs 3.24495 0.925809 3.52818 1.70E−04
    Homo sapiens mRNA for KIAA0930 3.65772 0.911608 3.09513 8.62E−05
    protein, partial cds. /PROD = KIAA0930
    protein
    Homo sapiens forkhead transcription 1.51993 −2.14186 −0.86243 3.75E−02
    factor FOXL2 (BPES), mRNA.
    /PROD = forkhead transcription factor
    FOXL2 /FL = gb: AF301906.1
    gb: NM_023067.1
    ESTs 1.9867 −1.5948 −0.2171 2.81E−03
    ESTs, Highly similar to AF174600 1 F- 3.06175 0.202953 2.35691 2.45E−02
    box protein Fbx20 (H. sapiens)
    Homo sapiens ankyrin 3, node of 3.11644 0.101116 2.09865 2.90E−03
    Ranvier (ankyrin G) (ANK3), transcript
    variant
    2, mRNA. /PROD = ankyrin 3,
    isoform 2 /FL = gb: NM_001149.1
    gb: U43965.1
    Homo sapiens tumor necrosis factor 2.39086 −1.04254 0.558779 1.62E−02
    receptor superfamily, member 6
    (TNFRSF6), mRNA. /PROD = apoptosis
    (APO-1) antigen 1 /FL = gb: NM_000043.1
    gb: M67454.1
    Homo sapiens thyrotropin-releasing 1.30849 −1.50688 0.822517 4.91E−02
    hormone degrading ectoenzyme
    (TRHDE), mRNA. /PROD = thyrotropin-
    releasing hormone
    degradingectoenzyme
    /FL = gb: AF126372.1 gb: NM_013381.1
    ESTs 4.65711 0.915563 2.32327 6.29E−03
    Homo sapiens mRNA; cDNA 2.36072 −2.02594 −1.25286 1.50E−02
    DKFZp564N1116 (from clone
    DKFZp564N1116)
    Homo sapiens placental protein 13-like 3.55894 0.434263 2.47085 3.48E−03
    protein (LOC56891), mRNA.
    /PROD = placental protein 13-like protein
    /FL = gb: NM_020129.1 gb: AF267852.1
    Homo sapiens, Similar to receptor 2.05578 0.178992 3.5007 4.46E−04
    tyrosine kinase-like orphan receptor 1,
    clone MGC: 12687, mRNA, complete
    cds. /PROD = Similar to receptor tyrosine
    kinase-like orphanreceptor 1
    /FL = gb: BC006374.1
    ESTs 0.159066 −2.96514 −0.84976 3.25E−02
    Homo sapiens, Similar to v-ets avian 2.22285 −1.58562 −0.1074 2.63E−02
    erythroblastosis virus E26 oncogene
    homolog
    1, clone MGC: 29755
    IMAGE: 3946751, mRNA, complete cds.
    /PROD = Similar to v-ets avian
    erythroblastosis virusE26 oncogene
    homolog 1 /FL = gb: BC017314.1
    ESTs, Highly similar to dedicator of cyto- 0.83871 −2.29829 −0.12951 1.85E−02
    kinesis 1 (H. sapiens)
    ESTs 1.1465 −1.7403 0.682768 3.99E−02
    ESTs 1.7466 −1.67509 0.283174 2.97E−03
    ESTs, Weakly similar to PMXB_HUMAN 1.03192 −2.44485 −0.51457 1.87E−03
    PAIRED MESODERM HOMEOBOX
    PROTEIN 2B (H. sapiens)
    ESTs 3.22222 −0.29339 1.59904 7.36E−03
    Homo sapiens cDNA FLJ10561 fis, 2.295 −1.60823 −0.08369 3.93E−03
    clone NT2RP2002672
    Homo sapiens, Similar to KIAA0441 1.38545 −1.60426 0.86909 2.33E−03
    gene product, clone MGC: 45124
    IMAGE: 5578893, mRNA, complete cds.
    /PROD = Similar to KIAA0441 gene
    product /FL = gb: BC036731.1
    Homo sapiens, clone IMAGE: 4067166, 0.195869 −2.50507 0.276993 1.16E−03
    mRNA
    ESTs 1.5857 −2.61787 −1.30022 1.12E−02
    Homo sapiens methyl-CpG binding 0.490598 −3.45465 −1.70163 3.90E−03
    protein MBD2 (MBD2) mRNA, complete
    cds. /PROD = methyl-CpG binding protein
    MBD2 /FL = gb: NM_003927.2
    gb: AF072242.1
    KIAA1151 protein 0.642527 −2.04288 1.00885 4.09E−03
    H. sapiens mRNA for B-HLH DNA 5.43034 1.56039 3.45122 1.12E−04
    binding protein. /PROD = B-HLH DNA
    binding protein /FL = gb: NM_000474.1
    Homo sapiens, Similar to hypothetical 0.520711 −1.99011 1.26899 2.63E−02
    protein FLJ32001, clone MGC: 39559
    IMAGE: 4828136, mRNA, complete cds.
    /PROD = Similar to hypothetical protein
    FLJ32001 /FL = gb: BC036200.1
    Homo sapiens Wilms tumor 1 (WT1), 2.40462 −2.96375 −2.43546 1.41E−02
    transcript variant D, mRNA.
    /PROD = Wilms tumor 1 isoform D
    /FL = gb: NM_024424.1 gb: NM_024426.1
    ESTs 1.53104 −1.54553 1.37309 8.14E−03
    Homo sapiens, Similar to cadherin 6, 2.54746 −0.69811 2.13419 1.45E−04
    type 2, K-cadherin (fetal kidney), clone
    MGC: 1470, mRNA, complete cds.
    /PROD = Similar to cadherin 6, type 2, K-
    cadherin (fetalkidney)
    /FL = gb: BC000019.1
    ESTs 0.133612 −3.77953 −1.56636 1.40E−02
    paternally expressed 3 2.87581 −1.67669 −0.0118 2.17E−02
    Homo sapiens Charot-Leyden crystal 3.30086 −0.41644 2.1096 2.08E−02
    protein (CLC), mRNA. /PROD = Charot-
    Leyden crystal protein
    /FL = gb: NM_001828.3 gb: L01664.1
    ESTs 3.26198 −0.72777 1.5682 3.16E−03
    Homo sapiens tachykinin, precursor 1 2.77265 −1.02081 1.48319 4.07E−04
    (substance K, substance P, neurokinin
    1, neurokinin 2, neuromedin L,
    neurokinin alpha, neuropeptide
    K, neuropeptide gamma) (TAC1),
    transcript variant beta, mRNA.
    /PROD = tachykinin 2 precursor, isoform
    beta /FL = gb: U3
    Homo sapiens PNAS-123 mRNA, 1.59659 −1.46656 1.84663 3.90E−03
    complete cds
    Homo sapiens hypothetical protein −0.359245 −3.46654 −0.06528 2.55E−03
    FLJ20075 (FLJ20075), mRNA.
    /PROD = hypothetical protein FLJ20075
    /FL = gb: NM_017655.1
    Human platelet-derived growth factor 2.44464 −0.5846 2.90484 3.63E−04
    alpha-receptor (PDGFRA) mRNA, exons
    13-16
    Homo sapiens cDNA: FLJ22547 fis, 4.29365 −0.10882 2.09548 1.54E−02
    clone HSI00356
    Homo sapiens HSPC156 protein 0.786188 −2.32161 1.29619 8.62E−05
    (HSPC156), mRNA. /PROD = HSPC156
    protein /FL = gb: NM_014178.1
    gb: AF161505.1
    Homo sapiens mRNA; cDNA 0.485642 −4.8524 −3.09356 2.02E−02
    DKFZp586P1124 (from clone
    DKFZp586P1124)
    caldesmon 1 /FL = gb: M64110.1 7.09236 2.67863 5.63436 7.47E−03
    gb: NM_004342.2
    Homo sapiens, Similar to hypothetical 1.69808 −2.74939 0.215566 1.92E−02
    protein FLJ10058, clone MGC: 34305
    IMAGE: 5167647, mRNA, complete cds.
    /PROD = Similar to hypothetical protein
    FLJ10058 /FL = gb: BC034293.1
    Homo sapiens, Similar to LIM homeobox −0.062884 −4.30121 −1.10961 1.16E−02
    protein 8, clone IMAGE: 4839343,
    mRNA.
    Homo sapiens chorionic 0.812166 −2.21846 2.21843 3.60E−03
    somatomammotropin hormone 2
    (CSH2), transcript variant 4, mRNA.
    /PROD = chorionic somatomammotropin
    hormone
    2, isoform4
    /FL = gb: NM_022646.1
    Homo sapiens HES-related repressor 2.80599 −1.19396 2.40562 2.44E−03
    protein 1 HERP1 mRNA, complete cds.
    /PROD = HES-related repressor protein 1
    HERP1 /FL = gb: NM_012259.1
    gb: AF311884.1 gb: AB044755.1
    gb: AF232238.1 gb: AF237949.1
    gb: AF173901.1
    Homo sapiens cadherin 6, type 2, K- 0.824752 −3.83509 −0.87634 2.49E−02
    cadherin (fetal kidney) (CDH6), mRNA.
    /PROD = cadherin 6, type 2, K-cadherin
    (fetal kidney) /FL = gb: D31784.1
    gb: NM_004932.1
    paternally expressed 3 2.87398 −4.12925 −3.49232 2.19E−02
    Homo sapiens cDNA FLJ11398 fis, 2.5268 −1.71619 1.84868 4.46E−04
    clone HEMBA1000637.
    Homo sapiens BCL2-interacting killer 4.30042 0.023414 3.56237 1.94E−03
    (apoptosis-inducing) (BIK), mRNA.
    /PROD = BCL2-interacting killer
    /FL = gb: NM_001197.2 gb: BC001599.1
    gb: U34584.1 gb: U49730.1
    Homo sapiens testis expressed 3.51154 −2.84891 −1.3824 2.18E−02
    sequence 14 (TEX14), mRNA.
    /PROD = testis expressed sequence 14
    /FL = gb: NM_031272.1
    Homo sapiens growth hormone 2 (GH2), 0.922569 −2.77587 1.4679 1.60E−03
    transcript variant 2, mRNA.
    /PROD = growth hormone 2, isoform 2
    precursor /FL = gb: J03756.1
    gb: NM_022557.1
    Homo sapiens, clone IMAGE: 4828836, 0.972907 −4.24111 −1.48398 1.63E−02
    mRNA.
    Homo sapiens galanin receptor 1 2.50745 −3.13141 −0.33802 3.38E−03
    (GALR1), mRNA. /PROD = galanin
    receptor
    1 /FL = gb: NM_001480.2
    gb: U23854.1 gb: L34339.1 gb: U53511.1
    Homo sapiens, serine (or cysteine) 1.96851 −4.13943 −1.56568 3.06E−02
    proteinase inhibitor, clade B (ovalbumin),
    member 3, clone MGC: 12244, mRNA,
    complete cds. /PROD = serine (or
    cysteine) proteinase inhibitor, cladeB
    (ovalbumin), member 3
    /FL = gb: NM_006919.1 gb: U19556.1
    gb: BC005224.1
    DKFZP566K1924 protein 2.88225 −2.59632 1.39062 0.00E+00
    ESTs, Moderately similar to 4.24074 −0.69782 3.87444 1.12E−04
    ALU2_HUMAN ALU SUBFAMILY SB
    SEQUENCE CONTAMINATION
    WARNING ENTRY (H. sapiens)
    Human mRNA upregulated during 0.494505 −4.62912 −0.18915 4.38E−02
    camptothecin-induced apoptosis of U937
    cells.
    nuclear receptor subfamily 1, group I, 1.77902 −4.02806 −0.19682 4.34E−02
    member 3
    ESTs −1.3795 −6.08172 −0.94606 6.91E−04
    Homo sapiens chorionic 1.12437 −3.12138 2.61605 1.12E−04
    somatomammotropin hormone 2
    (CSH2), transcript variant 1, mRNA.
    /PROD = chorionic somatomammotropin
    hormone
    2, isoform 1precursor
    /FL = gb: NM_020991.2 gb: BC002717.1
    Homo sapiens mRNA for SCCA2b, 3.32911 −3.16288 0.568045 1.93E−02
    complete cds. /PROD = SCCA2b
    /FL = gb: AB046400.1
    Homo sapiens KIAA0469 gene product 4.03097 −1.94826 2.56518 2.49E−04
    (KIAA0469), mRNA. /PROD = KIAA0469
    gene product /FL = gb: AB007938.1
    gb: NM_014851.1
    ESTs 4.45389 −2.40636 5.33359 5.65E−04
  • TABLE VII
    THE EFFECT OF WNT-3A TREAMENT ON CYTOKINE
    EXPRESSION IN POPULATIONS OF CELLS FROM THE HUMAN
    EMBRYONIC STEM CELL LINE H9.
    Cell lysate Cell conditioned media
    w/o wnt 1 w/o wnt 2 w/ wnt 1 w/ wnt 2 w/o wnt 1 w/o wnt 2 w/ wnt 1 w/ wnt 2
    POS 62,842.33 67,606.06 49,758.34 50,702.57 96,585.15 109,721.82 199,709.04 195,889.94
    NEG 23.38 34.49 370.82 400.66 58.06 34.45 216.90 105.20
    Angiogenin 38.50 132.91 619.40 750.85 13,726.35 11,749.55 57,442.48 54,969.04
    BDNF 167.50 167.82 763.51 951.45 689.22 563.39 770.08 715.86
    BLC 2.00 48.99 742.24 476.36 107.69 512.77 337.12 249.80
    BMP-4 247.00 273.70 348.44 304.48 583.40 541.83 1,158.54 771.19
    BMP-6 8.50 141.92 571.23 641.05 354.91 355.28 985.70 670.60
    CK beta 8-1 1.00 1.13 22.48 21.54 17.79 25.31 42.78 75.44
    CNTF 1.00 55.19 1,382.51 1,060.82 282.81 211.86 787.19 541.51
    EGF 32.50 82.22 742.24 984.39 1,681.85 1,406.12 23,331.57 22,768.53
    Eotaxin 1.00 7.32 301.87 253.80 207.89 199.67 622.91 291.71
    Eotaxin-2 262.00 336.77 1,155.31 960.31 890.56 744.31 2,156.21 1,445.14
    Eotaxin-3 184.50 284.96 934.12 847.56 975.77 665.56 2,140.81 1,480.35
    FGF-6 629.00 379.57 1,327.92 964.54 1,239.85 947.73 3,283.94 2,519.78
    FGF-7 31.00 222.45 1,075.02 941.73 449.49 352.47 1,086.66 861.72
    Flt-3 Ligand 44.00 180.21 857.45 795.19 315.58 277.47 511.67 792.98
    Fractalkine 23.50 62.51 792.02 456.51 120.80 145.30 359.37 367.15
    GCP-2 1.00 45.05 593.71 538.86 203.21 166.86 451.78 362.12
    GDNF 84.00 292.84 436.75 444.68 176.99 194.98 443.22 509.66
    GM-CSF 433.00 636.37 1,343.17 1,161.75 777.25 722.75 1,808.82 1,446.82
    I-309 152.50 246.10 708.52 743.25 1,828.87 179.05 318.30 288.36
    IFN-gamma 349.00 441.52 1,202.67 1,037.60 283.74 559.64 1,238.97 1,435.08
    IGFBP-1 1.50 3.38 59.41 85.73 33.71 24.37 227.60 155.91
    IGFBP-2 3,785.50 5,554.45 2,671.90 2,414.72 27,656.85 28,473.95 42,215.53 43,934.33
    IGFBP-4 5,553.00 6,043.27 4,459.85 4,173.19 10,091.09 6,029.45 18,988.34 13,380.14
    IGF-I 1.00 2.25 558.38 208.19 88.96 148.11 254.98 187.77
    IL-10 158.00 422.93 1,222.75 1,096.30 722.93 564.32 1,625.71 1,129.96
    IL-13 428.50 555.84 1,486.89 1,407.96 1,114.36 786.49 2,522.42 1,807.27
    IL-15 599.50 808.13 1,677.16 1,601.79 1,140.59 858.67 2,495.04 1,981.62
    IL-16 2.00 137.97 651.11 668.50 259.39 214.67 436.38 405.71
    IL-1alpha 1,236.00 1,216.99 1,612.93 1,421.89 10,155.70 5,842.90 3,999.26 3,545.79
    IL-1beta 1.00 1.13 352.05 312.08 117.99 75.93 316.59 278.30
    IL-1ra 33.00 25.91 302.68 198.48 142.34 93.74 354.23 581.75
    IL-2 210.50 681.99 966.23 1,133.88 188.22 363.72 758.10 881.84
    IL-3 3,146.50 1,976.13 2,119.94 1,497.48 4,598.86 5,260.77 11,345.78 12,174.73
    IL-4 17.50 74.34 625.42 638.94 357.72 239.04 804.30 673.95
    IL-5 400.50 555.84 1,245.23 1,167.24 1,056.31 791.18 2,120.27 1,713.38
    IL-6 828.50 516.98 1,121.19 842.07 7,377.28 9,527.88 9,774.82 9,991.93
    IL-7 190.00 313.12 1,212.31 531.68 456.98 430.27 681.09 1,017.63
    Leptin 900.50 617.79 1,234.39 1,091.65 2,999.42 2,016.38 7,238.71 4,886.99
    LIGHT 10.50 232.02 1,081.04 888.10 619.92 453.71 1,346.78 1,108.17
    MCP-1 242.50 282.71 1,022.03 1,076.03 572.17 500.58 2,015.89 1,567.53
    MCP-2 24.00 109.25 909.63 1,152.46 129.23 351.53 694.78 694.07
    MCP-3 1.00 168.38 1,184.61 1,255.50 401.73 169.67 701.62 583.42
    MCP-4 7.50 46.74 1,380.51 1,606.86 123.61 61.87 231.02 276.62
    M-CSF 1.00 63.64 852.63 867.83 529.09 269.04 740.98 684.01
    MDC 1.00 2.25 269.36 280.41 157.32 52.50 280.65 132.44
    MIG 291.00 226.39 1,113.16 1,137.26 272.50 486.52 617.77 1,222.17
    MIP-1-delta 1.50 1.13 491.75 322.22 144.21 66.56 474.02 291.71
    MIP-3-alpha 1.00 5.63 1,602.09 1,399.93 55.25 0.00 208.78 184.41
    NAP-2 1.00 6.19 465.66 177.37 97.39 110.61 371.35 343.68
    NT-3 1.00 1.13 316.73 275.76 97.39 171.55 571.57 427.51
    PARC 1.00 2.25 352.85 353.04 170.43 81.56 474.02 363.80
    PDGF-BB 437.00 1,644.99 517.84 597.56 4,729.96 8,521.09 2,635.37 2,005.09
    RANTES 1.00 89.54 724.98 637.25 393.31 347.78 682.80 855.01
    SCF 74.50 148.67 1,152.50 1,021.13 691.09 487.46 1,394.69 2,155.98
    SDF-1 23.50 51.25 940.14 1,294.78 382.07 123.74 326.85 616.95
    TARC 1.50 5.07 372.93 343.33 206.95 71.24 277.23 1,642.97
    TGF-beta 1 362.00 230.33 1,426.67 1,240.30 752.90 740.56 1,827.65 1,849.18
    TGF-beta 3 3.00 25.91 377.74 1,130.93 111.44 115.30 294.34 343.68
    TNF-alpha 431.00 579.49 2,851.74 1,285.91 1,205.20 929.91 2,609.70 2,167.71
    TNF-beta 241.00 342.40 1,049.33 1,026.62 675.17 671.19 1,701.01 1,637.94
    Internal Control 18,642.67 18,642.67 18,642.67 18,642.67 18,642.67 18,642.67 18,642.67 18,642.67
    POS 60,794.83 48,106.85 35,652.42 47,391.58 94,013.71 90,889.16 141,321.30 149,125.43
    NEG 11.13 5.53 11.22 30.45 24.93 17.75 36.22 33.27
    Acrp30 1,036.00 744.50 601.61 690.66 1,797.52 1,319.88 2,795.71 2,302.99
    AgRP 11.00 153.99 77.82 89.00 378.17 195.90 651.26 482.21
    Angiopoietin-2 28.50 10.43 357.97 771.04 655.82 632.60 65,959.24 71,664.21
    Amphiregulin 17.50 15.44 73.63 154.62 250.52 151.01 619.07 782.60
    Axi 637.50 795.83 88.59 139.03 401.31 324.05 610.40 748.34
    bFGF 59,820.50 54,057.29 11,722.95 21,398.52 7,948.03 4,993.83 1,226.99 1,241.09

Claims (20)

What is claimed is:
1. A method for differentiating human pluripotent stem cells into pancreatic endocrine cells comprising:
a. differentiating human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent cells with activin A;
b. differentiating the definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor; and
c. differentiating the pancreatic endoderm cells into cells expressing markers characteristics of the pancreatic endocrine lineage by treating the pancreatic endoderm cells with a gamma secretase inhibitor.
2. The method of claim 1, wherein step a. comprises treating the human pluripotent stem cells with activin A and (i) a GSK-3B inhibitor, (ii) a Wnt ligand, or (iii) both a GSK-3B inhibitor and a Wnt ligand.
3. The method of claim 2, wherein the GSK-3B inhibitor is GSK-3B inhibitor IX or GSK-3B inhibitor XI.
4. The method of claim 2, wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.
5. The method of claim 1, wherein the step of differentiating the human pluripotent stem cells into definitive endoderm cells is achieved by treating the human pluripotent stem cells with activin A for up to about eight days.
6. The method of claim 1, wherein the step a. comprises:
i) plating the human pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and lacking a feeder cell layer;
ii) culturing the pluripotent stem cells with activin A, a Wnt ligand, and a GSK-3B inhibitor in a first culture medium; and
iii) culturing the pluripotent stem cells with activin A in a second culture medium wherein the second culture medium does not contain a Wnt ligand, for a period of time sufficient to differentiate the pluripotent stem cells into definitive endoderm cells.
7. The method of claim 6, wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum.
8. The method of claim 1, wherein the fibroblast growth factor is selected from the selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.
9. The method of claim 1, wherein the fibroblast growth factor is FGF-2 or FGF-4.
10. The method of claim 1, wherein the step of differentiating the definitive endoderm cells into pancreatic endoderm cells is achieved by treating the definitive endoderm cells with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.
11. The method of claim 10, wherein the cells are treated for about one to about six days.
12. The method of claim 1, wherein the step of differentiating the definitive endoderm cells into pancreatic endoderm cells comprises:
(i) culturing human definitive endoderm cells in a medium supplemented with retinoic acid or a retinoic acid analog;
(ii) removing the retinoic acid or retinoic acid analog; and
(iii) subsequently culturing the cells in a medium supplemented with at least one fibroblast growth factor.
13. The method of claim 12, wherein the culture medium in step (i), step (iii) or both step (i) and (iii) is supplemented with about 0% to about 2% of serum.
14. The method of claim 1, wherein the gamma secretase inhibitor is L-685,458.
15. A method for generating pancreatic endocrine cells comprising treating pancreatic endoderm cells with a gamma secretase inhibitor, wherein the pancreatic endocrine cells are derived from human definitive endoderm cells.
16. The method of claim 15, wherein the gamma secretase inhibitor is L-685,458.
17. The method of claim 15, further comprising differentiating human definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor.
18. The method of claim 17, wherein the fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10.
19. The method of claim 15, further comprising differentiating the human definitive endoderm cells into pancreatic endoderm cells by a method comprising:
(i) culturing the human definitive endoderm cells in a medium supplemented with retinoic acid or a retinoic acid analog;
(ii) removing the retinoic acid or retinoic acid analog; and
(iii) subsequently culturing the cells in a medium supplemented with at least one fibroblast growth factor.
20. The method of claim 19, wherein the culture medium in step (i), step (iii) or both step (i) and (iii) is supplemented with about 0% to about 2% of serum.
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Families Citing this family (122)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6391005B1 (en) 1998-03-30 2002-05-21 Agilent Technologies, Inc. Apparatus and method for penetration with shaft having a sensor for sensing penetration depth
US8641644B2 (en) 2000-11-21 2014-02-04 Sanofi-Aventis Deutschland Gmbh Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means
US8337419B2 (en) 2002-04-19 2012-12-25 Sanofi-Aventis Deutschland Gmbh Tissue penetration device
US9226699B2 (en) 2002-04-19 2016-01-05 Sanofi-Aventis Deutschland Gmbh Body fluid sampling module with a continuous compression tissue interface surface
US7041068B2 (en) 2001-06-12 2006-05-09 Pelikan Technologies, Inc. Sampling module device and method
US7981056B2 (en) 2002-04-19 2011-07-19 Pelikan Technologies, Inc. Methods and apparatus for lancet actuation
AU2002348683A1 (en) 2001-06-12 2002-12-23 Pelikan Technologies, Inc. Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge
US9795747B2 (en) 2010-06-02 2017-10-24 Sanofi-Aventis Deutschland Gmbh Methods and apparatus for lancet actuation
US9427532B2 (en) 2001-06-12 2016-08-30 Sanofi-Aventis Deutschland Gmbh Tissue penetration device
EP1395185B1 (en) 2001-06-12 2010-10-27 Pelikan Technologies Inc. Electric lancet actuator
ES2336081T3 (en) 2001-06-12 2010-04-08 Pelikan Technologies Inc. SELF-OPTIMIZATION PUNCTURE DEVICE WITH MEANS OF ADAPTATION TO TEMPORARY VARIATIONS IN CUTANEOUS PROPERTIES.
US8579831B2 (en) 2002-04-19 2013-11-12 Sanofi-Aventis Deutschland Gmbh Method and apparatus for penetrating tissue
US8360992B2 (en) 2002-04-19 2013-01-29 Sanofi-Aventis Deutschland Gmbh Method and apparatus for penetrating tissue
US7226461B2 (en) 2002-04-19 2007-06-05 Pelikan Technologies, Inc. Method and apparatus for a multi-use body fluid sampling device with sterility barrier release
US7892183B2 (en) 2002-04-19 2011-02-22 Pelikan Technologies, Inc. Method and apparatus for body fluid sampling and analyte sensing
US7901362B2 (en) 2002-04-19 2011-03-08 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7547287B2 (en) 2002-04-19 2009-06-16 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US9248267B2 (en) 2002-04-19 2016-02-02 Sanofi-Aventis Deustchland Gmbh Tissue penetration device
US7331931B2 (en) 2002-04-19 2008-02-19 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7892185B2 (en) 2002-04-19 2011-02-22 Pelikan Technologies, Inc. Method and apparatus for body fluid sampling and analyte sensing
US7491178B2 (en) 2002-04-19 2009-02-17 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7674232B2 (en) 2002-04-19 2010-03-09 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US8784335B2 (en) 2002-04-19 2014-07-22 Sanofi-Aventis Deutschland Gmbh Body fluid sampling device with a capacitive sensor
US7229458B2 (en) 2002-04-19 2007-06-12 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7909778B2 (en) 2002-04-19 2011-03-22 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7297122B2 (en) 2002-04-19 2007-11-20 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US8221334B2 (en) 2002-04-19 2012-07-17 Sanofi-Aventis Deutschland Gmbh Method and apparatus for penetrating tissue
US9314194B2 (en) 2002-04-19 2016-04-19 Sanofi-Aventis Deutschland Gmbh Tissue penetration device
US8267870B2 (en) 2002-04-19 2012-09-18 Sanofi-Aventis Deutschland Gmbh Method and apparatus for body fluid sampling with hybrid actuation
US8702624B2 (en) 2006-09-29 2014-04-22 Sanofi-Aventis Deutschland Gmbh Analyte measurement device with a single shot actuator
US9795334B2 (en) 2002-04-19 2017-10-24 Sanofi-Aventis Deutschland Gmbh Method and apparatus for penetrating tissue
US7976476B2 (en) 2002-04-19 2011-07-12 Pelikan Technologies, Inc. Device and method for variable speed lancet
US7232451B2 (en) 2002-04-19 2007-06-19 Pelikan Technologies, Inc. Method and apparatus for penetrating tissue
US7175642B2 (en) 2002-04-19 2007-02-13 Pelikan Technologies, Inc. Methods and apparatus for lancet actuation
US8574895B2 (en) 2002-12-30 2013-11-05 Sanofi-Aventis Deutschland Gmbh Method and apparatus using optical techniques to measure analyte levels
EP1628567B1 (en) 2003-05-30 2010-08-04 Pelikan Technologies Inc. Method and apparatus for fluid injection
US7850621B2 (en) 2003-06-06 2010-12-14 Pelikan Technologies, Inc. Method and apparatus for body fluid sampling and analyte sensing
WO2006001797A1 (en) 2004-06-14 2006-01-05 Pelikan Technologies, Inc. Low pain penetrating
WO2005033659A2 (en) 2003-09-29 2005-04-14 Pelikan Technologies, Inc. Method and apparatus for an improved sample capture device
US9351680B2 (en) 2003-10-14 2016-05-31 Sanofi-Aventis Deutschland Gmbh Method and apparatus for a variable user interface
EP1706026B1 (en) 2003-12-31 2017-03-01 Sanofi-Aventis Deutschland GmbH Method and apparatus for improving fluidic flow and sample capture
US7822454B1 (en) 2005-01-03 2010-10-26 Pelikan Technologies, Inc. Fluid sampling device with improved analyte detecting member configuration
EP1751546A2 (en) 2004-05-20 2007-02-14 Albatros Technologies GmbH & Co. KG Printable hydrogel for biosensors
WO2005120365A1 (en) 2004-06-03 2005-12-22 Pelikan Technologies, Inc. Method and apparatus for a fluid sampling device
US9775553B2 (en) 2004-06-03 2017-10-03 Sanofi-Aventis Deutschland Gmbh Method and apparatus for a fluid sampling device
US8017395B2 (en) 2004-12-17 2011-09-13 Lifescan, Inc. Seeding cells on porous supports
US8652831B2 (en) 2004-12-30 2014-02-18 Sanofi-Aventis Deutschland Gmbh Method and apparatus for analyte measurement test time
CN101484575B (en) 2005-06-08 2013-10-02 森托科尔公司 Cellular therapy for ocular degeneration
US8741643B2 (en) 2006-04-28 2014-06-03 Lifescan, Inc. Differentiation of pluripotent stem cells to definitive endoderm lineage
US8685730B2 (en) 2006-05-02 2014-04-01 Wisconsin Alumni Research Foundation Methods and devices for differentiating pluripotent stem cells into cells of the pancreatic lineage
SE534150C2 (en) * 2006-05-02 2011-05-10 Wisconsin Alumni Res Found Procedure for differentiating stem cells into cells of the endodermal and pancreatic developmental line
EP2126045A4 (en) * 2007-01-30 2010-05-26 Univ Georgia Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (mmc)
US9080145B2 (en) 2007-07-01 2015-07-14 Lifescan Corporation Single pluripotent stem cell culture
RU2473685C2 (en) 2007-07-31 2013-01-27 Лайфскен, Инк. Differentiation of human embryo stem cells
ATE523585T1 (en) 2007-11-27 2011-09-15 Lifescan Inc DIFFERENTIATION OF HUMAN EMBRYONAL STEM CELLS
KR20190057164A (en) 2008-02-21 2019-05-27 얀센 바이오테크 인코포레이티드 Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
WO2009117439A2 (en) 2008-03-17 2009-09-24 The Scripps Research Institute Combined chemical and genetic approaches for generation of induced pluripotent stem cells
WO2009126900A1 (en) 2008-04-11 2009-10-15 Pelikan Technologies, Inc. Method and apparatus for analyte detecting device
US8338170B2 (en) 2008-04-21 2012-12-25 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
AU2008355123B2 (en) * 2008-04-21 2014-12-04 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
US8623648B2 (en) 2008-04-24 2014-01-07 Janssen Biotech, Inc. Treatment of pluripotent cells
JP2011520470A (en) 2008-05-22 2011-07-21 ヴェスタ セラピューティクス，インコーポレーテッド Method for differentiating mammalian progenitor cells into insulin-producing islet cells
US20090298178A1 (en) * 2008-06-03 2009-12-03 D Amour Kevin Allen Growth factors for production of definitive endoderm
JP5734183B2 (en) * 2008-06-30 2015-06-17 ヤンセン バイオテツク，インコーポレーテツド Differentiation of pluripotent stem cells
AU2009267167A1 (en) * 2008-06-30 2010-01-07 Centocor Ortho Biotech Inc. Differentiation of pluripotent stem cells
US9233926B2 (en) 2008-09-17 2016-01-12 Sanford-Burnham Medical Research Institute Compounds for stem cell differentiation
US9012217B2 (en) * 2008-09-17 2015-04-21 Burnham Institute For Medical Research Benzimidazole compounds differentiate a mammalian stem cell into mesodermal or cardiomyocyte cells
CN102272291B (en) * 2008-10-31 2018-01-16 詹森生物科技公司 Differentiation of the human embryo stem cell to pancreatic endocrine pedigree
RU2528861C2 (en) 2008-10-31 2014-09-20 Сентокор Орто Байотек Инк. Differentiation of human embryonic stem cell into pancreatic endocrine cell line
EP2356227B1 (en) 2008-11-14 2018-03-28 Viacyte, Inc. Encapsulation of pancreatic cells derived from human pluripotent stem cells
BRPI0920956A2 (en) 2008-11-20 2015-08-18 Centocor Ortho Biotech Inc Pluripotent stem cell culture in microvessels
RU2547925C2 (en) 2008-11-20 2015-04-10 Сентокор Орто Байотек Инк. Methods and compositions for cell fixation and culture on flat carriers
EP2367931B1 (en) 2008-12-03 2017-05-31 International Stem Cell Corporation Methods of deriving differentiated cells from stem cells
WO2010077955A1 (en) 2008-12-17 2010-07-08 The Scripps Research Institute Generation and maintenance of stem cells
US9375169B2 (en) 2009-01-30 2016-06-28 Sanofi-Aventis Deutschland Gmbh Cam drive for managing disposable penetrating member actions with a single motor and motor and control system
EP2412800A1 (en) 2010-07-29 2012-02-01 Koninklijke Nederlandse Akademie van Wetenschappen Liver organoid, uses thereof and culture method for obtaining them
US9752124B2 (en) 2009-02-03 2017-09-05 Koninklijke Nederlandse Akademie Van Wetenschappen Culture medium for epithelial stem cells and organoids comprising the stem cells
JP5458112B2 (en) * 2009-02-03 2014-04-02 コーニンクレッカ ネザーランド アカデミー ヴァン ウェテンシャッペン Culture medium for epithelial stem cells and organoids containing the stem cells
WO2010141622A2 (en) * 2009-06-02 2010-12-09 Cornell University Method to isolate, identify, and use embryonic stem cells directed to forebrain interneuron fate
SG177416A1 (en) 2009-07-20 2012-02-28 Janssen Biotech Inc Differentiation of human embryonic stem cells
WO2011011300A2 (en) 2009-07-20 2011-01-27 Centocor Ortho Biotech Inc. Differentiation of human embryonic stem cells
GB2485112B (en) * 2009-07-20 2014-02-26 Janssen Biotech Inc Differentiation of human embryonic stem cells
ES2638464T3 (en) 2009-10-16 2017-10-20 The Scripps Research Institute Induction of pluripotent cells
RU2701335C2 (en) 2009-12-23 2019-09-25 Янссен Байотек, Инк. Method for producing population of pancreatic endocrine cells co-expressing nkx6.1 and insulin, and method of treating diabetes
ES2633648T3 (en) * 2009-12-23 2017-09-22 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
CA2791476C (en) 2010-03-01 2020-06-30 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
CN102959076B (en) 2010-03-31 2015-09-16 斯克里普斯研究所 Reprogrammed cell
US8965476B2 (en) 2010-04-16 2015-02-24 Sanofi-Aventis Deutschland Gmbh Tissue penetration device
EP2561087A4 (en) * 2010-04-20 2013-10-16 Int Stem Cell Corp Physiological methods for isolation of high purity cell populations
RU2663339C1 (en) 2010-05-12 2018-08-03 Янссен Байотек, Инк. Differentiation of human embryo stem cells
WO2011159726A2 (en) 2010-06-14 2011-12-22 The Scripps Research Institute Reprogramming of cells to a new fate
EP2611907B1 (en) 2010-08-31 2016-05-04 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
CN103221536B (en) * 2010-08-31 2016-08-31 詹森生物科技公司 The differentiation of human embryo stem cell
PL2611910T3 (en) * 2010-08-31 2018-06-29 Janssen Biotech, Inc Differentiation of human embryonic stem cells
KR102703637B1 (en) 2010-12-22 2024-09-05 페이트 세러퓨틱스, 인코포레이티드 Cell culture platform for single cell sorting and enhanced reprogramming of iPSCs
CN103890167A (en) 2011-06-21 2014-06-25 诺沃—诺迪斯克有限公司 Efficient induction of definitive endoderm from pluripotent stem cells
EP2794857A4 (en) 2011-12-22 2015-07-08 Janssen Biotech Inc Differentiation of human embryonic stem cells into single hormonal insulin positive cells
CA2866590A1 (en) 2012-03-07 2013-09-12 Janssen Biotech, Inc. Defined media for expansion and maintenance of pluripotent stem cells
JP6450674B2 (en) * 2012-05-07 2019-01-09 ヤンセン バイオテツク，インコーポレーテツド Differentiation of human embryonic stem cells into the endoderm of the pancreas
EP3450542B1 (en) 2012-06-08 2021-09-01 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
CA2876671A1 (en) * 2012-06-14 2013-12-27 Janssen Biotech, Inc. Treatment of pluripotent cells
CN105143445B (en) 2012-11-29 2020-07-07 宝生物欧洲公司 Maturation of hepatocyte-like cells derived from human pluripotent stem cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
CA2896658C (en) 2012-12-31 2021-06-22 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using hb9 regulators
WO2014106141A1 (en) 2012-12-31 2014-07-03 Janssen Biotech, Inc. Suspension and clustering of human pluripotent cells for differentiation into pancreatic endocrine cells
RU2658488C2 (en) 2012-12-31 2018-06-21 Янссен Байотек, Инк. Method for obtaining cells expressing markers characteristic for pancreatic endocrine cells
WO2014130887A1 (en) * 2013-02-22 2014-08-28 Cedars-Sinai Medical Center Pancreatic insulin-producing beta-cell lines derived from human pluripotent stem cells
US9540613B2 (en) * 2013-04-29 2017-01-10 Wisconsin Alumni Research Foundation Methods for producing insulin-secreting beta cells from human pluripotent stem cells
EP3114214B1 (en) 2014-03-04 2023-11-01 Fate Therapeutics, Inc. Improved reprogramming methods and cell culture platforms
KR102162138B1 (en) 2014-05-16 2020-10-06 얀센 바이오테크 인코포레이티드 Use of small molecules to enhance mafa expression in pancreatic endocrine cells
RS58070B2 (en) * 2015-02-20 2022-10-31 Inst Nat Sante Rech Med Use of a laminin for differentiating pluripotent cells into hepatocyte lineage cells
CA2983764A1 (en) 2015-04-24 2016-10-27 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
KR20180055918A (en) 2015-10-16 2018-05-25 페이트 세러퓨틱스, 인코포레이티드 Platform for the induction and maintenance of ground-state versatility
WO2017154209A1 (en) * 2016-03-11 2017-09-14 株式会社ニコン Evaluation device, observation device, and program
MA45479A (en) 2016-04-14 2019-02-20 Janssen Biotech Inc DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN ENDODERMAL CELLS OF MIDDLE INTESTINE
MA45502A (en) 2016-06-21 2019-04-24 Janssen Biotech Inc GENERATION OF FUNCTIONAL BETA CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS WITH GLUCOSE-DEPENDENT MITOCHONDRIAL RESPIRATION AND TWO-PHASE INSULIN SECRETION RESPONSE
US10767164B2 (en) 2017-03-30 2020-09-08 The Research Foundation For The State University Of New York Microenvironments for self-assembly of islet organoids from stem cells differentiation
CA2983845C (en) 2017-10-26 2024-01-30 University Of Copenhagen Generation of glucose-responsive beta cells
WO2020027316A1 (en) * 2018-08-03 2020-02-06 国立大学法人京都大学 Cell production method
CN112961823B (en) * 2021-03-19 2024-01-23 上海爱萨尔生物科技有限公司 Culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells
WO2024151541A1 (en) 2023-01-09 2024-07-18 Sana Biotechnology, Inc. Type-1 diabetes autoimmune mouse
GB202308408D0 (en) 2023-06-06 2023-07-19 Cambridge Entpr Ltd Stem cells based three-dimensional embryo model

Family Cites Families (183)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3209652A (en) 1961-03-30 1965-10-05 Burgsmueller Karl Thread whirling method
AT326803B (en) 1968-08-26 1975-12-29 Binder Fa G MESHWARE AND METHOD OF MANUFACTURING THE SAME
US3935067A (en) 1974-11-22 1976-01-27 Wyo-Ben Products, Inc. Inorganic support for culture media
CA1201400A (en) 1982-04-16 1986-03-04 Joel L. Williams Chemically specific surfaces for influencing cell activity during culture
US4499802A (en) 1982-09-29 1985-02-19 Container Graphics Corporation Rotary cutting die with scrap ejection
US4537773A (en) 1983-12-05 1985-08-27 E. I. Du Pont De Nemours And Company α-Aminoboronic acid derivatives
US4557264A (en) 1984-04-09 1985-12-10 Ethicon Inc. Surgical filament from polypropylene blended with polyethylene
US5089396A (en) 1985-10-03 1992-02-18 Genentech, Inc. Nucleic acid encoding β chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid
US5215893A (en) 1985-10-03 1993-06-01 Genentech, Inc. Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid
US4737578A (en) 1986-02-10 1988-04-12 The Salk Institute For Biological Studies Human inhibin
US5863531A (en) 1986-04-18 1999-01-26 Advanced Tissue Sciences, Inc. In vitro preparation of tubular tissue structures by stromal cell culture on a three-dimensional framework
US5804178A (en) 1986-11-20 1998-09-08 Massachusetts Institute Of Technology Implantation of cell-matrix structure adjacent mesentery, omentum or peritoneum tissue
US5567612A (en) 1986-11-20 1996-10-22 Massachusetts Institute Of Technology Genitourinary cell-matrix structure for implantation into a human and a method of making
CA1340581C (en) 1986-11-20 1999-06-08 Joseph P. Vacanti Chimeric neomorphogenesis of organs by controlled cellular implantation using artificial matrices
NZ229354A (en) 1988-07-01 1990-09-26 Becton Dickinson Co Treating polymer surfaces with a gas plasma and then applying a layer of endothelial cells to the surface
EP0363125A3 (en) 1988-10-03 1990-08-16 Hana Biologics Inc. Proliferated pancreatic endocrine cell product and process
US5837539A (en) 1990-11-16 1998-11-17 Osiris Therapeutics, Inc. Monoclonal antibodies for human mesenchymal stem cells
US5795965A (en) 1991-04-25 1998-08-18 Chugai Seiyaku Kabushiki Kaisha Reshaped human to human interleukin-6 receptor
US5449383A (en) 1992-03-18 1995-09-12 Chatelier; Ronald C. Cell growth substrates
GB9206861D0 (en) 1992-03-28 1992-05-13 Univ Manchester Wound healing and treatment of fibrotic disorders
CA2114282A1 (en) 1993-01-28 1994-07-29 Lothar Schilder Multi-layered implant
JP3525221B2 (en) 1993-02-17 2004-05-10 味の素株式会社 Immunosuppressants
US5523226A (en) 1993-05-14 1996-06-04 Biotechnology Research And Development Corp. Transgenic swine compositions and methods
GB9310557D0 (en) 1993-05-21 1993-07-07 Smithkline Beecham Plc Novel process and apparatus
TW257671B (en) 1993-11-19 1995-09-21 Ciba Geigy
US6001647A (en) 1994-04-28 1999-12-14 Ixion Biotechnology, Inc. In vitro growth of functional islets of Langerhans and in vivo uses thereof
US5834308A (en) 1994-04-28 1998-11-10 University Of Florida Research Foundation, Inc. In vitro growth of functional islets of Langerhans
US6703017B1 (en) 1994-04-28 2004-03-09 Ixion Biotechnology, Inc. Reversal of insulin-dependent diabetes by islet-producing stem cells, islet progenitor cells and islet-like structures
US6083903A (en) 1994-10-28 2000-07-04 Leukosite, Inc. Boronic ester and acid compounds, synthesis and uses
CA2209124C (en) 1994-12-29 2010-03-23 Chugai Seiyaku Kabushiki Kaisha Antitumor agent effect enhancer containing il-6 antagonist
US5843780A (en) 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US5718922A (en) 1995-05-31 1998-02-17 Schepens Eye Research Institute, Inc. Intravitreal microsphere drug delivery and method of preparation
US5908782A (en) 1995-06-05 1999-06-01 Osiris Therapeutics, Inc. Chemically defined medium for human mesenchymal stem cells
US5965583A (en) 1997-04-24 1999-10-12 Ortho-Mcneil Pharmaceutical, Inc. Substituted imidazoles useful in the treatment of inflammatory disease
PT1028737E (en) 1997-07-03 2007-07-11 Osiris Therapeutics Inc Human mesenchymal stem cells from peripheral blood
US6670127B2 (en) 1997-09-16 2003-12-30 Egea Biosciences, Inc. Method for assembly of a polynucleotide encoding a target polypeptide
ES2340857T3 (en) 1997-09-16 2010-06-10 Centocor Ortho Biotech Inc. METHOD FOR COMPLETE CHEMICAL SYNTHESIS AND EXEMPTION OF GENES AND GENOMES.
AU729377B2 (en) 1997-10-23 2001-02-01 Asterias Biotherapeutics, Inc. Methods and materials for the growth of primate-derived primordial stem cells in feeder-free culture
US6372779B1 (en) 1997-12-29 2002-04-16 Ortho Pharmaceutical Corporation Anti-inflammatory compounds
JP4740452B2 (en) 1998-03-18 2011-08-03 オシリス セラピューティクス，インコーポレイテッド Methods, compositions and methods of using mesenchymal stem cells for the prevention and treatment of immune responses in transplantation
MY132496A (en) 1998-05-11 2007-10-31 Vertex Pharma Inhibitors of p38
US7410798B2 (en) 2001-01-10 2008-08-12 Geron Corporation Culture system for rapid expansion of human embryonic stem cells
US6667176B1 (en) 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
US6610540B1 (en) 1998-11-18 2003-08-26 California Institute Of Technology Low oxygen culturing of central nervous system progenitor cells
US6413556B1 (en) 1999-01-08 2002-07-02 Sky High, Llc Aqueous anti-apoptotic compositions
WO2000043500A2 (en) 1999-01-21 2000-07-27 Vitro Diagnostics, Inc. Immortalized cell lines and methods of making the same
US6815203B1 (en) 1999-06-23 2004-11-09 Joslin Diabetes Center, Inc. Methods of making pancreatic islet cells
US6333029B1 (en) 1999-06-30 2001-12-25 Ethicon, Inc. Porous tissue scaffoldings for the repair of regeneration of tissue
US6306424B1 (en) 1999-06-30 2001-10-23 Ethicon, Inc. Foam composite for the repair or regeneration of tissue
US6685936B2 (en) 1999-10-12 2004-02-03 Osiris Therapeutics, Inc. Suppressor cells induced by culture with mesenchymal stem cells for treatment of immune responses in transplantation
US20030082155A1 (en) 1999-12-06 2003-05-01 Habener Joel F. Stem cells of the islets of langerhans and their use in treating diabetes mellitus
AU778155B2 (en) 1999-12-13 2004-11-18 Scripps Research Institute, The Markers for identification and isolation of pancreatic islet alpha and beta cell progenitors
US7005252B1 (en) 2000-03-09 2006-02-28 Wisconsin Alumni Research Foundation Serum free cultivation of primate embryonic stem cells
US7439064B2 (en) 2000-03-09 2008-10-21 Wicell Research Institute, Inc. Cultivation of human embryonic stem cells in the absence of feeder cells or without conditioned medium
US6436704B1 (en) 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
US6458589B1 (en) 2000-04-27 2002-10-01 Geron Corporation Hepatocyte lineage cells derived from pluripotent stem cells
EP2295539A1 (en) 2000-06-26 2011-03-16 Nc Medical Research Inc. Cell fractions containing cells capable of differentiating into neural cells
EP1333833B1 (en) 2000-10-23 2011-08-24 GlaxoSmithKline LLC Novel trisubstituted-8H-pyrido[2,3-d]pyrimidin-7-one compound for the treatment of CSBP/p38 kinase mediated diseases
MXPA03005139A (en) 2000-12-08 2004-01-29 Ortho Mcneil Pharm Inc Macroheterocylic compounds useful as kinase inhibitors.
YU46603A (en) 2000-12-08 2006-05-25 Ortho-Mcneil Pharmaceutical Inc. Indazolyl-substituted pyrroline compounds as kinase inhibitors
US6599323B2 (en) 2000-12-21 2003-07-29 Ethicon, Inc. Reinforced tissue implants and methods of manufacture and use
US20040121460A1 (en) 2001-01-24 2004-06-24 Lumelsky Nadya L Differentiation of stem cells to pancreatic endocrine cells
CA2435146C (en) 2001-01-25 2011-03-29 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Formulation of boronic acid compounds
US6656488B2 (en) 2001-04-11 2003-12-02 Ethicon Endo-Surgery, Inc. Bioabsorbable bag containing bioabsorbable materials of different bioabsorption rates for tissue engineering
JP2004527249A (en) 2001-04-19 2004-09-09 デヴェロゲン アクチエンゲゼルシャフト フュア エントヴィックルングスビオローギッシェ フォルシュング Method of differentiating stem cells into insulin producing cells
CN100516204C (en) 2001-04-24 2009-07-22 味之素株式会社 Stem cells and method of separating the same
EP1393066A4 (en) 2001-05-15 2006-01-25 Rappaport Family Inst For Res Insulin producing cells derived from human embryonic stem cells
US6626950B2 (en) 2001-06-28 2003-09-30 Ethicon, Inc. Composite scaffold with post anchor for the repair and regeneration of tissue
KR100418195B1 (en) 2001-07-05 2004-02-11 주식회사 우리기술 Apparatus and method for multi-testing insulation of power cables
GB0117583D0 (en) 2001-07-19 2001-09-12 Astrazeneca Ab Novel compounds
AU2002319780A1 (en) 2001-08-06 2003-02-24 Bresagen, Ltd. Alternative compositions and methods for the culture of stem cells
US6617152B2 (en) 2001-09-04 2003-09-09 Corning Inc Method for creating a cell growth surface on a polymeric substrate
WO2003033697A1 (en) 2001-10-18 2003-04-24 Ixion Biotechnology, Inc. Conversion of liver stem and progenitor cells to pancreatic functional cells
EP1442115B9 (en) 2001-11-15 2009-12-16 Children's Medical Center Corporation Methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof
AU2002357135C1 (en) 2001-12-07 2009-01-22 Macropore Biosurgery, Inc. Systems and methods for treating patients with processed lipoaspirate cells
GB2415432B (en) 2001-12-07 2006-09-06 Geron Corp Islet cells from human embryonic stem cells
AU2002218893A1 (en) 2001-12-21 2003-07-09 Thromb-X Nv Compositions for the in vitro derivation and culture of embryonic stem (es) cell lines with germline transmission capability
US20030162290A1 (en) 2002-01-25 2003-08-28 Kazutomo Inoue Method for inducing differentiation of embryonic stem cells into functioning cells
WO2003087349A1 (en) 2002-04-17 2003-10-23 Otsuka Pharmaceutical Co., Ltd. METHOD OF FORMING PANCREATIC β CELLS FROM MESENCHYMAL CELLS
US20040161419A1 (en) 2002-04-19 2004-08-19 Strom Stephen C. Placental stem cells and uses thereof
ATE387444T1 (en) 2002-05-08 2008-03-15 Janssen Pharmaceutica Nv SUBSTITUTED PYRROLINES AS KINASE INHIBITORS
US20060003446A1 (en) 2002-05-17 2006-01-05 Gordon Keller Mesoderm and definitive endoderm cell populations
WO2003102134A2 (en) 2002-05-28 2003-12-11 Becton, Dickinson And Company Pancreatic acinar cells into insulin producing cells
AU2003238874A1 (en) 2002-06-05 2003-12-22 Janssen Pharmaceutica N.V. Bisindolyl-maleimid derivatives as kinase inhibitors
GB0212976D0 (en) 2002-06-06 2002-07-17 Tonejet Corp Pty Ltd Ejection method and apparatus
CN1171991C (en) 2002-07-08 2004-10-20 徐如祥 Culture process of human nerve stem cell
US6877147B2 (en) 2002-07-22 2005-04-05 Broadcom Corporation Technique to assess timing delay by use of layout quality analyzer comparison
US7838290B2 (en) 2002-07-25 2010-11-23 The Scripps Research Institute Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
EP1539930A4 (en) 2002-07-29 2006-08-09 Es Cell Int Pte Ltd Multi-step method for the differentiation of insulin positive, glucose
WO2004016747A2 (en) 2002-08-14 2004-02-26 University Of Florida Bone marrow cell differentiation
WO2004023100A2 (en) 2002-09-06 2004-03-18 Amcyte Inc. Cd56 positive human adult pancreatic endocrine progenitor cells
US9969977B2 (en) 2002-09-20 2018-05-15 Garnet Biotherapeutics Cell populations which co-express CD49c and CD90
US20040062753A1 (en) 2002-09-27 2004-04-01 Alireza Rezania Composite scaffolds seeded with mammalian cells
US20060252150A1 (en) 2002-11-08 2006-11-09 Linzhao Cheng Human embryonic stem cell cultures, and compositions and methods for growing same
US7144999B2 (en) 2002-11-23 2006-12-05 Isis Pharmaceuticals, Inc. Modulation of hypoxia-inducible factor 1 alpha expression
US20060040385A1 (en) 2002-12-05 2006-02-23 Technion Research & Development Foundation Ltd. Cultured human pancreatic islets, and uses thereof
DK1572984T3 (en) 2002-12-16 2016-06-13 Technion Res & Dev Foundation FEEDER CELL-FREE, XENOPHRIC CULTIVATION SYSTEM FOR HUMAN EMBRYONAL STEM CELLS
WO2005045001A2 (en) 2003-02-14 2005-05-19 The Board Of Trustees Of The Leland Stanford Junior University Insulin-producing cells derived from stem cells
US20070155661A1 (en) 2003-02-14 2007-07-05 The Board Of Trustees Of The Leland Standord Junior University Methods and compositions for modulating the development of stem cells
US20070020242A1 (en) 2003-03-27 2007-01-25 Ixion Biotechnology, Inc. Method for transdifferentiation of non-pancreatic stem cells to the pancreatic pathway
US20060194315A1 (en) 2003-03-31 2006-08-31 Condie Brian G Compositions and methods for the control, differentiaton and/or manipulation of pluripotent cells through a gamma-secretase signaling pathway
US20090203141A1 (en) 2003-05-15 2009-08-13 Shi-Lung Lin Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents
PL1641913T3 (en) 2003-06-27 2016-06-30 Depuy Synthes Products Inc Postpartum cells derived from umbilical cord tissue, and methods of making and using the same
IL161903A0 (en) 2003-07-17 2005-11-20 Gamida Cell Ltd Ex vivo progenitor and stem cell expansion for usein the treatment of disease of endodermally- deri ved organs
ITRM20030395A1 (en) 2003-08-12 2005-02-13 Istituto Naz Per Le Malattie Infettive Lazz CULTURE GROUND FOR MAINTENANCE, PROLIFERATION AND DIFFERENTIATION OF MAMMALIAN CELLS.
US7569385B2 (en) 2003-08-14 2009-08-04 The Regents Of The University Of California Multipotent amniotic fetal stem cells
US7157275B2 (en) 2003-08-15 2007-01-02 Becton, Dickinson And Company Peptides for enhanced cell attachment and growth
WO2005021728A2 (en) 2003-08-27 2005-03-10 Stemcells California, Inc. Enriched pancreatic stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for these populations
JP2007515433A (en) 2003-12-17 2007-06-14 アラーガン　インコーポレイテッド Methods of treating retinoid responsive disorders using selective inhibitors of CYP26A and CYP26B
US20060030042A1 (en) 2003-12-19 2006-02-09 Ali Brivanlou Maintenance of embryonic stem cells by the GSK-3 inhibitor 6-bromoindirubin-3'-oxime
US7625753B2 (en) 2003-12-23 2009-12-01 Cythera, Inc. Expansion of definitive endoderm cells
US20050266554A1 (en) 2004-04-27 2005-12-01 D Amour Kevin A PDX1 expressing endoderm
DK1709159T3 (en) 2003-12-23 2019-07-29 Viacyte Inc DEFINITIVE ENDODERM
TWI334443B (en) 2003-12-31 2010-12-11 Ind Tech Res Inst Method of single cell culture of undifferentiated human embryonic stem cells
US20050233446A1 (en) 2003-12-31 2005-10-20 Parsons Xuejun H Defined media for stem cell culture
US20080241107A1 (en) 2004-01-23 2008-10-02 Copland Iii John A Methods and Compositions For Preparing Pancreatic Insulin Secreting Cells
GB2441530B (en) 2004-02-12 2009-09-23 Univ Newcastle Stem Cells
US20060281174A1 (en) 2004-03-09 2006-12-14 Gang Xu Methods for generating insulin-producing cells
AU2005221079B2 (en) 2004-03-10 2010-07-22 Regents Of The University Of California Compositions and methods for growth of embryonic stem cells
EP1730268A2 (en) 2004-04-01 2006-12-13 Wisconsin Alumni Research Foundation Differentiation of stem cells to endoderm and pancreatic lineage
KR101278421B1 (en) 2004-04-27 2013-07-15 비아싸이트, 인크. Pdx1 expressing endoderm
EP1786896B1 (en) 2004-07-09 2018-01-10 Viacyte, Inc. Methods for identifying factors for differentiating definitive endoderm
US8187878B2 (en) 2004-08-13 2012-05-29 University Of Georgia Research Foundation, Inc. Methods for increasing definitive endoderm differentiation of pluripotent human embryonic stem cells with PI-3 kinase inhibitors
US20080268533A1 (en) 2004-08-25 2008-10-30 University Of Georgia Research Foundation, Inc. Methods and Compositions Utilizing Myc and Gsk3Beta to Manipulate the Pluripotency of Embryonic Stem Cells
DE102004043256B4 (en) 2004-09-07 2013-09-19 Rheinische Friedrich-Wilhelms-Universität Bonn Scalable process for culturing undifferentiated stem cells in suspension
DK3196296T3 (en) 2004-09-08 2019-02-04 Wisconsin Alumini Res Foundation Cultivation of human embryonic stem cells
KR101264940B1 (en) 2004-09-08 2013-05-15 위스콘신 얼럼나이 리서어치 화운데이션 Medium and culture of embryonic stem cells
US7124937B2 (en) 2005-01-21 2006-10-24 Visa U.S.A. Inc. Wireless payment methods and systems
JP2008528038A (en) * 2005-01-31 2008-07-31 エス セル インターナショナル ピーティーイー リミテッド Directed differentiation of embryonic stem cells and their use
AU2006218359A1 (en) 2005-03-04 2006-09-08 John O'neil Adult pancreatic derived stromal cells
GB0505970D0 (en) 2005-03-23 2005-04-27 Univ Edinburgh Culture medium containing kinase inhibitor, and uses thereof
EP1876893B1 (en) 2005-04-15 2012-04-11 Geron Corporation Cancer treatment by combined inhibition of proteasome and telomerase activities
EP1874367B1 (en) 2005-04-26 2011-07-06 Arhus Universitet Biocompatible material for surgical implants and cell guiding tissue culture surfaces
WO2006135824A1 (en) 2005-06-10 2006-12-21 Irm Llc Compounds that maintain pluripotency of embryonic stem cells
WO2006138433A2 (en) 2005-06-14 2006-12-28 The Regents Of The University Of California Induction of cell differentiation by class i bhlh polypeptides
WO2006137787A1 (en) 2005-06-21 2006-12-28 Ge Healthcare Bio-Sciences Ab Method for cell culture
UA92608C2 (en) 2005-06-30 2010-11-25 Янссен Фармацевтика Н.В. Cyclic anilino - pyridinotriazines as gsk-3 inhibitors
AU2006274438A1 (en) 2005-07-29 2007-02-01 Australian Stem Cell Centre Limited Compositions and methods for growth of pluripotent cells
US20080194021A1 (en) 2005-07-29 2008-08-14 Mays Robert W Use of a Gsk-3 Inhibitor to Maintain Potency of Culture Cells
JP2009506770A (en) 2005-09-02 2009-02-19 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ Induction of progenitor cell lines
US9422521B2 (en) 2005-09-12 2016-08-23 Es Cell International Pte Ltd. Differentiation of pluripotent stem cells with a kinase inhibitor or PGI2
WO2007047509A2 (en) 2005-10-14 2007-04-26 Regents Of The University Of Minnesota Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype
DK2674485T3 (en) 2005-10-27 2019-08-26 Viacyte Inc PDX-1 EXPRESSING DORSAL AND VENTRAL FORTARM ENDODERM
WO2007082963A1 (en) 2006-01-18 2007-07-26 Fundación Instituto Valenciano De Infertilidad Human embryo stem-cell lines and methods for using same
EP1994141B1 (en) 2006-02-23 2017-11-15 ViaCyte, Inc. Compositions and methods useful for culturing differentiable cells
EP2650360B1 (en) 2006-03-02 2019-07-24 Viacyte, Inc. Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production
US7695965B2 (en) 2006-03-02 2010-04-13 Cythera, Inc. Methods of producing pancreatic hormones
US8741643B2 (en) 2006-04-28 2014-06-03 Lifescan, Inc. Differentiation of pluripotent stem cells to definitive endoderm lineage
AU2007244675A1 (en) 2006-04-28 2007-11-08 Lifescan, Inc. Differentiation of human embryonic stem cells
SE534150C2 (en) 2006-05-02 2011-05-10 Wisconsin Alumni Res Found Procedure for differentiating stem cells into cells of the endodermal and pancreatic developmental line
WO2007139929A2 (en) 2006-05-25 2007-12-06 The Burnham Institute For Medical Research Methods for culture and production of single cell populations of human embryonic stem cells
US8415153B2 (en) 2006-06-19 2013-04-09 Geron Corporation Differentiation and enrichment of islet-like cells from human pluripotent stem cells
CN100494359C (en) 2006-06-23 2009-06-03 中日友好医院 Method for in vitro amplifying and in 3D solid culturing for nerve stem cell
CA2893679C (en) 2006-06-26 2018-10-30 Lifescan, Inc. Conditioned medium for pluripotent stem cell culture
US20080003676A1 (en) 2006-06-26 2008-01-03 Millipore Corporation Growth of embryonic stem cells
GB2454386B (en) 2006-07-06 2011-07-06 Es Cell Int Pte Ltd Method for embryonic stem cell culture on a positively charged support surface
AU2007277364B2 (en) 2006-07-26 2010-08-12 Viacyte, Inc. Methods of producing pancreatic hormones
KR101331510B1 (en) 2006-08-30 2013-11-20 재단법인서울대학교산학협력재단 Media compostions containing low concentrations of glucose useful for human embryonic stem cells, differentiation method of human embryonic stem cells into insulin-producing cells or cell clusters using thereof, and insulin-producing cells or cell clusters differentiated thereby
JP2008099662A (en) 2006-09-22 2008-05-01 Institute Of Physical & Chemical Research Method for culturing stem cell
WO2008039521A2 (en) 2006-09-26 2008-04-03 Nmt Medical, Inc. Method for modifying a medical implant surface for promoting tissue growth
WO2008048647A1 (en) 2006-10-17 2008-04-24 Cythera, Inc. Modulation of the phosphatidylinositol-3-kinase pathway in the differentiation of human embryonic stem cells
US8835163B2 (en) 2006-10-18 2014-09-16 The Board Of Trustees Of The University Of Illinois Embryonic-like stem cells derived from adult human peripheral blood and methods of use
TW200836749A (en) 2007-01-09 2008-09-16 Vioquest Pharmaceuticals Inc Compositions including triciribine and bortezomib and derivatives thereof and methods of use thereof
EP2126045A4 (en) 2007-01-30 2010-05-26 Univ Georgia Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (mmc)
GB0703188D0 (en) 2007-02-19 2007-03-28 Roger Land Building Large scale production of stem cells
CA2693156C (en) 2007-07-18 2018-03-06 Alireza Rezania Differentiation of human embryonic stem cells
RU2473685C2 (en) 2007-07-31 2013-01-27 Лайфскен, Инк. Differentiation of human embryo stem cells
EP2190413B1 (en) 2007-08-24 2015-01-28 Stichting Het Nederlands Kanker Instituut Compositions for the treatment of neoplastic diseases
ATE523585T1 (en) 2007-11-27 2011-09-15 Lifescan Inc DIFFERENTIATION OF HUMAN EMBRYONAL STEM CELLS
SG154367A1 (en) 2008-01-31 2009-08-28 Es Cell Int Pte Ltd Method of differentiating stem cells
EP2250252A2 (en) 2008-02-11 2010-11-17 Cambridge Enterprise Limited Improved reprogramming of mammalian cells, and the cells obtained
KR20190057164A (en) 2008-02-21 2019-05-27 얀센 바이오테크 인코포레이티드 Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
EP2271747B1 (en) 2008-03-17 2016-09-07 Agency for Science, Technology And Research Microcarriers for pluripotent stem cell culture
AU2008355123B2 (en) 2008-04-21 2014-12-04 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
US8728812B2 (en) 2008-04-22 2014-05-20 President And Fellows Of Harvard College Compositions and methods for promoting the generation of PDX1+ pancreatic cells
US20090298178A1 (en) 2008-06-03 2009-12-03 D Amour Kevin Allen Growth factors for production of definitive endoderm
JP5734183B2 (en) 2008-06-30 2015-06-17 ヤンセン バイオテツク，インコーポレーテツド Differentiation of pluripotent stem cells
DE102008032236A1 (en) 2008-06-30 2010-04-01 Eberhard-Karls-Universität Tübingen Isolation and / or identification of stem cells with adipocytic, chondrocytic and pancreatic differentiation potential
US20100028307A1 (en) 2008-07-31 2010-02-04 O'neil John J Pluripotent stem cell differentiation
CN102272291B (en) 2008-10-31 2018-01-16 詹森生物科技公司 Differentiation of the human embryo stem cell to pancreatic endocrine pedigree
US8008075B2 (en) 2008-11-04 2011-08-30 Viacyte, Inc. Stem cell aggregate suspension compositions and methods of differentiation thereof
SG177416A1 (en) 2009-07-20 2012-02-28 Janssen Biotech Inc Differentiation of human embryonic stem cells
SG183400A1 (en) 2010-03-02 2012-09-27 Univ Singapore Culture additives to boost stem cell proliferation and differentiation response

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