WO2015065524A2 - Mise en suspension et agrégation de cellules souches pluripotentes humaines aux fins de différenciation en cellules endocrines du pancréas - Google Patents

Mise en suspension et agrégation de cellules souches pluripotentes humaines aux fins de différenciation en cellules endocrines du pancréas Download PDF

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WO2015065524A2
WO2015065524A2 PCT/US2014/038993 US2014038993W WO2015065524A2 WO 2015065524 A2 WO2015065524 A2 WO 2015065524A2 US 2014038993 W US2014038993 W US 2014038993W WO 2015065524 A2 WO2015065524 A2 WO 2015065524A2
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cells
cell
differentiation
stage
pluripotent
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PCT/US2014/038993
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WO2015065524A3 (fr
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Benjamin Fryer
Daina LANIAUSKAS
Marcia BLACKMOORE
Haiyun Wang
Kostadinka LILOVA
Shelley Nelson
Elizabeth ROSOCHA
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Janssen Biotech, Inc.
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Priority claimed from US13/998,974 external-priority patent/US10377989B2/en
Priority to MX2016005657A priority Critical patent/MX2016005657A/es
Priority to KR1020187034291A priority patent/KR20180128529A/ko
Priority to CN201480059910.5A priority patent/CN105793413A/zh
Priority to JP2016527338A priority patent/JP2016534731A/ja
Priority to EP14857034.4A priority patent/EP3063268A4/fr
Priority to AU2014342995A priority patent/AU2014342995C1/en
Priority to CA2928639A priority patent/CA2928639A1/fr
Application filed by Janssen Biotech, Inc. filed Critical Janssen Biotech, Inc.
Priority to KR1020167014409A priority patent/KR20160079072A/ko
Priority to SG11201603045VA priority patent/SG11201603045VA/en
Priority to RU2016121409A priority patent/RU2689710C2/ru
Publication of WO2015065524A2 publication Critical patent/WO2015065524A2/fr
Publication of WO2015065524A3 publication Critical patent/WO2015065524A3/fr
Priority to PH12016500782A priority patent/PH12016500782A1/en
Priority to AU2018208707A priority patent/AU2018208707A1/en

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Definitions

  • the present invention is in the field of cell differentiation including preparing embryonic stem cells and other pluripotent cells that maintain pluripotency in aggregated cell cluster for differentiation to endoderm progenitor cells, pancreatic endocrine cells, mesoderm cells or ectoderm cells.
  • the invention discloses a method of generating clusters of pluripotent stem cells and maintaining them in suspension culture for differentiation to pancreatic endoderm, pancreatic endocrine precursor cells, and single-hormone pancreatic endocrine 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, 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.
  • the endoderm is partitioned into anterior-posterior domains that can be recognized by the expression of a panel of factors that uniquely mark anterior, mid, and posterior regions of the endoderm. For example, HHEX, and SOX2 identify the anterior region while CDX1, 2, and 4 identify the posterior region of the endoderm.
  • FGFs secreted factors
  • Wnts Wnts
  • TGF-Bs retinoic acid
  • RA retinoic acid
  • BMP ligands and their antagonists BMP ligands and their antagonists.
  • FGF4 and BMP are reported to promote CDX2 expression in the presumptive hindgut endoderm and repress expression of the anterior genes HHEX and SOX2 (2000 Development, 127: 1563-1567). WNT signaling has also been shown to work in parallel to FGF signaling to promote hindgut development and inhibit foregut fate (2007 Development, 134:2207-2217).
  • secreted retinoic acid by mesenchyme regulates the foregut-hindgut boundary 2002 Curr Biol, 12: 1215-1220).
  • the level of expression of specific transcription factors may be used to designate the identity of a tissue.
  • the gut tube becomes regionalized into broad domains that can be observed at the molecular level by restricted gene expression patterns.
  • the regionalized pancreas domain in the gut tube shows a very high expression of PDXl and very low expression of CDX2 and SOX2.
  • PDXl, NKX6.1, PTFIA, and KX2.2 are highly expressed in pancreatic tissue; and expression of CDX2 is high in intestine tissue.
  • pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm.
  • Dorsal and ventral pancreatic domains arise from the foregut epithelium.
  • Foregut also gives rise to the esophagus, trachea, lungs, thyroid, stomach, liver, pancreas, and bile duct system.
  • pancreatic-duodenal homeobox gene PDXl a gene that influences pancreatic-duodenal homeobox gene.
  • pancreas fails to develop beyond the formation of ventral and dorsal buds.
  • PDXl expression marks a critical step in pancreatic organogenesis.
  • the mature pancreas contains both, exocrine tissue and endocrine tissue arising from the differentiation of pancreatic endoderm.
  • 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 (Nature Biotechnol 2005, 23 : 1534-1541 ; U.S. Patent No. 7,704,738).
  • pancreatic islet cells [Oil] Fisk et al. report a system for producing pancreatic islet cells from human embryonic stem cells (U.S. Patent No. 7,033,831). Small molecule inhibitors have also been used for induction of pancreatic endocrine precursor cells. For example, small molecule inhibitors of TGF- ⁇ receptor and BMP receptors (Development 201 1, 138:861-871; Diabetes 201 1, 60:239-247) have been used to significantly enhance the number of pancreatic endocrine cells. In addition, small molecule activators have also been used to generate definitive endoderm cells or pancreatic precursor cells (Curr Opin Cell Biol 2009, 21 :727- 732; Nature Chem Biol 2009, 5:258-265).
  • U.S. Patent App. Pub. No. 2008/0159994 discloses a method of culturing human embryonic stem cells encapsulated within alginate beads in a three- dimensional culture system.
  • Figure la shows micrographs of Dispase ® -treated cells of the human embryonic stem (“hES") cell line HI immediately after lifting (left hand panel) and after 24 hours in non-adherent static culture (right hand panel) according to Example 1.
  • the cells after lifting (left hand panel) resembled fragments of monolayer with an average fragment diameter of about 20-30 microns each fragment consisting of clumps of cells.
  • the cells assumed a cluster-like configuration.
  • Figure lb shows the results of flow cytometry for CD9, SSEA4, CXCR4, TRA-1-60 and TRA-1-81 for the Dispase ® -treated cells of the human embryonic stem cell line HI after culturing for 4 days in a 125 mL spinner flask containing 25 mL mTeSR ® l media according to Example 1.
  • the cells exhibited high expression for markers of pluripotency (CD9, SSEA4, TRA-1-60 and TRA-1-81) with almost no expression of CXCR4, a marker for differentiation.
  • Figure lc shows micrographs of the Dispase ® -treated cells of the human embryonic stem cell line HI after 72 and 96 hours of differentiation at the end of stage 1. Visible in Figure lc are loose cell aggregates after 72 hours at 4X magnification (left hand panel), 96 hours at 4X magnification (center panel) and 96 hours at 10X magnification (right hand panel).
  • Figure Id shows flow cytometry results for the Dispase ® -treated cells of the human embryonic stem cell line HI at the end of stage 1 differentiation for the markers CD9, CD 184 (CXCR4) and CD99 (see Example 1). As shown in Figure Id, expression of CD9, a marker for pluripotency, was nearly eliminated, while the expression of markers of definitive endoderm differentiation CXCR4 (CD 184) and CD99 were quite high.
  • Figure le shows quantitative reverse transcription polymerase chain reaction (qRT- PCR) results for expression of selected genes associated with pluripotency and genes associated with definitive endoderm for the Dispase ® -treated cells of the human embryonic stem cell line HI at the end of stage 1 compared to undifferentiated HI (WA01) hES cells (see Example 1).
  • the cells at the end of stage 1 showed a dramatic decrease in the expression of pluripotency genes (CD9, NANOG, and POU5F1/OCT4) and a large increase in genes associated with definitive endoderm (CXCR4, CERBERUS (CER1), GSC, FOXA2, GATA4, GATA6, MNX1, and SOX 17) versus undifferentiated WA01 hES cells.
  • Figure If shows micrographs of the Dispase ® -treated cells of the human embryonic stem cell line HI as the cells further differentiated from definitive endoderm toward the pancreatic endoderm (see Example 1). Clear morphological changes to cells and cell clusters are visible as differentiation progresses from stage 2, day 1 (top left hand panel) to stage 2, day 3 (top right hand panel) to stage 3, day 4 (lower left hand panel) and stage 4, day 1 (lower right hand panel).
  • Figure 2a shows flow cytometry data for EDTA-treated cells of the human embryonic stem cell line HI after 2 days of culture in stirred suspension culture post-EDTA treatment, and prior to transition to differentiation culture, for markers associated with pluripotency and differentiation according to Example 2.
  • the data showed high expression for the markers of pluripotency (CD9, SSEA4, TRA-1-60, and TRA-1-81) with almost no expression of a marker for differentiation (CXCR4).
  • Figure 2b shows micrographs of the EDTA-treated cells of the human embryonic stem cell line HI differentiated into stage 1, day 3 cells grown in spinner flask and stage 2 day 2, stage 4 day 1 and stage 4 day 3 cells grown in spinner flasks or Erlenmeyer flasks according to Example 2.
  • Suspension differentiated cultures formed substantially uniform and homogenous populations of cells in spherical aggregates.
  • Figure 2c shows flow cytometry data for the EDTA-treated cells of the human embryonic stem cell line HI at the end of stage 1 for cell surface markers of pluripotency and endoderm differentiation.
  • expression of CD9 a marker for pluripotency
  • CXCR4 CD 184
  • Figure 2d shows qRT-PCR results for expression of selected genes associated with pluripotency and genes associated with definitive endoderm for the EDTA-treated cells of the human embryonic stem cell line HI at the end of stage 1 compared to undifferentiated HI (WA01) hES cells (see Example 2).
  • Figure 2d shows a decrease in the expression of pluripotency genes (CD9, Nanog, and POU5F1/OCT4) and a large increase in genes associated with definitive endoderm (CXCR4, CERBERUS (“CER1”), FOXA2, GATA4, GATA6, MNX1, and SOX 17).
  • Figure 2e shows flow cytometry data for markers indicative of differentiation (NKX6.1, CDX2, SOX2, and Chromagranin) for the EDTA-treated cells of the human embryonic stem cell line HI which were differentiated from stage 1 to pancreatic endoderm cells by suspension in spinner flasks or Erlenmeyer flasks according to Example 2.
  • the flow cytometry data shows high levels of NKX6.1, a transcription factor required for functional ⁇ cells, and high levels of endocrine pancreas markers such as synaptophysin (data not shown) and chromogranin with both suspension formats.
  • Figure 2f shows qRT-PCR results for expression of selected genes associated with differentiation for the EDTA-treated cells of the human embryonic stem cell line HI which were further differentiated from stage 1 to pancreatic endoderm cells by suspension in spinner flasks or Erlenmeyer flasks according to Example 2. The data is compared to expression in WA01 hES cells. The RT-PCR results show high levels of expression of pancreatic precursor genes.
  • Figure 3 a shows a micrograph of cells of the human embryonic stem cell line HI, which had been lifted from a static culture following treatment with Accutase ® . As shown in
  • Figure 3b shows phase contrast micrographs of cells of the human embryonic stem cell line HI, which had been lifted from a static culture following treatment with Accutase ® and which were then expanded in suspension culture for three days. Visible in Figure 3b is the formation of a substantially uniform, spherical population of cell clusters.
  • Figure 3c shows a micrograph of clusters of cells of the human embryonic stem cell line HI, which had been lifted from a static culture following treatment with Accutase ® , which were then expanded in suspension culture for three days, and which were then serially passaged using Accutase ® dissociation.
  • Figure 4a shows micrographs of suspension cultured human embryonic stem cells of the cell line HI using a directed differentiation protocol at different stages of differentiation. Visible in Figure 4a are micrographs of the cells at each stage of differentiation.
  • Figure 4b shows the results of flow cytometry for markers of differentiation (CXCR4, CD56 and PDX1) for suspension cultured human embryonic stem cells of the cell line HI using a directed differentiation protocol at different stages of differentiation (hours after beginning differentiation). At the end of the differentiation process on day 4 of stage 4, a high percentage of the cells were positive for PDX1 expression.
  • markers of differentiation CXCR4, CD56 and PDX1
  • Figure 4c shows the non-fasting blood glucose levels of SCID-Bg Mice transplanted with differentiated cells encapsulated in a TheraCyteTM device.
  • Figure 5 a shows flow cytometry data for the EDTA-treated cells of the human embryonic stem cell line HI prior to transition to differentiation culture for markers associated with pluripotency and differentiation. As shown in Figure 5a, high expression of the pluripotency markers CD9, SSEA4, TRA-1-60 and TRA-1-80 was observed.
  • Figure 5b shows phase contrast images of the cells and flow cytometry data for CXCR4/CD184 and CD99 (markers of differentiation) and CD9 (a pluripotency marker) for three different feed settings during stage 1.
  • the conditions tested were as follows: (A) media change 24 hours after initiation of differentiation, no media change at 48 hours; (B) media change 24 hours after initiation of differentiation and glucose bolus addition at 48 hours; and (C) no media change throughout stage 1 with glucose and GDF8 bolus added 24 hours after initiation of differentiation, then a glucose bolus added at 48 hours post initiation.
  • Figure 5c shows phase contrast images of the differentiated cells exhibiting pancreatic endoderm morphology, which were differentiated using the following feed settings during the formation of definitive endoderm: (A) media change 24 hours after initiation of differentiation, no media change at 48 hours; (B) media change 24 hours after initiation of differentiation and glucose bolus addition at 48 hours; and (C) no media change throughout stage 1 with glucose and GDF8 bolus added 24 hours after initiation of differentiation, then a glucose bolus added at 48 hours post initiation.
  • Figure 5d shows the results of flow cytometry for select markers of pancreatic gene expression (NKX6.1 and chromogranin) and select non-pancreatic genes (CDX2 and SOX2) for differentiated cell as the end of stage 4, which were differentiated using the following feed settings during formation of definitive endoderm: (A) media change 24 hours after initiation of differentiation, no media change at 48 hours; (B) media change 24 hours after initiation of differentiation and glucose bolus addition at 48 hours; and (C) no media change throughout stage 1 with glucose and GDF8 bolus added 24 hours after initiation of differentiation, then a glucose bolus added at 48 hours post initiation.
  • NKX6.1 and chromogranin select markers of pancreatic gene expression
  • CDX2 and SOX2 select non-pancreatic genes
  • Figure 5e shows qRT-PCR results for select pancreatic and non-pancreatic gene expression for differentiated cells as the end of stage 4, which were differentiated using the following feed settings during formation of definitive endoderm: (A) media change 24 hours after initiation of differentiation, no media change at 48 hours; (B) media change 24 hours after initiation of differentiation and glucose bolus addition at 48 hours; and (C) no media change throughout stage 1 with glucose and GDF8 bolus added 24 hours after initiation of differentiation, then a glucose bolus added at 48 hours post initiation. The data are shown as fold difference in expression versus undifferentiated HI (WA01) hES cells (baseline expression of 1).
  • Figure 5f shows the expression of C-peptide in SCID-Bg mice that were implanted with cells differentiated according to condition A (media change 24 hours after initiation of differentiation, no media change at 48 hours). Each SCID-Bg mouse was implanted with 5 million of the cells under the kidney capsule. As shown in Figure 5f, by 12 weeks post implantation, human c-peptide was detectable at levels above lng/mL, and at 16 weeks c- peptide levels were an average of 2.5ng/mL.
  • Figure 5g shows the effect of glucose treatment for selected SCID-Bg mice pre- and post-administration (e.g. implantation) of cells differentiated according to condition A (media change 24 hours after initiation of differentiation, no media change at 48 hours).
  • glucose treatment induced a significant increase in circulating human c-peptide from an average of 0.93ng/mL in a fasted state to 2.39ng/mL in a fed state.
  • Figure 5h shows the effect of streptozotocin (STZ) administration (i.e. STZ-induced diabetes) on SCID-Bg mice that had been administered cells differentiated according to condition A (media change 24 hours after initiation of differentiation, no media change at 48 hours).
  • STZ streptozotocin
  • animals with a graft of functional GSIS competent tissue i.e. those that had been administered the cells maintained normal blood glucose levels unlike the untreated controls which developed frank diabetes.
  • Figure 6a shows micrographs of cells of the human embryonic stem cell line HI grown on Cytodex ® 3 microcarrier beads prior to differentiation.
  • Figure 6b shows micrographs of cells of the human embryonic stem cell line HI grown on Cytodex ® 3 microcarrier beads at various stages of differentiation.
  • Figure 6c shows the cell count (cells/cm 2 ) as a function of days of differentiation for cells of the human embryonic stem cell line HI grown and differentiated on plates in media containing Activin A (AA) and W T3A (WTN3A/AA plate), microcarriers in media containing Activin A and W T3A (WTN3A/AA microcarriers), plates in media containing MCX and GDF8 (MCX/GDF8 plate) and microcarriers in media containing MCX and GDF8 (MCX/GDF8 microcarriers).
  • Figure 6d shows the cell count (cells/ml) as a function of days of differentiation for cells of the human embryonic stem cell line HI grown and differentiated on plates in media containing Activin A and W T3A (WTN3A/AA plate), microcarriers in media containing Activin A and W T3A (WTN3A/AA microcarrier), plates in media containing MCX and GDF8 (MCX/GDF8 plate) and microcarriers in media containing MCX and GDF8
  • Figure 6e shows flow cytometry results for the first stage of differentiation of cells grown on a microcarrier culture or planar culture in the presence of: (a) W T3A and AA; or (2) MCX and GDF8 as a dot plot of cell expression of CXCR4/CD 184 (Y-axis) and CD9 (X- axis).
  • Figure 6f shows flow cytometry results for the first stage of differentiation of cells grown on a microcarrier culture or planar culture in the presence of: (a) W T3A and AA; or (2) MCX and GDF8 as total expression of each of the markers (CXCR4 and CD9).
  • Figure 6g shows qRT-PCR results for expression of selected genes associated with differentiation for cells of the human embryonic stem cell line HI, which were differentiated by growth on planar culture or on microcarrier beads in suspension culture in the presence of: (a) W T3A and AA; or (2) MCX and GDF8.
  • Figure 7 shows the cell counts at various stages of differentiation in a Bioreactor from stage 1, day 1 to stage 4, day 3 for cells differentiated according to the protocol of Example 7. Cell counts are shown as million cells/ml as determined by an image-based cytometer ( ucleoCounter®).
  • Figure 8 shows the average daily bioreactor medium pH levels as a function of time (days of differentiation) during the differentiation protocol of Example 7. pH levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 9 shows the average daily bioreactor medium lactate levels as a function of time (days of differentiation) during the differentiation protocol of Example 7. Lactate levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 10 shows the average daily bioreactor medium glucose levels as a function of time (days of differentiation) during the differentiation protocol of Example 7. Glucose levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 11 shows the undifferentiated gene expression, as determined by qRT-PCR, for stage 0, day 1 (i.e. twenty-four hours after inoculation) cells differentiated according to the protocol of Example 7 for the pluripotency array, which contains select genes associated with pluripotency.
  • Figure 12 shows the undifferentiated gene expression, as determined by qRT-PCR, for stage 0, day 1 (i.e. twenty-four hours after inoculation) cells for the definitive endoderm ("DE") array, which contains select genes associated with definitive endoderm (see Example V).
  • Figure 13 shows the undifferentiated gene expression, as determined by qRT-PCR, for stage 0, day 3 (i.e. seventy-two hours after inoculation) cells for the pluripotency array, which contains select genes associated with pluripotency (see Example 7).
  • Figure 14 shows the undifferentiated gene expression, as determined by qRT-PCR, for stage 0, day 3 (i.e. seventy-two hours after inoculation) cells for the DE array, which contains select genes associated with DE (see Example 7).
  • Figure 15 shows the results of fluorescence-activated cell sorting (FACS) for CD9, CD 184/CXCR4, SSEA4, TRA-1-60 and TRA-1-81 for undifferentiated stage 0, day 3 (i.e. seventy-two hours after inoculation) cells (see Example 7). The results are also shown in Table 8.
  • FACS fluorescence-activated cell sorting
  • Figure 16 shows the undifferentiated gene expression, as determined by qRT-PCR, for select genes of stage 0, day 1 (i.e. twenty-four hours after inoculation) and stage 0, day 3 (i.e. seventy-two hours after inoculation) cells differentiated according to the protocol of Example 7. Specifically, Figure 16 shows a modest increase in gene expression for GATA4, GSC, MIXL1, and T and a >100x increase in GATA2 expression during the stage 0 process prior to directed differentiation.
  • Figure 17 shows the undifferentiated gene expression, as determined by qRT-PCR, for the DE array, which contains select genes associated with DE, for stage 0, day 1 (i.e. twenty- four hours after inoculation) and stage 0, day 3 (i.e. seventy -two hours after inoculation) cells differentiated according to the protocol of Example 7. Specifically, Figure 17 shows a >100x increase in CERl, FGF 17, and FGF4 expression during the stage 0 process prior to directed differentiation.
  • Figures 18 and 19 show the gene expression for stage 1, day 1 cells differentiated according to the protocol of Example 7.
  • Figure 18 shows the gene expression, as determined by qRT-PCR, for the pluripotency array, which contains select genes associated with pluripotency, for stage 1, day 1 cells.
  • Figure 19 shows the gene expression, as determined by qRT-PCR, for the DE array, which contains select genes associated with DE, for stage 1, day 1 cells.
  • Figures 18 and 19 illustrate significant alterations in gene expression patterns such as a ⁇ 700x increase in FOXA2 expression and a lOOOx increase in CERl, EOMES, FGF17, FGF4, GATA4, GATA6, GSC, MIXL1, and T expression.
  • Figures 20 and 21 show the gene expression for stage 1, day 3 cells differentiated according to the protocol of Example 7.
  • Figure 20 shows the gene expression, as determined by qRT-PCR, for the pluripotency array, which contains select genes associated with pluripotency, for stage 1, day 3 cells.
  • Figure 21 shows the gene expression, as determined by qRT-PCR, for the DE array, which contains select genes associated with DE, for stage 1, day 3 cells.
  • Figure 22 shows the results of FACS for CD9, CD184 (also known as CXCR4) and CD99 for stage 1, day 3 cells differentiated according to the protocol of Example 7.
  • Figure 23 shows the gene expression, as determined by qRT-PCR, for the DE array, which contains select genes associated with DE, for stage 1, day 3; stage 2, day 1 ; and stage 2, day 3 cells differentiated according to the protocol of Example 7.
  • Figure 23 shows that FTNF4a and GATA6 expression levels at stage 2 days 1 and 3 increased, while genes expressed at high levels on day 3 of stage 1 (CXCR4, EOMES, FGF 17, FGF4, MNX1, PRDM1, SOX17, and VWF) showed reduced expression by the end of stage 2.
  • Figure 24 shows the gene expression of the foregut genes AFP, PDX1, and PROX1, as determined by qRT-PCR, for stage 2, day 1 cells and stage 2, day 3 cells differentiated according to the protocol of Example 7. As shown in Figure 24, the expression of these genes increased.
  • Figure 25 shows the results of FACS for PDX1, FOXA2, chromogranin, NKX2.2 and SOX2 for stage 3, day 3 cells grown in stage 3 medium (Table 7) differentiated according to the protocol of Example 7. As shown in Figure 25, the cells expressed markers consistent with an endodermal pancreatic lineage as measured by PDX1 and FOXA2 expression (90.9% ⁇ 11.9SD PDX1 positive and 99.2% ⁇ 0.6SD FOXA2 positive).
  • Figure 26 shows the gene expression, as determined by qRT-PCR, for the stage 4 array, which contains select genes associated with stage 4, for stage 3, day 1 and stage 3, day 3 cells differentiated according to the protocol of Example 7.
  • Figure 26 illustrates that these cells exhibit increased levels of a host of genes commonly expressed in the pancreas (ARX, GAST, GCG, INS, ISLl, NEURODl, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF IA, and SST).
  • Figure 27 shows the results of FACS for NKX6.1, chromagranin (CHGA), CDX2, SOX2, NKX2.2, PDX1, FOXA2 and NEUROD for stage 4, day 3 cells differentiated according to the protocol of Example 7. As shown in Figure 27, stage 4 day 3 the cells retained high levels of PDX1 and FOXA2 expression and further developed an expression pattern consistent with a mix of pancreatic endocrine cells (28.1% ⁇ 12.5SD chromogranin positive) and pancreatic progenitor cells (58.3% ⁇ 9.7SD positive for NKX6.1).
  • Figure 28 shows the gene expression, as determined by qRT-PCR, for the stage 4 array, which contains select genes associated with stage 4, for stage 3, day 3; stage 4, day 1 and stage 4, day 3 cells differentiated according to the protocol of Example 7.
  • Figure 28 shows an increased expression level of genes commonly expressed in the pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD 1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF 1A, and SST).
  • Figure 29 shows the average results of FACS for NKX6.1, chromagranin (CHGA), CDX2, SOX2, NKX2.2, PDXl, FOXA2 and NEUROD for stage 4, day 3 cells differentiated according to the protocol of Example 7. Specifically, Figure 29 shows the average FACS expression pattern of pancreatic precursors generated at a 3L scale from different seed material lots.
  • Figure 30 shows the average results of FACS for NKX6.1, chromagranin (CHGA), CDX2, SOX2, NKX2.2, PDXl, FOXA2 and NEUROD for stage 4, day 3 cells differentiated according to the protocol of Example 7. Prior to differentiation in stage 4, day 3 cells, the cells were expanded to form ISM and then grown at stage 0 in either a custom in-house medium "IH3" or Essential8TM, both of which were supplemented with 0.5% BSA. The cells grown in the IH3 medium are the "IH3-P grown cells" and the cells grown in Essential8TM are the “EZ8 grown cells.” No significant difference in expression patterns was observed between the cells grown in the different media.
  • IH3 custom in-house medium
  • Essential8TM both of which were supplemented with 0.5% BSA.
  • the cells grown in the IH3 medium are the "IH3-P grown cells” and the cells grown in Essential8TM are the "EZ8 grown cells.” No significant difference in expression patterns was observed between the cells grown in the different
  • Figure 31 shows the average results of FACS for NKX6.1, chromagranin (CHGA), CDX2, SOX2, NKX2.2, PDXl, FOXA2 and NEUROD for stage 4, day 3 cells, which were previously grown at different pH levels in stage 0 (see Example 7). No significant change in the stage 4, day 3 cell profile was observed.
  • Figure 32 compares the results of FACS for NKX6.1 , chromogranin (CHGA), CDX2, SOX2, NKX2.2, PDXl, FOXA2 and NEUROD for stage 4, day 3 cells, which were not treated with Anti-Foam C, and stage 4, day 3 cells, which were treated with Anti-Foam C emulsion (94 ppm) (see Example 7).
  • Anti-Foam C emulsion (Sigma Cat#A801 1) was not observed to affect the profile of stage 4 day 3 cells.
  • Figures 33 to 35 show the gene expression, as determined by qRT-PCR, for select genes for cells differentiated according to the protocol of Example 8.
  • Figure 33 shows the gene expression, as determined by qRT-PCR, for select genes of cells, twenty-four hours prior to the start of differentiation (see Example 8).
  • cells from the bioreactor retained expression for genes characteristic of pluripotency (POU5F1, NANOG, SOX2, and ZFP42) and showed minimal or no induction of genes characteristic of differentiation (AFP, and FOXA2: ⁇ 50 fold increase; FOXD3, GATA2, GATA4, GSC, HAND2, MIXL1, and T: ⁇ 10 fold increased expression).
  • Figure 34 shows the gene expression, as determined by qRT-PCR, for select genes of cells twenty- four hours after the start of differentiation.
  • Figure 35 shows the gene expression, as determined by qRT-PCR, for select genes of cells seventy-two hours after the start of differentiation.
  • Figure 36(a) to 36(e) show the gene expression, as determined by qRT-PCR, for select genes for cells differentiated from stage 2 to stages 3 and 4 according to the protocol of Example 8. Specifically, these Figures show the gene expression of the cells at stage 2, day 1; stage 2, day 2; stage 2, day 3; stage 3, day 3; and, depending on the gene, stage 4, day 1.
  • Figure 36(a) shows the gene expression for AFP, ATOH1, and CDX2.
  • Figure 36(b) shows the gene expression for GAST, HAND1, HHEX, and FTNF4a.
  • Figure 36(c) shows the gene expression for NKX2.2, NKX6.1, OSRl, and PDXl .
  • Figure 36(d) shows the gene expression for PROX1, PFTla, SOX17, and SOX2.
  • Figure 36(e) shows the gene expression for SOX9. The data are shown as difference in expression versus undifferentiated HI (WA01) hES cells (baseline expression of 1).
  • Figure 37 show the gene expression, as determined by qRT-PCR, for select genes for cells at stage 4, day 3 of differentiation according to the protocol in Example 8.
  • day 3 day 3 the cells have differentiated into pancreatic progenitor cells characterized by high expression levels of PDXl (>lxl0 6 fold induction) and other pancreatic genes (>1000 fold induction of ARX, GCG, GAST, INS, ISL, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PTFla, and SST) and near total loss of OCT4/POU5F1 expression as compared to undifferentiated HI human embryonic stem cells.
  • PDXl >lxl0 6 fold induction
  • pancreatic genes >1000 fold induction of ARX, GCG, GAST, INS, ISL, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PTFla, and SST
  • Figure 38 shows the daily cell counts during the differentiation protocol according to Example 8. Specifically, Figure 38 shows cell density as a function of the process day.
  • Figure 38 shows the cell counts for differentiation protocols of two reactor runs (PRD1205 and PRD1207) carried out at pH 6.8 and 7.2. For comparison, the cell counts for cell drift are also shown.
  • Figure 39(a) to Figure 39(d) illustrate the in vivo bioactivity of stage 4 day 3 cells, which were differentiated according to the protocol of Example 8 and were implanted into SCID-Bg mice.
  • the cells were implanted subcutaneously via a TheraCyteTM device, under the kidney capsule or implanted after incubation in an ultra-low attachment dish.
  • the mice were monitored for blood glucose and C-peptide levels every four weeks following graft implantation.
  • Figure 39(a) shows the C-peptide levels after implantation of 5 x 10 6 or 10 x 10 6 stage 4 day 3 cells in a TheraCyteTM device as a function of time.
  • Figure 39(b) shows the non-fasting glucose levels in animals after implantation of 5 x 10 6 or 10 x 10 6 stage 4 day 3 cells in a TheraCyteTM device.
  • the mice in Figure 39(b) were treated with STZ to ablate host ⁇ -cell function prior to implantation.
  • Figure 39(c) shows the C-peptide level produced after implantation of previously-cyropreserved stage 4 day 3 cells in a TheraCyteTM device as a function of time (weeks post implantation).
  • Figure 39(d) compares the C-peptide levels of mice treated by a kidney graft of never cryopreserved/fresh stage 4, day 3 cells or cryopreserved stage 4, day 3 cells implanted immediately after thaw (DO) or 1 day after thaw (Dl).
  • Figure 40A to Figure 40D show FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 9 which were treated at stage 1, day 1 with: MCX compound and GDF-8 (Figure 40A); MCX only ( Figure 40B); W T3A and Activin A (Figure 40C); and WNT3A only ( Figure 40D).
  • Figure 40A shows that in suspension culture, addition of 3 ⁇ MCX in the absence of a TGF- ⁇ family member on day one of differentiation generates definitive endoderm at levels comparable to that obtained when cells are treated with 3 ⁇ MCX plus lOOng/ml GDF-8 or 20ng/ml WNT- 3a plus lOOng/ml Activin A on day one.
  • Figures 41A to 4 ID show FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 10, which were treated with various amounts of MCX at stage 1, day 1. Specifically, the cells at stage 1, day lwere treated with: 4 ⁇ of MCX ( Figure 41A); 3 ⁇ of MCX ( Figure 41B); 2 ⁇ of MCX ( Figure 41C); and 1.5 ⁇ of MCX ( Figure 4 ID).
  • Figure 42A and Figure 42B show FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 11. Specifically, these Figures show the role of media exchange frequency in suspension culture.
  • Figure 42A shows FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 10 with full media exchange at stage 1.
  • Figure 42B shows FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 10 without a media exchange on day 3.
  • Figure 43 A and Figure 43B show FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 12. Specifically, these Figures show the role of GlutaMAXTM in suspension culture. The cells were cultured at stage 1 in a medium supplemented with IX GlutaMAXTM ( Figure 43 A) or free of GlutaMAXTM or any glutamine (0 M GlutaMAXTM) ( Figure 43B). The data suggest that in the suspension culture system, addition of GlutaMAXTM does not appear to influence the efficiency with which definitive endoderm is generated
  • Figures 44A to 44D show the effects of various amounts of sodium bicarbonate on cells differentiated according to the protocol of Example 13.
  • Figure 44A and Figure 44B show FACS plots for CXCR4, CD99, and CD9 of cells differentiated for three days according to the protocol of Example 13 with either 3.64 g/1 ( Figure 44A) or 2.49 g/1 ( Figure 44B) added at stage 1.
  • Figure 44C and Figure 44D show phase contrast micrographs of cells differentiated for three days according to the protocol of Example 13 with either 3.64 g/1 ( Figure 44C) or 2.49 g/1 ( Figure 44D) added at stage 1.
  • Figure 45 shows daily cell counts for cell density as a function of differentiation for cells differentiated according to the protocol of Example 14. The cells counts were obtained using an image -based cytometer (NucleoCounter®).
  • Figure 46 shows the average daily bioreactor medium pH levels as a function of time (days of differentiation) during the differentiation protocol of Example 14. pH levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 47 shows the average daily bioreactor medium glucose levels as a function of time (days of differentiation) during the differentiation protocol of Example 14. Glucose levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 48 shows the average daily bioreactor medium lactate levels as a function of time (days of differentiation) during the differentiation protocol of Example 14. Lactate levels were determined by a NOVA BioProfile® FLEX (Nova Biomedical Corporation, Waltham, MA).
  • Figure 49 shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for the pluripotency array, which contains select genes associated with pluripotency, for stage 0, day 1 to 3 and stage 1, day 1 to day 3 cells differentiated according to the protocol of Example 14.
  • Figure 50 shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for the DE array, which contains select genes associated with DE, for stage 0, day 1 to 3, stage 1, day 1 to day 3 and stage 2, day 1 to day 3 cells differentiated according to the protocol of Example 14.
  • Figure 51 shows the results of FACS for markers associated with pluripotency (CD184/CXCR4, SSEA4, TRA-1-60 and TRA-1-81) for stage 0, cells prior to being differentiated according to the protocol of Example 14. Specifically, Figure 51 shows high expression of markers associated with pluripotency.
  • Figure 52 shows FACS plots for the definitive endoderm markers CXCR4, CD99, and CD9 of cells differentiated to the end of stage 1 according to the protocol of Example 14.
  • Figure 53 shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for GAPDH, AFP, HHEX, FTNF4a, PDXl, and PROX1 for stage 2, day 1; stage 2, day 2 and stage 2, day 3 cells differentiated according to the protocol of Example 14.
  • Figure 53 shows an increase in expression of foregut genes (AFP, HHEX, PDXl, and PROX1).
  • Figure 54 shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for GAPDH, AFP, CDX2, GAST, H F4A, NKX2- 2, OSR1, PDXl and PFT1A for stage 2, day 1 to day 3 and stage 3, day 1 to day 3 cells differentiated according to the protocol of Example 14.
  • expression for PDXl increased 60 fold from 12,000x over control at the end of stage 2 day 3 to 739,000x over control at the end of stage 3, day 3.
  • Figure 55 shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for certain genes for stage 3, day 1 to 3 and stage 4, day 1 to day 3 cells differentiated according to the protocol of Example 14.
  • the top panel of Figure 55 shows the gene expression for GAPDH, AFP, ALB, ARX, CDX2, CHGA, GAST, GCG, IAAP, INS, ISL1, and MAFB.
  • the bottom panel of Figure 55 shows the gene expression of MAFB, MUCS, NEUROD1, NEUROG3, NKX2-2, NKX6-1, PAX4, PDXl, POUSF1, PTF1A, SST and Z1C1.
  • Figure 56 shows end stage micrographs for cells differentiated according to the protocol of Example 14. Visible in Figure 56 are representative micrographs (4X) of cell clusters at stage 0 and at the end of differentiation of stages 1 to 4.
  • Figures 57 to 80 show the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for cells differentiated according to various embodiments of the protocol of Example 15 after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation for the following genes: AFP ( Figure 57); CD99 (Figure 58); CD9 (Figure 59); CDH1 ( Figure 60); CDH2 ( Figure 61); CDX2 ( Figure 62); CER1 ( Figure 63); CXCR4 ( Figure 64); FGF17 (Figure 65); FGF4 (Figure 66); FOXA (Figure 67); GADPH ( Figure 68); GATA4 ( Figure 69); GATA6 ( Figure 70); GSC ( Figure 71); KIT ( Figure 72); MIXL1 ( Figure 73); MNX1 ( Figure 74); NANOG ( Figure 75); OTX2 ( Figure 76); POUF5F1 ( Figure 77); SOX17 ( Figure 78); SOX7 ( Figure 79
  • Figure 81 shows the percentage of cells in G0/G1 of Cell Cycle for cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15. Specifically, Figure 81 shows the results for clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only.
  • Neat Neat
  • 3 ⁇ MCX plus lOOng/ml GDF-8 Catalog # 120-00, Peprotech
  • 3 ⁇ MCX only 3 ⁇ MCX
  • Figure 82 shows the effects of EDU treatment on the cell clusters differentiated according to the protocol of Example 15.
  • the left hand panel of shows percentage of cells in G2/M of Cell Cycle for cells after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15.
  • the left hand panel shows the results for clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only. In one set of data, these clusters were also treated with EDU.
  • the right hand panel of Figure 82 shows the % Cells that are EDU positive 0 hours, 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15.
  • Figure 83 shows the general operational parameters used in the protocols of Example 15.
  • Figure 84 shows the amount of EDU incorporation of cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15. Specifically, Figure 84 shows the results for EDU incubated cells clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only.
  • Neat Neat
  • 3 ⁇ MCX plus lOOng/ml GDF-8 Catalog # 120-00, Peprotech
  • 3 ⁇ MCX only 3 ⁇ MCX
  • Figure 85 shows the percentage of cells in G0/G1 of Cell Cycle for cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15. Specifically, Figure 85 shows the results for clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only.
  • Neat Neat
  • 3 ⁇ MCX plus lOOng/ml GDF-8 Catalog # 120-00, Peprotech
  • 3 ⁇ MCX only 3 ⁇ MCX only
  • Figure 86 shows the percentage of cells in S-phase of Cell Cycle for cells after 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15. Specifically, Figure 86 shows the results for clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only.
  • Neat Neat
  • 3 ⁇ MCX plus lOOng/ml GDF-8 Catalog # 120-00, Peprotech
  • 3 ⁇ MCX only 3 ⁇ MCX only
  • Figure 87 shows the percentage of cells in S-phase of Cell Cycle for cells after hours, 6 hours, 24 hours, 30 hours, 48 hours, and 72 hours of differentiation according to various embodiments of the protocol of Example 15. Specifically, Figure 87 shows the results for clusters that were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml WNT-3A only.
  • Neat Neat
  • 3 ⁇ MCX plus lOOng/ml GDF-8 Catalog # 120-00, Peprotech
  • 3 ⁇ MCX only 3 ⁇ MCX
  • Figures 88A to 88E show the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for cells differentiated according to various embodiments of the protocol of Example 15 after 0 hours, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours of differentiation.
  • Figure 88A shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for CD99, CD9, CDH1, and CDH2.
  • Figure 88A shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for CXD2, CERl, CXCR4, and FGF17.
  • Figure 88C shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for FGF4, FOXA, GATA4, and GATA6.
  • Figure 88D shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for GSC, KIT, MIXLl and MNX1.
  • Figure 88E shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for NANOG, OTX2, POUF5F1, and SOX17.
  • Figure 88F shows the gene expression, as determined by qRT-PCR as a fold expression versus undifferentiated cells, for SOX7 and T. The underlying data for Figures 88A to 88F is shown in Figure 58 to 67 and 69 to 80.
  • Figure 89 shows the gene expression pattern, as determined by qRT-PCR, of pluripotent cells cultured in ectodermal differentiation medium according to the protocol of Example 16. As shown in Figure 89, the cells differentiated towards the neural cell lineage. Specifically, the left panel of Figure 89 shows the gene expression pattern for an induced pluripotent stem cell line generated from umbilical tissue cells (UTC). The right panel of Figure 89 shows the gene expression pattern for the WB0106 sub-clone of the HI hES cell line.
  • UTC umbilical tissue cells
  • Figure 90 shows the gene expression pattern, as determined by qRT-PCR, of pluripotent cells cultured in mesodermal differentiation medium according to the protocol of Example 16. As shown in Figure 90, the cells differentiated towards cardiac cell lineage. Specifically, the left panel of Figure 90 shows the gene expression pattern for an induced pluripotent stem cell line generated from umbilical tissue cells (UTC). The right panel of Figure 90 shows the gene expression pattern for the WB0106 sub-clone of the HI hES cell line.
  • UTC umbilical tissue cells
  • Figure 91 shows the gene expression pattern, as determined by qRT-PCR, of pluripotent cells cultured in ectodermal differentiation medium according to the protocol of Example 16. As shown in Figure 91, the cells differentiated towards neural cell lineage. Specifically, the left panel of Figure 91 shows the gene expression pattern for an induced pluripotent stem cell line generated from umbilical tissue cells (UTC). The right panel of Figure 91 shows the gene expression pattern for the WB0106 sub-clone of the HI hES cell line.
  • UTC umbilical tissue cells
  • Figure 92 shows the protein expression pattern for PAX6, SOX2, and
  • POU5F1/OCT4 as determined by FACS, of pluripotent cells cultured for three days in ectodermal differentiation medium according to the protocol of Example 16.
  • the left panels of Figure 92 show the expression pattern for PAX6, SOX2, and POU5F1/OCT4 for an induced pluripotent stem cell line generated from umbilical tissue cells (UTC).
  • the right panel of Figure 92 shows the protein expression pattern for PAX6, SOX2, and
  • Figure 93 shows the gene expression pattern, as determined by qRT-PCR, of pluripotent cells cultured in mesodermal differentiation medium according to the protocol of Example 16. As shown in Figure 93, the cells differentiated towards cardiac cell lineage. Specifically, the left panel of Figure 93 shows the gene expression pattern for an induced pluripotent stem cell line generated from umbilical tissue cells (UTC). The right panel of Figure 93 shows the gene expression pattern for the WB0106 sub-clone of the HI hES cell line.
  • UTC umbilical tissue cells
  • Figure 94 shows micrographs for cells differentiated in mesodermal differentiation medium according to the protocol of Example 16. As shown in Figure 94, the cells differentiated towards cardiac cell lineage. Specifically, the left hand panels of Figure 94 show micrographs of cells of the WB0106 sub-clone of the HI hES cell line at day 3, day 5 and day 10 of differentiation. The right hand panel of Figure 94 shows a micrograph of induced pluripotent stem cell line generated from umbilical tissue cells (UTC IPSCs) after 10 days of differentiation.
  • UTC IPSCs umbilical tissue cells
  • Figure 95 shows micrographs for cells differentiated in ectodermal differentiation medium according to the protocol of Example 16. As shown in Figure 95, the cells differentiated towards the neural cell lineage. Specifically, the left hand panels of Figure 95 show micrographs of cells of the WB0106 sub-clone of the HI hES cell line at day 3, day 5 and day 10 of differentiation. The right hand panel of Figure 95 shows a micrograph of induced pluripotent stem cell line generated from umbilical tissue cells (UTC iPCS) after 10 days of differentiation.
  • UTC iPCS umbilical tissue cells
  • This application is directed to preparing embryonic stem cells and other pluripotent cells that maintain pluripotency in aggregated cell cluster for differentiation to endoderm progenitor cells, pancreatic endocrine cells, mesoderm cells or ectoderm cells.
  • endoderm progenitor cells pancreatic endocrine cells
  • mesoderm cells or ectoderm cells For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.
  • Stem cells are undifferentiated cells defined by their ability, at the single cell level, to both self-renew and differentiate. Stem cells may 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). Stem cells also give rise to tissues of multiple germ layers following transplantation and contribute substantially to most, if not all, tissues following injection into blastocysts.
  • culture refer generally to cells taken from a living organism and grown under controlled conditions ("in culture” or “cultured”).
  • a primary cell culture is a culture of cells, tissues, or organs taken directly from an organism before the first subculture.
  • Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate one or both of cell growth and division, resulting in a larger population of the cells.
  • the rate of cell proliferation is sometimes measured by the amount of time needed for the cells to double in number (referred to as doubling time).
  • “Expanding”, as used herein is the process of increasing the number of pluripotent stem cells by culturing, such as by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 75%, 90%, 100%, 200%, 500%, 1000% or more, and levels within these percentages. It is appreciated that the number of pluripotent stem cells which can be obtained from a single pluripotent stem cell depends on the proliferation capacity of the pluripotent stem cell.
  • the proliferation capacity of the pluripotent stem cell can be calculated by the doubling time of the cell, i.e., the time needed for a cell to undergo a mitotic division in the culture, and the period that the pluripotent stem cell can be maintained in the undifferentiated state, which is equivalent to the number of passages multiplied by the days between each passage.
  • Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, a nerve cell or a muscle cell.
  • a differentiated cell or a 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
  • 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 to what cells it can give rise. The lineage of a cell places the cell within a hereditary scheme of development and
  • 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
  • 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 as compared to an undifferentiated cell.
  • 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.
  • a cell is "positive for” a specific marker or “positive” when the specific marker is sufficiently detected in the cell.
  • the cell is "negative for” a specific marker, or “negative” when the specific marker is not sufficiently detected in the cell.
  • positive by FACS is usually greater than 2%, whereas the negative threshold by FACS is usually less than 1%.
  • Positive by PCR is usually less than 34 cycles (Cts); whereas negative by PCR is usually more than 34.5 cycles.
  • cell density and “seeding density” are used interchangeably herein and refer to the number of cells seeded per unit area of a solid or semisolid planar or curved substrate.
  • suspension culture refers to a culture of cells, single cells or clusters, suspended in medium rather than adhering to a surface.
  • serum free refers to being devoid of human or animal serum. Accordingly, a serum free culture medium does not comprise serum or portions of serum.
  • 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 at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )), GATA4, GATA6, M X1, SOX 17, CXCR4, Cerberus, OTX2, brachyury, goosecoid, C-Kit, CD99, and MIXL1.
  • FOXA2 also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )
  • GATA4 also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )
  • GATA4 also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )
  • GATA4 also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )
  • GATA4 also known as hepatocyte nuclear factor 3- ⁇ (HNF3 )
  • GATA4 also known as hepatocyte nuclear factor 3- ⁇ (
  • definitive endoderm cells Markers characteristic of the definitive endoderm cells include CXCR4, FOXA2 and SOX 17. Thus, definitive endoderm cells may be characterized by their expression of CXCR4, FOXA2, and SOX 17. In addition, depending on the length of time cells are allowed to remain in stage 1, an increase in HNF4a may be observed.
  • Pantendocrine cells refer to cells capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide.
  • markers characteristic of pancreatic endocrine cells include one or more of NGN3, NeuroDl, ISLl, PDXl, NKX6.1, PAX4, ARX, NKX2.2, and PAX6.
  • Pancreatic endocrine cells expressing markers characteristic of ⁇ cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDXl, NKX2.2, NKX6.1, NeuroDl, ISLl , HNF3 , MAFA, PAX4, and PAX6.
  • Glucose and "D-Glucose” are used interchangeably herein and refer to dextrose, a sugar commonly found in nature.
  • NeuroD NeuroD and NeuroDl which identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.
  • LDN and LDN-193189 refer ((6-(4-(2-(piperidin-l-yl)ethoxy)phenyl)-3- (pyridin-4-yl)pyrazolo[l,5-a]pyrimidine, hydrochloride; DM-3189)), a BMP receptor inhibitor available under the trademark STEMOLECULETM from Stemgent, Inc., Cambridge, MA, USA.
  • Pluripotent stem cells may express one or more of the designated TRA-1-60 and TRA-1-81 antibodies (Thomson et al. 1998, Science 282: 1145-1147). Differentiation of pluripotent stem cells in vitro results in the loss of TRA-1-60, and TRA-1-81 expression. 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, Inc., Burlingame, CA). Undifferentiated pluripotent stem cells also typically express OCT4 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 stem cells can be confirmed, for example, by injecting cells into severe combined immune-deficiency ("SCID") mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining histologically for evidence of cell types from these 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.
  • SCID severe combined immune-deficiency
  • 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 cells may be readily expanded in culture using various feeder layers or by using matrix protein coated vessels. Alternatively, chemically defined surfaces in combination with defined media such as mTeSR®l media (StemCell Technologies, Vancouver, BC, Canada) may be used for routine expansion of the cells.
  • defined media such as mTeSR®l media (StemCell Technologies, Vancouver, BC, Canada) may be used for routine expansion of the cells.
  • Culturing in a suspension culture according to the method of some embodiments of the invention is effected by seeding the pluripotent stem cells in a culture vessel at a cell density that promotes cell survival and proliferation, but limits differentiation. Typically, a seeding density that maintains undifferentiation of cells is used. It will be appreciated that although single-cell suspensions of stem cells may be seeded, small clusters of cells may be advantageous.
  • the culture medium can be replaced or replenished on a daily basis or at a pre-determined schedule such as every 1-5 days.
  • Large clusters of pluripotent stem cells may cause cell differentiation, thus, measures may be taken to avoid large pluripotent stem cell aggregates.
  • the formed pluripotent stem cell clusters are dissociated, for example, every 2-7 days and the single cells or small clumps of cells are either split into additional culture vessels (i.e., passaged) or retained in the same culture vessel and processed with replacement or additional culture medium.
  • Large pluripotent stem cell clumps including a pellet of pluripotent stem cells resulting from centrifugation, can be subjected to one or both of enzymatic digestion and mechanical dissociation.
  • Enzymatic digestion of pluripotent stem cell clumps can be performed by subjecting the clump to an enzyme, such as type IV Collagenase, Dispase or Accutase ® .
  • Mechanical dissociation of large pluripotent stem cell clumps can be performed using a device designed to break the clumps to a predetermined size. Additionally, or alternatively, mechanical dissociation can be manually performed using a needle or pipette.
  • the culture vessel used for culturing the pluripotent stem cells in suspension according to the method of some embodiments of the invention can be any tissue culture vessel (e.g., with a purity grade suitable for culturing pluripotent stem cells) having an internal surface designed such that pluripotent stem cells cultured therein are unable to adhere or attach to such a surface (e.g., non-tissue culture treated vessel, to prevent attachment or adherence to the surface).
  • culturing according to some embodiments of the invention is effected using a controlled culturing system (preferably a computer-controlled culturing system) in which culture parameters such as temperature, agitation, pH, and oxygen are automatically monitored and controlled using a suitable device. Once the desired culture parameters are determined, the system may be set for automatic adjustment of culture parameters as needed to enhance pluripotent stem cell expansion and differentiation.
  • the pluripotent stem cells may be cultured under dynamic conditions (i.e., under conditions in which the pluripotent stem cells are subject to constant movement while in the suspension culture, e.g. a stirred suspension culture system) or under non-dynamic conditions (i.e., a static culture) while preserving their, proliferative, pluripotent capacity and karyotype stability over multiple passages.
  • dynamic conditions i.e., under conditions in which the pluripotent stem cells are subject to constant movement while in the suspension culture, e.g. a stirred suspension culture system
  • non-dynamic conditions i.e., a static culture
  • the pluripotent stem cells can be cultured in petri dishes, T-flasks, HyperFlasks® (Corning Incorporated, Corning, NY), CellStacks® (Corning Incorporated, Corning, NY) or Cell Factories (NUNCTM Cell
  • the pluripotent stem cells can be cultured in a suitable vessel, such as spinner flasks or Erlenmeyer flasks, stainless steel, glass or single use plastic shaker or stirred tank vessels.
  • the culture vessel can be connected to a control unit and thus present a controlled culturing system.
  • the culture vessel e.g., spinner flask or Erlenmeyer flask
  • the culture vessel may be agitated continuously or intermittently.
  • the cultured vessel is agitated sufficiently to maintain the pluripotent stem cells in suspension.
  • the pluripotent stem cells may be cultured in any medium that provides sufficient nutrients and environmental stimuli to promote growth and expansion. Suitable media include E8TM, IH3 and mTeSR ® l or mTeSR ® 2. The media may be changed periodically to refresh the nutrient supply and remove cellular by-products. According to some
  • the culture medium is changed daily.
  • pluripotent stem cell may be used in the methods of the invention.
  • 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 to 12 weeks gestation.
  • pre-embryonic tissue such as, for example, a blastocyst
  • embryonic tissue such as, for example, a blastocyst
  • fetal tissue fetal tissue taken any time during gestation, typically but not necessarily, before approximately 10 to 12 weeks gestation.
  • Non-limiting examples are established lines of human embryonic stem cells (hESCs) or human embryonic germ cells, such as, for example the human embryonic stem cell lines HI, H7, and H9 (WiCell Research Institute, Madison, WI, USA).
  • hESCs human embryonic stem cells
  • H7, and H9
  • IPS inducible pluripotent cells
  • reprogrammed pluripotent cells that can be derived from adult somatic cells using forced expression of a number of pluripotent related transcription factors, such as OCT4, NANOG, Sox2, KLF4, and ZFP42 (Annu Rev Genomics Hum Genet 201 1, 12: 165-185).
  • the human embryonic stem cells used in the methods of the invention may also be prepared as described by Thomson et al. (U.S. Patent No. 5,843,780; Science, 1998, 282: 1145-1147; Curr Top Dev Biol 1998, 38: 133-165; Proc Natl Acad Sci U.S.A. 1995, 92:7844-7848).
  • mutant human embryonic stem cell lines such as, for example, BGOlv (BresaGen, Athens, Ga.), or cells derived from adult human somatic cells, such as, for example, cells disclosed in Takahashi et al, Cell 131 : 1-12 (2007).
  • Pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in Li et al. (Cell Stem Cell 4: 16-19, 2009); Maherali et al. (Cell Stem Cell 1 : 55-70, 2007); Stadtfeld et al. (Cell Stem Cell 2: 230-240); Nakagawa et al. (Nature Biotechnology 26: 101-106, 2008); Takahashi et al.
  • pluripotent stem cells include induced pluripotent cells (IPS, Cell, 126(4): 663-676).
  • Other sources of cells suitable for use in the methods of invention include human umbilical cord tissue-derived cells, human amniotic fluid-derived cells, human placental-derived cells, and human parthenotes.
  • the umbilical cord tissue-derived cells may be obtained using the methods of U.S. Patent No. 7,510,873, the disclosure of which is incorporated by reference in its entirety as it pertains to the isolation and characterization of the cells.
  • the placental tissue-derived cells may be obtained using the methods of U.S. App. Pub. No. 2005/0058631, the disclosure of which is incorporated by reference in its entirety as it pertains to the isolation and characterization of the cells.
  • the amniotic fluid-derived cells may be obtained using the methods of U.S. App. Pub. No.
  • 2007/0122903 the disclosure of which is incorporated by reference in its entirety as it pertains to the isolation and characterization of the cells.
  • pluripotent stem cells 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 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the following: ABCG2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1- 60, TRA-1-81.
  • the pluripotent stem cells suitable for use in the methods of the invention express one or more (e.g.
  • the pluripotent stem cells suitable for use in the methods of the invention express one or more (e.g. 1, 2 or all) of CD9, NANOG and POU5F1/OCT4 as detected by RT-PCR.
  • Exemplary pluripotent stem cells include the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line HI (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line SA002 (Cellartis, Sweden).
  • the pluripotent stem cells are human embryonic stem cells, for example, HI hES cells.
  • pluripotent stem cells of non-embryonic origin are used.
  • the present invention in some of the embodiments as described below, relates to isolating and culturing stem cells, in particular culturing stem cell clusters, which retain pluripotency in a dynamic suspension culture system. Pluripotent cell clusters may be differentiated to produce functional ⁇ cells.
  • the pluripotent stem cells used in the methods of the present invention are preferably expanded in dynamic suspension culture prior to differentiation toward a desired end point.
  • the pluripotent stem cells can be cultured and expanded as clusters of cells in suspension in a suitable medium without loss of pluripotency.
  • Such culturing may occur in a dynamic suspension culture system wherein the cells or cell clusters are kept moving sufficiently to prevent loss of pluripotency.
  • Useful dynamic suspension culture systems include systems equipped with means to agitate the culture contents, such as via stirring, shaking, recirculation or the bubbling of gasses through the media. Such agitation may be intermittent or continuous, as long as sufficient motion of the cell clusters is maintained to facilitate expansion and prevent premature differentiation.
  • the agitation comprises continuous stirring such as via an impeller rotating at a particular rate.
  • the impeller may have a rounded or flat bottom.
  • the stir rate of the impeller should be such that the clusters are maintained in suspension and settling is minimized.
  • the angle of the impeller blade may be adjusted to aid in the upward movement of the cells and clusters to avoid settling.
  • the impeller type, angle and rotation rate may all be coordinated such that the cells and clusters are in what appears as a uniform colloidal suspension.
  • Suspension culturing and expansion of pluripotent stem cell clusters may be accomplished by transfer of static cultured stem cells to an appropriate dynamic culture system such as a disposable plastic, reusable plastic, stainless steel or glass vessel, e.g. a spinner flask or an Erlenmeyer flask.
  • a dynamic culture system such as a disposable plastic, reusable plastic, stainless steel or glass vessel, e.g. a spinner flask or an Erlenmeyer flask.
  • stem cells cultured in an adherent static environment i.e., plate or dish surface
  • a chelating agent or enzyme include, but are not limited to, type I Collagenase, Dispase ® (Sigma Aldrich LLC, St.
  • Accutase ® is a cell detachment solution comprising collagenolytic and proteolytic enzymes (isolated from crustaceans) and does not contain mammalian or bacterial derived products. Therefore, in one embodiment, the enzyme is a collagenolytic enzyme or a proteolytic enzyme or a cell detachment solution comprising collagenolytic and proteolytic enzymes.
  • Suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA).
  • the pluripotent stem cell cultures are incubated with the enzyme or chelating agent, preferably until colony edges began to curl and lift, but prior to full detachment of colonies from the culture surface.
  • the cell cultures are incubated at room temperature.
  • the cells are incubated at a temperature of more than 20°C, more than 25°C, more than 30°C or more than 35°C, for example, at a temperature of between about 20°C and about 40°C, between about 25°C and about 40°C, between about 30°C and about 40°C, for example, about 37°C.
  • the cells are incubated for at least about 1, at least about 5, at least about 10, at least about 15, at least about 20 minutes, for example between about 1 and about 30 minutes, between about 5 and about 30 minutes, between about 10 and about 25 minutes, between about 15 and about 25 minutes, for example, about 20 minutes.
  • the method involves the step of removing the enzyme or chelating agent from the cell culture after treatment.
  • the cell culture is washed once or twice or more, after removal of the enzyme or chelating agent.
  • the cell culture is washed with an appropriate culture medium, such as mTeSR®l (Stem Cell Technologies, Vancouver, BC, Canada).
  • a Rho-kinase inhibitor for example, Y-27632, Axxora Catalog#ALX-270-333, San Diego, CA.
  • the Rho-kinase inhibitor may be at a concentration of about 1 to about 100 ⁇ , about 1 to 90 ⁇ , about 1 to about 80 ⁇ , about 1 to about 70 ⁇ , about 1 to about 60 ⁇ , about 1 to about 50 ⁇ , about 1 to about 40 ⁇ , about 1 to about 30 ⁇ , about 1 to about 20 ⁇ , about 1 to about 15 ⁇ , about 1 to about 10 ⁇ , or about 10 ⁇ .
  • the Rho-kinase inhibitor is added at least 1 ⁇ , at least 5 ⁇ or at least 10 ⁇ .
  • the cells may be lifted from the surface of the static culture system with a scraper or rubber policeman.
  • Media and cells may be transferred to a dynamic culture system using a glass pipette or other suitable means.
  • the media in the dynamic culture system is changed daily.
  • the invention provides, in one embodiment, methods of culturing and expanding pluripotent stem cells in a three-dimensional suspension culture.
  • the methods provide for the culturing and expanding pluripotent stem cells by forming aggregated cell clusters of these pluripotent stem cells.
  • the cell clusters may form as a result of treating pluripotent stem cell cultures with an enzyme (e.g. a neutral protease, for example Dispase ® ) or a chelating agent prior to culturing the cells.
  • the cells may preferably be cultured in a stirred or shaken suspension culture system.
  • the invention further provides for formation of cells expressing markers characteristic of the pancreatic endoderm lineage from such clusters of pluripotent stem cells.
  • the cell clusters are aggregated pluripotent stem cells.
  • the aggregated stem cells express one or more markers of pluripotency, for example, one or more (e.g. 1, 2, 3 or all) of the markers CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of one or more markers for differentiation, for example, lack expression of CXCR4.
  • the aggregated stem cells express the markers for pluripotency CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of a marker for differentiation CXCR4.
  • One embodiment is a method of culturing pluripotent stem cells as cell clusters in suspension culture.
  • the cell clusters are aggregated pluripotent stem cells, cultured in a dynamic stirred or shaken suspension culture system.
  • the cell clusters may be transferred from a planar adherent culture using an enzyme, such as a neutral protease, for example Dispase, as a cell lifting agent to a stirred or shaken suspension culture system.
  • exemplary suitable enzymes include, but are not limited to, type IV Collagenase, Dispase ® or Accutase ® .
  • the cells maintain pluripotency in a stirred or shaken suspension culture system, in particular a stirred suspension culture system.
  • Another embodiment of the invention is a method of culturing pluripotent stem cells as cell clusters in suspension culture, wherein the cell clusters are aggregated pluripotent stem cells transferred from a planar adherent culture using a chelating agent, for example EDTA, and cultured in a stirred or shaken suspension culture system.
  • the cell clusters maintain pluripotency in a stirred or shaken suspension culture system, in particular a stirred
  • Another embodiment of the invention is a method of culturing pluripotent stem cells as cell clusters in suspension culture, wherein the cell clusters are aggregated pluripotent stem cells transferred from a planar adherent culture using the enzyme Accutase ® , and cultured in a stirred or shaken suspension culture system.
  • the cell clusters maintain pluripotency in the dynamically agitated suspension culture system.
  • the cell clusters of the invention may be differentiated into mesoderm cells, such as cardiac cells, ectoderm cells, such as neural cells, single hormone positive cells or pancreatic endoderm cells.
  • the method may further include differentiation, for example differentiation of the pancreatic endoderm cells into pancreatic precursor cells and pancreatic hormone expressing cells.
  • pancreatic precursor cells are characterized by expression of ⁇ cell transcription factors PDX1 and NKX6.1.
  • the step of differentiation is carried out after at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 196 hours or more, preferably about 48 hours to about 72 hours in the suspension culture system. Differentiation may be carried out using a stage-wise progression of media components, such as that described in the examples (e.g. see Table A and Tables la and lc).
  • a three-dimensional cell cluster is produced by growing pluripotent stem cells in a planar adherent culture; expanding the pluripotent stem cells to aggregated cell clusters; and transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension culture using an enzyme or chelating agent.
  • a further preferred embodiment is a method of expanding and differentiating pluripotent stem cells in a dynamically agitated suspension culture system by growing pluripotent stem cells in a planar adherent culture; expanding the pluripotent stem cells to aggregated cell clusters; and transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension culture using an enzyme or chelating agent; and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system to generate a pancreatic precursor cell population.
  • Another embodiment is a transplantable stem cell derived cell product comprising differentiated stem cells prepared from suspension of expanded pluripotent stem cell clusters that are differentiated to pancreatic precursor cells. More particularly, a transplantable stem cell derived product is produced by growing pluripotent stem cells in a planar adherent culture; expanding the pluripotent stem cells to aggregated cell clusters; and transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension culture using an enzyme or chelating agent; and differentiating the pluripotent cell clusters in a dynamically agitated suspension culture system.
  • the transplantable stem cell derived cell product is preferably used to treat diabetes.
  • the method includes transplantation into a diabetic animal for further in vivo maturation to functional pancreatic endocrine cells.
  • Another embodiment is a method of expanding and differentiating pluripotent stem cells in a suspension culture system comprising growing pluripotent stem cells in a planar adherent culture; removing the pluripotent stem cells from the planar adherent culture using an enzyme; adhering the pluripotent stem cells to microcarriers in static culture; expanding the pluripotent cells in a dynamically agitated suspension culture system; and differentiating the pluripotent cells in a dynamically agitated suspension culture system to generate a pancreatic precursor cell population.
  • the microcarriers may be of any form known in the art for adhering cells, in particular the microcarriers may be beads.
  • the microcarrier can be comprised of natural or synthetically-derived materials. Examples include collagen-based microcarriers, dextran- based microcarriers, or cellulose-based microcarriers.
  • microcarrier beads may be modified polystyrene beads with cationic trimethyl ammonium attached to the surface to provide a positively charged surface to the microcarrier.
  • the bead diameter may range from about 90 to about 200 ⁇ , alternately from about 100 to about 190 ⁇ , alternatively from about 1 10 to about 180 ⁇ , alternatively from about 125 to 175 ⁇ in diameter.
  • Microcamer beads may also be a thin layer of denatured collagen chemically coupled to a matrix of cross-linked dextran.
  • Microcarrier beads may be glass, ceramics, polymers (such as polystyrene), or metals. Further, microcarriers may be uncoated, or coated, such as with silicon or a protein such as collagen.
  • the microcarrier can be comprised of, or coated with, compounds that enhance binding of the cell to the microcarrier and enhance release of the cell from the microcarrier including, but not limited to, sodium hyaluronate, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, fibronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin, and collagen.
  • compounds that enhance binding of the cell to the microcarrier and enhance release of the cell from the microcarrier including, but not limited to, sodium hyaluronate, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, fibronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin, and collagen.
  • Examples further include microcarriers that possess a microcurrent, such as microcarriers with a particulate galvanic couple of zinc and copper that produces low levels of biologically relevant electricity; or microcarriers that are paramagnetic, such as paramagnetic calcium-alginate microcarriers.
  • the population of pancreatic endoderm cells is obtained by a stepwise differentiation of pluripotent cell clusters.
  • the pluripotent cells are human embryonic pluripotent stem cells.
  • 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.
  • the present invention relates to a stepwise method of differentiating pluripotent cells comprising culturing stage 3-5 cells in a dynamic suspension culture.
  • the pancreatic endoderm population generated is transplanted into diabetic animals for further in vivo maturation to functional pancreatic endocrine cells.
  • the invention also provides for systems or kits for use in the methods of the invention.
  • the invention also provides a cell or population of cells obtainable by a method of the invention.
  • the invention also provides a cell or population of cells obtained by a method of the invention.
  • the invention provides methods of treatment.
  • the invention provides methods for treating a patient suffering from, or at risk of developing, diabetes.
  • the invention also provides a cell or population of cells obtainable or obtained by a method of the invention for use in a method of treatment.
  • the invention provides a cell or population of cells obtainable or obtained by a method of the invention for use in a method of treating a patient suffering from, or at risk of developing, diabetes.
  • the diabetes may be Type 1 or Type 2 diabetes.
  • the method of treatment comprises implanting cells obtained or obtainable by a method of the invention into a patient.
  • the method of treatment comprises differentiating pluripotent stem cells in vitro into stage 1, stage 2, stage 3, stage 4, or stage 5 cells, for example as described herein, and implanting the differentiated cells into a patient.
  • the method further comprises the step of culturing pluripotent stem cells, for example as described herein, prior to the step of differentiating the pluripotent stem cells.
  • the method further comprises the step of differentiating the cells in vivo, after the step of implantation.
  • the patient is a mammal, preferably a human.
  • the 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. 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 growth factors known in the art 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.
  • the method of treatment further comprises incorporating the cells into a three-dimensional support prior to implantation.
  • 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.
  • one or more of the following may be used in the methods of the invention.
  • Lipid range From about 0.1% to about 0.2%, from about
  • MCX 1 About 3 ⁇ , about 2 ⁇ , about 1 ⁇ to about 5 ⁇ , about 2 ⁇ to about 4 ⁇ , about 1 ⁇ to about 3 ⁇ , about 2 ⁇ to about 3 ⁇
  • Oxygen Range 1-5 from hypoxia to about 30% of ambient, from about 10% to about 25% of ambient, from about 15% to about 30% of ambient
  • Retinoic Acid 3 About 2 ⁇ , from about 1 ⁇ to about 3 ⁇ , form about 1.5 ⁇ to about 2.5 ⁇
  • SANT 3, 4 About 0.25 ⁇ , from about 0.1 ⁇ to about 0.3 ⁇ , from about 0.2 to about 0.3 ⁇ . from about 0.1 ⁇ ⁇ about 0.25 ⁇
  • TppB or TPB 4 About 500 nM, about 100 nM, from about 50 nM to about 550 nM, from about 50 nM to about 150 nM, from about 200 nM to about 500 nM, from about 300 nM to about 550 nM, about 50nM, from about 25nM to about 75nM
  • Wnt3A 1 About 20 ng/ml, from about 10 ng/ml to about 25 ng/ml, from about 18 ng/ml to about 30 ng/ml, from about 18 ng/ml to about 22 ng/ml
  • Dispase ® was then removed and the culture dish was washed twice with mTeSR®l (Stem Cell Technologies, Vancouver, BC, Canada) media containing 10 ⁇ Y-27632 (Axxora Catalog#ALX-270-333, San Diego, CA). The mTeSR®l media containing 10 ⁇ Y-27632 was then added to the culture dish at
  • HI hES cells can be successfully transferred as cell clusters to suspension culture from a planar adherent culture format with Dispase ® as a cell lifting agent and maintain pluripotency in a stirred (dynamic) suspension culture system.
  • This example can also be carried out in shaken rather than stirred suspension systems with plates and Erlenmeyer flasks with comparable results.
  • the pluripotent cell aggregates were differentiated with a stage-wise progression of media components to induce the cells to form a pancreatic fate.
  • the spinner agitation was turned up for differentiation of the aggregates to a speed of 65 rpm.
  • the media and components are shown in Table 1 a.
  • stage 1 samples were run for flow cytometry and PCR. Suspension differentiated cultures formed a uniform and homogeneous population of cells in loose aggregates at the end of stage 1 (Figure lc), with expression of a marker for pluripotency (CD9) nearly eliminated, while the markers for definitive endoderm differentiation were quite high, 97.2% positive for CXCR4 (CD184) and 97.3% positive for CD99 ( Figure Id).
  • the definitive endoderm clusters were then further differentiated toward a primitive foregut by removing the TGF- ⁇ family member, GDF8, and adding FGF7 to the media. After three days culture with FGF7, the clusters were differentiated to a pancreatic PDX1 expressing fate by addition of all-trans-retinoic acid to either a media containing high glucose (25mM) and low concentration of lipid rich bovine serum albumin (AlbuMAX® (Life Technologies Corporation, Carlsbad, CA)) or a media containing a relatively low glucose concentration (8mM) and 2% fatty acid free bovine serum albumin. The detailed addition of components to these media is listed in Table la.
  • pancreatic precursor cells were analyzed for expression of markers of pancreatic precursor cells. It was observed that the clusters differentiated with either condition - low glucose + 2% FAF-BSA (A) or high glucose + 0.1% AlbuMAX® (B) - as measured by flow cytometry expressed high levels of NKX6.1, a transcription factor required for functional ⁇ cells, and high levels of endocrine pancreas markers such as synaptophysin and chromogranin (Table lb). These results were consistent with RT-PCR results which showed high levels of multiple pancreatic precursor genes expressed in samples from both condition A and B (data not shown).
  • stage 4 Cells differentiated according to condition B through stage 4 were held for an additional 5 days in stage 5 media containing an ALK5 inhibitor (see Table lc). This additional maturation in culture resulted in a significant increase in endocrine marker expression: INS, GCG, SST, PPY, and PCSK1.
  • the cell clusters were then implanted into the kidney capsule of SCID-Bg mice according to IACUC approved study protocol, and the mice were followed for 20 weeks with fasted/fed c-peptide measured every 2 to 4 weeks. After 4 weeks post implantation, following a 20 hour fast and then glucose stimulation, c- peptide was not detectable.
  • mice positive showed some (0.087 & 0.137 ng/mL) human c-peptide, and by 10 weeks, 5 of 5 mice were positive (0.085 - 0.291 ng/mL) for c-peptide.
  • differentiated cell clusters that were implanted and allowed to mature in vivo expressed insulin in response to glucose challenge at physiologically appropriate levels.
  • Fatty Acid Free Bovine Serum 2% Fatty Acid Free Bovine Serum Albumin (FAF- Supplement Albumin (FAF-BSA) and 2mM L- BSA) and 2mM L-Glutamine (A)
  • mTeSR®l media containing 10 ⁇ Y-27632 was added as needed to make a concentration of cells at 1.0 to 1.5 million cells/ml.
  • Cells were not centrifuged, as the clusters were loosely aggregated and would disassociate to single cells if centrifuged to a pellet and re-suspended by pipette. Instead, media and cells in the tube were gently swirled until a uniform suspension was formed. If desired, one can also lengthen the period of EDTA treatment and take cells to near a single cell suspension.
  • the cell suspension was then transferred to two non-tissue culture treated 6 well dishes (Becton Dickinson, Catalog# Falcon 351 146, Franklin Lakes, NJ) in a 37°C humidified 5% C0 2 incubator at 3 ml/well with a glass pipette. Cells were incubated in suspension for 2 hours at which point aggregates were observed. The aggregates were then triturated by gentle pipetting with a glass pipette to disrupt large aggregates and create a homogeneous, uniform cluster suspension, then incubated undisturbed overnight.
  • Becton Dickinson Catalog# Falcon 351 146, Franklin Lakes, NJ
  • the pluripotent cell aggregates were differentiated with a stage-wise progression of media components to induce the cells to form a pancreatic fate.
  • the spinner agitation was maintained at a speed of 55 rpm.
  • the media and components are shown in Table 2a.
  • stage 1 samples were run for flow cytometry and PCR. Suspension differentiated cultures formed a uniform and homogeneous population of cells in loose aggregates at the end of stage 1 (Figure 2b), with expression of a marker for pluripotency (CD9) nearly eliminated, while CXCR4 (CD 184), a marker for definitive endoderm differentiation, was quite high, 95.9% ⁇ 1.8sd ( Figure 2c) across three spinner flasks.
  • the definitive endoderm clusters from spinner flasks were then pooled and distributed to either another spinner flask or an Erlenmeyer flask (shaken agitation system) and directed for further differentiation toward a primitive foregut by removing GDF8, and adding FGF7 to the media.
  • the clusters were differentiated to a pancreatic PDX1 expressing fate by addition of all-trans -retinoic acid to a media containing a relatively low glucose concentration (8mM) and 2% fatty acid free bovine serum albumin. The detailed addition of components to these media is listed in Table 2a.
  • the samples were analyzed for expression of markers of pancreatic precursor cells.
  • Accutase ® is a cell detachment solution comprised of collagenolytic and proteolytic enzymes (isolated from crustaceans) and does not contain mammalian or bacterial derived products.) Accutase ® was then removed and after 3 more minutes (6 1/2 minutes total Accutase ® exposure), the plate was rinsed with mTeSR®l media containing 10 ⁇ Y- 27632 and dislodged cells were collected in a 50 ml conical tube using a glass pipet. One additional rinse of the plate with mTeSR®l media containing 10 ⁇ Y-27632 was performed and pooled with dislodged cells.
  • Phase contrast microscope images of the clusters show a uniform, spherical population of clusters that formed after 90 minutes in static suspension culture and expanded over three days in culture (Figure 3b).
  • the cells were assayed for pluripotency by flow cytometry results for the markers CD9, SSEA4, TRA-1-60, TRA-1-81, and CXCR4.
  • the cells maintained high expression of markers for pluripotency (CD9, SSEA4, TRA-1-60, TRA-1-81) and almost no expression for CXCR4, a marker of differentiation (Table 3).
  • HI hES cells can be transferred to suspension culture from a planar adherent culture format using an enzymatic lifting method, such as Accutase ® , and will maintain pluripotency in a dynamic agitated suspension culture system.
  • an enzymatic lifting method such as Accutase ®
  • the pluripotent clusters were then serially passaged using Accutase ® dissociation for an additional 20 passages.
  • 50 million cells were gravity settled for 2 minutes in a 50 ml conical tube, washed twice with PBS and treated with a half strength solution of Accutase ® in a 37°C water bath with gentle swirling of the tube at two and four minutes after addition of Accutase ® .
  • Accutase ® was aspirated from the tube without disturbing the cell pellet.
  • the cells were then incubated 3 more minutes (9 minutes total Accutase ® exposure).
  • the tube was then rinsed with mTeSR®l media containing 10 ⁇ Y-27632, triturated twice using a glass pipet, and the suspended cells passed through a 70 micron cell strainer (BD Falcon, Cat#352350, Franklin Lakes, NJ). Two additional rinses of the tube with mTeSR®l media containing 10 ⁇ Y-27632 were performed and passed through the cell strainer.
  • Pluripotency was confirmed through the first 20 passages as shown in Table 3, with stable high levels of pluripotency markers maintained throughout the culture, as measured by flow cytometry.
  • cells were differentiated to a pancreatic precursor in a dynamic agitated suspension culture system through a step-wise progression of different media containing morphogens or growth factors intended to recapitulate normal pancreatic development. This process gives rise to a pancreatic precursor cell population characterized by a high PDX1 and NKX6.1 co-expression. When these cells were transplanted, they matured further to functional glucose stimulated insulin secreting tissue able to secrete insulin in response to glucose and maintain normal blood glucose in a streptozotocin induced model of diabetes. See Figure 4c and Table 4c.
  • pancreatic precursor cells In order to generate these pancreatic precursor cells, HI human embryonic stem cells that had been expanded and maintained in a dynamic agitated suspension culture system for 16 passages were differentiated using the method described in Example 3. In summary, the cells were expanded for 30 passages, tested for pluripotency for the first 20 of these passages; the cells were differentiated on the 16 th passage. Pluripotent cells in cluster format were transferred from mTeSR®l media to FBC solution (Table 4a) at 4°C for 3 hours. Cell clusters were then moved to a 3 liter glass suspension bioreactor regulated by a Sartorius Stedim Biostat B-DCU (Goettingen, Germany) control unit and suspended in differentiation media at 0.55 million cells/mL according to Table 4b.
  • Pluripotent cells in cluster format were transferred from mTeSR®l media to FBC solution (Table 4a) at 4°C for 3 hours. Cell clusters were then moved to a 3 liter glass suspension bioreactor regulated by a Sartorius
  • the cells were maintained 14 days in the closed sterile suspension bioreactor regulated for temperature, pH, and dissolved oxygen (DO) (FermProbe ® pH electrode 225mm, Model # F-635, and dissolved oxygen OxyProbe ® 12mm Sensor, model number D-145 from Broadley- James Corporation, Irvine CA).
  • DO dissolved oxygen
  • media bicarbonate levels were maintained at 3.64g/L with pH maintained at pH 7.4 by regulation of CO2 flow in a total media volume of ⁇ 1.6 liters.
  • the bioreactor head space was continuously perfused with CO2, air, and O2, under control of the Sartorious control system with a 20% dissolved oxygen set-point for stage 1 and a 30% dissolved oxygen set-point for stage 2 onward with a constant gas flow of 150cc/minute.
  • Oxygen flow was regulated in response to dissolved oxygen content and CO2 flow was regulated in response to pH.
  • Temperature was maintained at 37°C throughout the run by an electric heated jacket.
  • encapsulation device (TheraCyteTM, Irvine CA) that was implanted subcutaneously (6 animals per condition).
  • the implanted cells expressed significant levels of circulating human C-peptide (>0.1ng/mL) as detected by ELISA (human c-peptide custom ELISA Mercodia cat# 10-1141-01) in response to fasting and then feeding and by 16-20 weeks animals had over lng/mL of circulating c-peptide (Table 4c).
  • a human embryonic stem cell derived cell product to treat diabetes can be prepared from suspension of expanded and differentiated stem cells.
  • the product can be generated in a scalable, stirred, closed loop bioreactor system and the cell product can be processed with a closed loop wash and concentration as required for commercial cGMP manufacturing.
  • This human embryonic stem cell derived cell product can treat diabetes in a widely used animal model of diabetes as shown by GSIS competence, ability to regulate blood glucose, and the return to a diabetic state upon removal of the cell therapy.
  • Pluripotency of the cellular aggregates was measured by flow cytometry as shown in Figure 5a and high expression of the pluripotency markers CD9, SSEA4, TRA-1-61, and TRA-1-80, indicating the cells were highly pluripotent, was observed.
  • These pluripotent cells were then differentiated to a pancreatic precursor in a dynamically agitated suspension culture system through a step-wise progression of different media containing small molecules and growth factors intended to recapitulate morphogen drivers of normal pancreatic development. This process produces a pancreatic precursor cell population characterized by co-expression of the pancreatic cell transcription factors, PDX1 and NKX6.1. When these cells are transplanted they mature further to functional glucose stimulated insulin secreting tissue which can correct high blood glucose in a streptozotocin induced model of diabetes.
  • pluripotent cells in cluster format maintained in mTeSR®l media were transferred to a 0.2 liter glass stirred suspension bioreactor (Dasgip, Catalog#DS0200 TBSC, Shrewsbury, MA) with controller regulated temperature, pH, and dissolved oxygen.
  • Pluripotent cell clusters were cultured in the bioreactor for two days. At that time (stage 1, day 0) the media was exchanged and differentiation was initiated as the cell aggregates were suspended at approximately 0.7 million cells/mL in differentiation media according to Table 5a. The cells were then maintained in this closed sterile suspension bioreactor for 14 days.
  • stages 1, day 0 the media was exchanged and differentiation was initiated as the cell aggregates were suspended at approximately 0.7 million cells/mL in differentiation media according to Table 5a.
  • the cells were then maintained in this closed sterile suspension bioreactor for 14 days.
  • SCIO is an Alk5 inhibitor having the chemical name 4- ⁇ [2-(5-Chloro-2-fluorophenyl)-5-(l-methylethyl)pyrimidin-4- yl]amino ⁇ -N-(2-hydroxypropyl)pyridine-3-carboxamide and CAS number 674794-97-9.
  • the chemical structure of SCIO is shown below:
  • stage 1 Several different feed settings were tested during stage 1 : (a) media change 24 hours after initiation of differentiation, no media change at 48 hours; (b) media change 24 hours after initiation of differentiation and glucose bolus addition at 48 hours; and (c) no media change throughout stage 1 with glucose and GDF8 bolus added 24 hours after initiation of differentiation, then a glucose bolus added at 48 hours post initiation.
  • Condition C expressed significantly higher levels of genes indicative of an alternative non-pancreatic fate, e.g. CDX2, AFP, and Albumin (Figure 5e).
  • the cells differentiated according to condition A were removed from the bioreactor, washed with MCDB 131 media containing 0.1% FAF-BSA and implanted in SCID-Bg mice. Each mouse was transplanted with 5 million cells directly under the kidney capsule. Every 4 weeks after implantation blood draws were performed and blood glucose and c-peptide were measured. By 12 weeks post implantation, human c-peptide was detectable by ELISA at levels above Ing/mL, and at 16 weeks c-peptide levels were an average of 2.5ng/mL (Figure 5f). At 20 weeks post- implantation c-peptide was measured in the animals in a fasted and then fed state.
  • Glucose treatment induced a significant increase in circulating human c-peptide from 0.93ng/mL in a fasted state to 2.39ng/mL in a fed state ( Figure 5g) indicating that the transplanted cells had matured to functional GSIS competent tissue.
  • the animals were given a streptozotocin (STZ) administration (mouse ⁇ cells are more sensitive to and preferentially destroyed by STZ compared to human ⁇ cells) to induce a diabetic state, the animals with a graft of functional GSIS competent tissue maintained normal blood glucose levels unlike the untreated controls which developed frank diabetes (Figure 5h).
  • Cytodex ® 3 Microcarrier beads (C3) (Sigma-Aldrich Co LLC, St. Louis, MO, Catalog # C3275) were prepared for culture by soaking 400mg of the beads in 20ml volume silicon coated glass scintillation vials containing 15ml Dulbecco's PBS (DPBS), for 4-24 hours.
  • Cytodex ® 3 consists of a thin layer of denatured collagen chemically coupled to a matrix of cross-linked dextran. The denatured collagen layer on Cytodex ® 3 is susceptible to digestion by a variety of proteases, including trypsin and collagenase, and provides the ability to remove cells from the microcarriers while maintaining maximum cell viability, function, and integrity.
  • the beads were autoclaved and rinsed with sterile DPBS and re- suspended in mouse embryonic fibroblast conditioned media (MEF-CM) supplemented with 10 ⁇ Y-27632.
  • MEF-CM mouse embryonic fibroblast conditioned media
  • the beads were then transferred to 125ml Corning® glass spinner flasks (Corning Incorporated, Corning, NY) at a density of lOOmg beads/flask.
  • the spinner containing beads and MEF-CM with Y-27632 was equilibrated in a humidified 5% CO 2 incubator at 37°C for at least 60min.
  • the cells were grown in this manner for 5 days with a daily 50mL media exchange of MEF-CM. After 5 days in culture, the flasks contained 53xl0 6 cells ( ⁇ 12xl0 6 SD). As a control, one million HI hES cells were also seeded to 6 well tissue culture polystyrene dishes coated with a 1 :30 dilution of MatrigelTM and maintained with a daily media change of MEF-CM.
  • Phase contrast images of the cells on micro-carriers were taken and a time course of the cell morphology as pluripotent culture before differentiation of the cells was initiated is shown in Figure 6a.
  • a time course showing the culture differentiating is shown in Figure 6b.
  • a cell count was also taken at various time points through the experiment, and the results are presented as a function of surface area (cells/cm 2 in Figure 6c) or media volume (cells/mL in Figure 6d) for the media formulations in either a planar culture or a suspended microcarrier culture.
  • pancreatic precursor cell population characterized by co-expression of the pancreatic cell transcription factors, PDX1 and NKX6.1.
  • PDX1 and NKX6.1 pancreatic cell transcription factors
  • B27 is Gibco® B-27® Supplement (Life Technologies Corporation, Carlsbad, CA).
  • the MCX compound is 14-Prop-2-en-l-yl- 3,5,7, 14, 17,23,27-heptaazatetracyclo[19.3.1.1-2,6- ⁇ . l ⁇ 8, 12. ⁇ ]heptacosa- 1(25),2(27),3,5,8(26),9, 11,21,23- ⁇ - aen-16-one, which has the following formula (Formula 1):
  • cyclic aniline-pyridinotriazines may also be used instead of the above- described MCX compound.
  • Such compounds include but are not limited to 14-Methyl- 3,5,7, 14, 18,24,28-heptaazatetracyclo[20.3.1.1 ⁇ 2,6 ⁇ .- 1-8, 12 ⁇ ]octacosa- l(26),2(28),3,5,8(27),9,l l,22,24-nonaen-17-on- e and 5-Chloro-l,8, 10, 12, 16,22,26,32- octaazapentacyclo[24.2.2.1 ⁇ 3,7 ⁇ -l ⁇ 9, 13 ⁇ . l ⁇ 14, 18 ⁇ ]tritriaconta-3(33),4,6,9(32),10- ,12, 14(31),15,17-nonaen-23-one.
  • Formula 3 Formula 3
  • the Cyp26 inhibitor used at Stage 4 in this example was N- ⁇ 4-[2-Ethyl-l-(lH-l,2,4- triazol-l-yl)butyl]phenyl ⁇ -l,3-benzothiazol-2-amine, which has a CAS number of 201410- 53-9 and the following structure.
  • Cypi This Cyp26 inhibitor is also known as "Cypi.”
  • the structure and synthesis of this Cyp26 inhibitor are disclosed in U.S. Patent No. 7,378,433, the disclosure of which is incorporated in its entirety as it pertains to Cyp26 inhibitors and their synthesis.
  • a sub-clone of the HI (WA01) hES cell line - WB0106 was used for this example.
  • WB0106 was derived at the WiCell Research Institute (Madison, WI) from HI line seed material termed DDL- 13.
  • the WB0106 sub-clone of the HI line was derived from a DDL- 13 vial thawed at passage 23 into mTeSR ® l medium on a MatrigelTM substrate, and was subsequently passaged using EDTA.
  • WB0106 was frozen at passage 28 and was selected for these studies on the basis of a normal karyotype (FISH and G-band), ability to differentiate to pancreatic progenitor cells, and competency to form clusters and expand in suspension culture.
  • FISH and G-band normal karyotype
  • a WB0106 WCB vial was then thawed into medium on a substrate of MatrigelTM in a T225 flask (Corning Incorporated, Corning, NY) and at the first passage the cells were expanded into multiple T225 flasks. At the second passage the cells from multiple T225 flasks were combined and used to seed a single 2-Layer Cell StackTM (CS2). Once the CS2 was 70% confluent, C-Flex® tubing assembly caps with adjacent pump tubing were attached to the media ports to close the system.
  • T225 flask Corning Incorporated, Corning, NY
  • the cells in the CellSTACK® formed rounded spherical aggregates (clusters) of pluripotent cells.
  • the medium supplemented with 0.5% BSA containing the suspended cell clusters were transferred from the CellSTACK® to a 1 liter disposable spinner flask (Corning;
  • the cell clusters were disassociated with Accutase ® treatment for passaging and further expansion.
  • the passage process was initiated by removing the 1 liter disposable spinner flask from the humidified 5% CO 2 incubator. The flask was placed on a spinner plate inside of a biosafety cabinet to maintain a homogeneous suspension of cells. The cell suspension was removed from the spinner flask by lOOmL pipette and distributed evenly between four 175mL conical polycarbonate tubes (ThermoFisher-Nalgene; Buffalo, NY) and centrifuged for 5 minutes at 80-200 rcf.
  • the spent medium was aspirated without disturbing the cell pellets. Then 25mL of DPBS without calcium or magnesium (DPBS 7 ) was added to each tube, and the cells were combined into one conical tube and centrifuged for 5 minutes at 80-200 rcf.
  • the DPBS 7" was aspirated from the conical tube and 30mL of a 50% Accutase ® /50% DPBS 7" solution was added to the tube.
  • the cell clusters were pipetted up and down 1-3 times, and then intermittently swirled for 4 minutes, then centrifuged for 5 minutes at 80-200 rcf.
  • the Accutase ® was then aspirated as completely as possible without disturbing the cell pellet and the conical tube was continuously and gently tapped for 3-5 minutes until the cell suspension appeared a uniform milky white.
  • lOmL of medium supplemented with 0.5% BSA containing lOmicromolar Rho Kinase inhibitor, Y-27632 was added to the cell suspension and triturated 2-4 times to inactivate the residual Accutase ® .
  • the cell density in the lOOmL volume of the filtered cell suspension was determined with a NC-100 NucleoCounter ® (ChemoMetec A/S, Allerod, Denmark) and additional medium was added to give a final cell concentration of 1 x 10 6 cells/mL in medium supplemented with 0.5% BSA containing lOmicromolar Rho Kinase inhibitor, Y-27632. Then 225mL (225 million cells) of the cell suspension was transferred to a 1 liter disposable spinner flask and incubated for 1 hour without agitation in a humidified 5% CO 2 incubator.
  • the flask was then removed from the incubator and agitated at 100 rpm on a spinner plate in a biosafety cabinet for 1-3 minutes. While the cell suspension was mixing, an additional 225mL of medium supplemented with 0.5% BSA containing lOmicromolar Rho Kinase inhibitor, Y-27632, was added to the cell suspension. The spinner flask was then returned to the humidified 5% CO 2 incubator for 30 minutes. The flask was then removed from the incubator and agitated at 100 rpm on a spinner plate in a biosafety cabinet for 1-3 minutes.
  • suspension-passaged and cultured cells could then be cryopreserved and stored for future use.
  • the cell clusters were dissociated with Accutase ® as described above for suspension passaging, except cells were not passed through a 40 micron cell strainer.
  • the cell count for the lOOmL cell suspension generated from each 1 liter disposable flask was determined.
  • the cell suspensions were then combined and centrifuged for 5 minutes at 80-200 rcf. The medium from the centrifuge tube was then removed as completely as possible without disturbing the cell pellet.
  • CryoStor®10 (Stem Cell Technologies, Inc., Vancouver, BC, Canada) was then added in a drop-wise manner to achieve a final concentration of 150 million cells per mL and the cell solution was held in an ice bath during transfer to a 1.8mL Corning® cryo vial (Corning Incorporated, Corning, NY) or 15mL Miltenyi cryo bag (Miltenyi Biotec Inc. Auburn, CA).
  • the suspension expanded cells were then frozen in a vial at high density in a controlled rate freezer as follows. The chamber was pre-cooled to 4°C and the temperature was held until sample vial temperature reached 6°C.
  • the chamber temperature was then lowered 2°C/min until the sample reached -7°C. Once the sample vial reached -7°C, the chamber was cooled 20°C/min until the chamber reached -45°C. The chamber temperature was then allowed to briefly rise at 10°C/min until the chamber temperature reached -25°C, and the chamber was then further cooled at 0.8°C/min until the sample vial reached -45°C. The chamber temperature was then cooled at 35°C/min until the chamber reached -160°C. The chamber temperature was then held at -160°C for at least 10 minutes, after which the vials were transferred to gas phase liquid nitrogen storage.
  • the cells were then centrifuged at 80-200rcf for 5 minutes.
  • the supernatant from the tube was aspirated, 10ml fresh medium (IH3 or E8TM) containing 0.5 %BSA and supplemented with lOmicromolar Rho Kinase inhibitor, Y-27632 was added and the volume containing the cells was pipetted into a media transfer bottle (Cap2V8®, SaniSure, Moorpark, CA).
  • the bottle contents were then pumped directly into the bioreactor via a sterile C-flex tubing weld by peristaltic pump.
  • the bioreactor In preparation for pluripotent stem cell inoculation the bioreactor was prepared with 1.5L of medium (IH3 or E8TM supplemented with 0.5% BSA and containing lOmicromolar Rho Kinase inhibitor, Y-27632), pre-warmed to 37°, stirred at 70 rpm, regulated to 6.8-7.1 pH by CO2, with a dissolved oxygen set-point of 30% (CO2, air, O2, and 2 regulated). Immediately post-inoculation the bioreactor was sampled for cell count, and medium volume was adjusted as needed to give a final cell concentration of 0.225 x 10 6 cells/mL.
  • medium IH3 or E8TM supplemented with 0.5% BSA and containing lOmicromolar Rho Kinase inhibitor, Y-27632
  • the cells inoculated into the stirred tank bioreactor formed cell clusters in the continuously stirred tank, and were maintained in pluripotency medium (IH3 or E8TM, supplemented with 0.5% BSA) in the reactor for three days total.
  • Medium was changed daily, with a partial media exchange performed 24 hours after inoculation as 1-1.3 liter of spent medium was removed and 1.5 liters of fresh medium added.
  • Forty-eight hours after inoculation 1.5-1.8 liters of spent medium was removed and 1.5 liters of fresh medium was added.
  • pluripotent cell differentiation was initiated by removing >90% of the spent medium and adding differentiation medium (Table 7).
  • the cells were maintained for 12 or more days in the closed sterile suspension bioreactor regulated for temperature (37°), pH (7.4 for differentiation), and dissolved oxygen (10% DO set-point for stage 1 and 30% DO set-point all other times, C0 2 , 0 2 , N 2 , and air regulated).
  • the impeller was stopped 5-20 minutes prior to medium removal via dip-tube to allow clusters to settle.
  • Medium in the bioreactor was removed or added to/from a closed bottle or bag by peristaltic pump through a dip tube connected to C- Flex® tubing using a TerumoTM tube welder to maintain a closed system.
  • the impeller and heater were re-energized once sufficient medium was added to the vessel to fully submerge the impeller.
  • stage specific changes in pH and metabolism matched stage changes in mRNA expression patterns.
  • a test of bioreactor cell samples was carried out using four Applied Biosystems® Low Density Arrays (Life Technologies Corporation, Carlsbad, CA) designated Pluripotency, Definitive Endoderm (DE), Gut Tube (GT), or stage 4 (S4) the results were compared to a historical undifferentiated HI (WB0106) hES cell sample as control to standardize expression across all runs and arrays.
  • stage 0 72 hours after reactor inoculation
  • the cells were moved into differentiation medium (Table 7) containing MCX and GDF8.
  • Twenty-four hours after this media change significant alterations in gene expression patterns were noted ( Figures 18 and 19), such as a ⁇ 700x increase in FOXA2 expression and a lOOOx increase in CER1, EOMES, FGF 17, FGF4, GATA4, GATA6, GSC, MIXLl, and T expression.
  • EOMES FGF 17, FGF4, GATA4, GATA6, GSC, MIXLl, and T expression.
  • CDX2 levels were elevated at stage 1 day 1 versus undifferentiated cells (470x increase in expression vs. control), however this was a transient increase in expression and CDX2 levels dropped 94% from stage 1, day 1 to stage 1 day 3 returning to levels comparable to those observed prior to induction of differentiation ( Figures 14, 19, and 21).
  • the cells expressed a profile consistent with specification to definitive endoderm, as CXCR4 levels peaked and FOXA2 and SOX 17 were expressed at >1000x over historical control. Consistent with definitive endoderm, it was also noted that the genes CER1, EOMES, FGF 17, FGF4, GATA4, GATA6, GSC, MIXLl, and T dropped from elevated levels observed at stage 1 day 1 ( Figures 20 and 21).
  • stage 2 Following the completion of definitive endoderm formation (stage 1) the medium was changed to one containing FGF7, a morphogen used to induce primitive foregut formation (stage 2). Consistent with formation of primitive foregut, HNF4a and GATA6 expression levels at stage 2 days 1 and 3 were increased, while genes expressed at high levels on day 3 of stage 1 (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX 17, and VWF) showed reduced expression by the end of stage 2 ( Figure 23). The expression of foregut genes (AFP, PDX1, and PROX1) was increased (Figure 24).
  • stage 3 medium (Table 7). Once in this medium the cells expressed markers consistent with an endodermal pancreatic lineage as measured by PDX1 and FOXA2 expression (90.9% ⁇ 11.9SD PDX1 positive and 99.2% ⁇ 0.6SD FOXA2 positive) shown in Figure 25. These results were confirmed by data from samples analyzed by qRT-PCR for gene expression. Gene expression for PDX1 increased 5 fold in 24 hours from the end of stage 2 day3 (38,000x vs. HI) to the end of stage 3 day l(200,000x vs. HI) and doubled again 48 hours later on stage 3 day 3 (435,000x vs. HI).
  • stage 4 day 3 the cells retained high levels of PDX1 and FOXA2 expression and further developed an expression pattern consistent with a mix of pancreatic endocrine cells (28.1% ⁇ 12.5SD chromogranin positive) and pancreatic progenitor cells (58.3% ⁇ 9.7SD positive for NKX6.1) as shown in Figure 27.
  • This stage specific marker expression pattern indicated an efficient stage-wise differentiation from a pluripotent population to pancreatic precursor cells. The results observed with flow cytometry, were further confirmed with data from qRT-PCR.
  • the product cells were cryopreserved.
  • the cells were washed in MCDB131 with 3.63 g/L sodium bicarbonate or MCDB131 with 3.63 g/L sodium bicarbonate, glucose (8mM final), and lx Glutamax, and then transferred to cold ( ⁇ 4°C) cryopreservation media comprised of 57.5% MCDB131 with 2.43g/L sodium bicarbonate, 30% Xeno-free KSR, 10% DMSO, and 2.5% HEPES (final concentration 25mM).
  • the cells were then frozen in a controlled rate freezer (CRF) using a cooling profile that maintained the cell clusters in cryopreservation media at ambient temperature for a maximum of 15 minutes, reduced to a temperature of 4°C for 45min, and further reduced by 2.00 °C/min to -7.0°C (sample).
  • the sample was then quickly cooled, reducing the temperature of the chamber at a rate of 25.0 °C /min to -45.0°C.
  • a compensation increase was then provided by increasing the chamber temp 10.0 °C /min to -25.0°C (chamber).
  • the sample was then cooled at 0.2 °C /min until the temperature reached -40.0°C.
  • the chamber was then cooled to -160°C at a rate of 35.0°C /min and held at that temperature for 15 minutes.
  • the samples were moved to a gas phase liquid nitrogen storage container at the termination of the CRF run.
  • the cells could be thawed by removal from vapor phase liquid nitrogen storage and transferring the vial to a 37°C water bath.
  • the vial was gently swirled in the water bath for less than 2 minutes until a small ice crystal remained in the vial.
  • the vial contents were then transferred to a 50ml conical and diluted drop-wise over two minutes using MCDB131 media with 2.43 g/L sodium bicarbonate and 2% BSA to a final volume of 20ml total.
  • the total cell number was then determined by NucleoCounter ® and the cell suspension transferred to an ultra-low attachment culture dish for lhour.
  • the cells were then isolated from the media in a 50ml conical, the supernatant removed and cells re-suspended in stage 4 media for analysis or in vivo study.
  • vialed cells were transferred to an empty 125mL glass Corning® spinner flask (Corning, Incorporated, Corning, NY) and lOmL MCDB131 medium containing 2.43g/L sodium bicarbonate and 2% BSA was added to the flask in a drop-wise manner. The final volume was then adjusted to 80mL of the same medium. The total cell number was determined by NucleoCounter ® and the cell suspension stirred at 40-65 rpm overnight (12-28 hours). The cells were then characterized or used for in vivo study.
  • composition of IH3 media is shown below as well as in U.S. Pub. App. No. 2013/0236973, the disclosure of which is incorporated in its entirety as it pertains to suitable cell culture media.
  • the amount of BSA in IH3 media may vary.
  • hES human embryonic stem cell line HI, (WA01 cells, WiCell, Madison WI)
  • a second rinse volume was added, collected, and pooled with the first rinse.
  • 1.6-2.0 x 10 9 cells were recovered from the CS2s in a final volume of 2 liters.
  • 2.0 -2.5 x 10 8 cells per layer were transferred into four CS2s or eight 1 layer Cell StacksTM and incubated at 37° for 2 hours in a humidified 5% CO 2 incubator in a volume of 200mL per layer.
  • the CellSTACKs® were then incubated overnight at 37° for 18 hours in a humidified 5% CO 2 incubator.
  • the 2 liters of cells and media from the Cell Stacks were then pooled and transferred, 1 liter each, into two 3 liter DASGIP bioreactors along with 1.5 liter of fresh mTeSR® medium per bioreactor.
  • the cells were maintained for two additional days with mTeSR® medium before initiating
  • the cells were maintained 14 or 15 days total (2 days mTeSR® + 12 or 13 days of staged differentiation) in the closed sterile suspension bioreactor regulated for temperature (37°), pH (drift, or regulated by CO 2 to 6.8 or 7.2 for pluripotent cells and 7.4 for differentiation), and dissolved oxygen (30% DO set-point, CO 2 / air regulated).
  • the impeller was stopped for 5-20 minutes prior to each media exchange to allow clusters to settle.
  • Medium was removed or added by peristaltic pump through a dip tube connected to C-Flex® tubing (Cole-Parmer North America, Vernon Hills, IL) using a TerumoTM tube welder to maintain a closed system.
  • the impeller and heat jacket were re-energized once sufficient medium was added to submerge the impeller.
  • stage 1 day 3 formation of definitive endoderm
  • stage 1 At the end of stage 1 the cell culture medium was changed from one containing GDF8 to a medium containing FGF7.
  • stage 2 Several different gene expression patterns were noted: an increase in expression of certain genes over the course of stage 2 (AFP, ATOH1, HHEX, OSR1, PDX1, PROX1, SOX2, and SOX9), a decrease in expression (HAND1 and SOX17), stable high expression throughout (HNF4a), or low/no expression (CDX2, GAST, NKX2.2, NKX6.1, and PTFla) ( Figure 36a-e).
  • stage 3 By the end of stage 3 the cells had specified to a pancreatic lineage as measured by PDX1 expression demonstrated by >100,000 fold increase in mRNA vs. undifferentiated control ( Figure 36) and 76-98% of the cells expressing PDX1 by flow cytometry (Table 1 1). Also observed was induction of other genes of the pancreas (GAST, NKX2.2, NKX6.1, PROX1, PTFla, and SOX9) and gut such as AFP and CDX2; indicating the cells had begun to specify to a more mature fate.
  • pancreatic progenitor cells Chromogranin positive and pancreatic progenitor cells (33-52% positive for NKX6.1) as shown in Tables 1 1 and 12.
  • This stage specific marker expression pattern indicated an efficient stage-wise differentiation from a pluripotent population to pancreatic progenitor cells characterized by high expression levels of PDX1 (>lxl0 6 fold induction) and other pancreatic genes (>1000 fold induction of ARX, GCG, GAST, INS, ISL, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PTFla, and SST) and near total loss of OCT4/POU5F 1 expression as compared to undifferentiated HI human embryonic stem cells (Figure 37).
  • cryopreservation media comprised of 57.5% MCDB131 with 2.43g/L sodium bicarbonate, 30% Xeno-free KSR, 10% DMSO, and 2.5% HEPES (final concentration 25mM).
  • a compensation increase was then provided by increasing the chamber temp 10.0 °C /min to -25.0°C (chamber).
  • the sample was then cooled at 0.2 °C /min until the temperature reached -40.0°C.
  • the chamber was then cooled to -160°C at a rate of 35.0°C /min and held at that temperature for 15 minutes.
  • the samples were moved to a gas phase liquid nitrogen storage container at the termination of the CRF run.
  • the cells were then isolated from the media in a 50ml conical, the supernatant removed and cells re-suspended in stage 4 media. The cells were then either implanted into an animal subcutaneously via TheraCyteTM device or under the kidney capsule or the cells were incubated in an ultra-low attachment culture dish overnight and then implanted into an animal. [0259] The animals were monitored for blood glucose and C-peptide levels every four weeks following graft implantation.
  • cryopreserved pancreatic precursor cells can function comparably to non-cryopreserved controls when tested in an animal model.
  • Basal media in Table 10 above may optionally include 5 mM glucose at stages 1- when Glutamax is not used in supplement.
  • Cypi ([100 nM]) may optionally be added at stage 4 in Table 10 shown above.
  • Max shear stress imposed on cell aggregates is not a result of turbulent eddies
  • Max shear stress imposed on cell aggregates is not a result of aggregate-aggregate or aggregate -impeller collision
  • Endoderm role of MCX/GDF8 in Suspension Culture
  • stage 1 basal medium MCDB-131 medium supplemented in this manner will be referred to as "stage 1 basal medium” for the purposes of this application.
  • Clusters in this medium were treated on the first day of differentiation with either 3 ⁇ MCX (a GSK3B inhibitor, 14-Prop-2-en-l-yl-3,5,7, 14, 17,23,27- heptaazatetracyclo [19.3. l . l ⁇ 2,6 ⁇ . l ⁇ 8, 12 ⁇ ]heptacosa-l(25),2(27),3,5,8(26),9,l 1,21,23- nonaen-16-one, U.S. Patent Application No.
  • Clusters from pluripotent human embryonic stem cell line HI were seeded at cell densities ranging from 0.25 x 10 6 to 2 x 10 6 cells/ml in Erlenmeyer/shaker flasks or spinner flasks in stage 1 basal media as described in Example 9.
  • Clusters were treated with stage 1 basal medium containing 1.5, 2, 3, or 4 ⁇ MCX on day one of differentiation and with fresh stage 1 basal medium containing lOOng/ml GDF-8 on day 2. No media exchange was performed on day three. Samples were collected for flow cytometry and PCR analysis at the end of day three of differentiation.
  • concentrations the effects of MCX can be modulated such that concentrations > 4 micromolar may be used. Conversely, concentrations ⁇ 1.5 micromolar may be used to generate definitive endoderm when used with lower BSA concentrations. Table 14
  • Clusters from pluripotent human embryonic stem cell line HI were seeded at cell densities ranging from 0.25 x 10 6 to 2 x 10 6 cells/ml in Erlenmeyer/shaker flasks or spinner flasks in stage 1 basal media as described in Example 9.
  • Clusters were treated with stage 1 basal medium containing 3 ⁇ MCX on day one of differentiation and with fresh stage 1 basal medium containing lOOng/ml GDF-8 on day 2.
  • Control cultures received a media exchange on day three; to a separate vessel, no media exchange was performed on day three. Samples were collected for flow cytometry and PCR analysis at the end of day three of differentiation.
  • stage 1 basal media described in Example 9
  • GlutaMAXTM which was supplemented with 3 ⁇ MCX on day one of differentiation and with fresh stage 1 basal medium containing lOOng/ml GDF-8 on day 2. No media exchange was performed on day three. Samples were collected for flow cytometry and PCR analysis at the end of day three of differentiation.
  • Clusters from pluripotent human embryonic stem cell line HI were seeded at cell densities ranging from 0.25 x 10 6 to 2 x 10 6 cells/ml in Erlenmeyer/shaker flasks or spinner flasks in either stage 1 basal media as described in Example 9 (containing 3.64g/l sodium bicarbonate), or in a modified stage 1 basal media which contained 2.43g/l sodium bicarbonate.
  • Clusters were treated with stage 1 basal medium containing MCX and GDF-8 as described in Example 12. Samples were collected for flow cytometry at the end of day three of differentiation. Phase contrast images were also captured on each day of differentiation.
  • Cell therapies will require large numbers (>10 8 ) of cells per dose.
  • This example demonstrates a process capable of differentiating induced pluripotent stem cell (iPS cell) masses at 3 to 5 orders of magnitude greater than possible with current cell therapy manufacturing practices.
  • an iPS cell line was used - UTC (derived from umbilical tissue cells previously described in US patent application 13/330,931 (U.S. Pub. App. 2013/0157365), the disclosure of which is incorporated by reference as it pertains to deriving iPS cell lines).
  • the cells were derived on mouse embryonic feeder cells using plasmid transfection in a "foot-print" free manner and cryo-preserved at passage 15.
  • Pre-Pre MCB Pre-Pre Master Cell Bank
  • WCB working cell bank
  • CS1 1 -Layer Cell StackTM
  • the cells were transferred in medium supplemented with 0.5% BSA and containing lOmicromolar of the Rho Kinase inhibitor, Y-27632, to a 1 liter disposable spinner flask (Corning; Corning, NY) at a concentration of 1 x 10 6 cells/mL in 225mL liter.
  • the cells were allowed to cluster in static suspension for 60 minutes in a humidified 5% CO 2 incubator, then agitated for 5 minutes at 55-65 rpm and 225mL additional medium supplemented with 0.5% BSA and containing lOmicromolar of the Rho Kinase inhibitor, Y-27632 was added.
  • the cells were allowed to settle in static culture for 30 additional minutes, and then 150mL additional medium supplemented with 0.5% BSA and containing lOmicromolar of the Rho Kinase inhibitor, Y-27632, was added to the spinner flask. Thereafter the cells were continuously stirred at 50-70 rpm in a humidified 5% C0 2 incubator. Twenty-four hours later the spinner flask was removed from the incubator and the clusters allowed to settle for 5-10 minutes. The medium was then aspirated until 200mL remained in the vessel and 400mL of additional fresh culture medium was then added to the spinner flask. This process was repeated at the end of day 2 (48 hours after transfer).
  • CryoStor®10 Cold ( ⁇ 4°C) CryoStor®10 was then added in a drop-wise manner to achieve a final concentration of 150 million cells per mL and the cell solution was held in an ice bath during transfer to a 1.8mL corning cryo vial (Corning; Corning, NY) or 15mL Miltenyi cryo bag(Miltenyi Biotec Inc. Auburn, CA).
  • the suspension expanded cells were then frozen in a vial at high density in a controlled rate freezer as follows.
  • the chamber was pre-cooled to 4°C and the temperature was held until sample vial temperature reached 6°C.
  • the chamber temp was then ramped down at 2°C/min until the sample reached -7°C.
  • the chamber was cooled 20°C/min until the chamber reached -45°C.
  • the chamber temperature was then allowed to briefly rise at 10°C/min until the chamber temperature reached -25°C, and the chamber was then further cooled at 0.8°C/min until the sample vial reached -45°C.
  • the chamber temperature was then cooled at 35°C/min until the chamber reached -160°C.
  • the chamber temperature was then held at -160°C for at least 10 minutes, after which the vials were transferred to gas phase liquid nitrogen storage.
  • cryo-preserved cells were removed from the liquid nitrogen storage, thawed and used to seed a closed 0.2 liter glass bioreactor (DASGIP; Julich, Germany).
  • Cryo-vials were removed from gas phase liquid nitrogen storage and placed directly in a 37°C water bath for 105 seconds. The thawed vial contents were then transferred via 2mL glass pipette to a 50mL conical tube. Then 9mL of E8TM containing 0.5%BSA supplemented with lOmicromolar Rho Kinase inhibitor, Y- 27632 was then added to the tube in a drop wise manner.
  • the cells were then centrifuged at 80-200rcf for 5 minutes. Afterwards, the supernatant was aspirated from the tube and, 10ml of fresh E8 containing 0.5%BSA and supplemented with lOmicromolar Rho Kinase inhibitor, Y-27632 was added. This volume containing the cells was pipetted into a media transfer bottle (Cap2V8 ® , SaniSure, Moorpark, CA) and the bottle contents were pumped directly into the bioreactor via a sterile C-flex tubing weld by peristaltic pump. In preparation for pluripotent stem cell inoculation the bioreactor was prepared with 0.15L of E8TM
  • the cells inoculated into the stirred tank bioreactor formed cell clusters in the continuously stirred tank. After inoculation, the cell clusters were maintained in E8TM medium, supplemented with 0.5% BSA, in the reactor for three days. The medium was changed daily; 24 hours after inoculation 90% of spent medium was removed and 0.15 liters of fresh medium added. Forty-eight hours after inoculation, 90% of spent medium was removed and 0.15 liters of fresh medium was added. At 72 hours after inoculation, pluripotent cell differentiation was initiated by removing >90% of the spent medium and adding differentiation medium (Table 18).
  • the cells were maintained for 12 or more days in the closed sterile suspension bioreactor regulated for temperature (37°C), pH (7.4 for differentiation), and dissolved oxygen (10% DO set-point for stage 1 and 30% DO set-point all other times, CO2, O2, N 2 , and air regulated).
  • the impeller was stopped 5-20 minutes prior to medium removal via dip-tube to allow clusters to settle.
  • Medium in the bioreactor was removed or added to/from a closed bottle or bag by peristaltic pump through a dip tube connected to C- Flex ® tubing using a TerumoTM tube welder to maintain a closed system.
  • the impeller and heater were re-energized once sufficient medium was added to the vessel to fully submerge the impeller.
  • stage specific changes in pH and metabolism matched stage changes in mRNA expression patterns as measured by qRT-PCR the following was done.
  • Four Applied Biosystems Low Density Arrays were used (LifeTM , Carlsbad, CA) designated Pluripotency, Definitive Endoderm (DE), Gut Tube (GT), or stage 4 (S4). Results are presented as fold differences versus undifferentiated UTCiPS cell sample as control to standardize expression across all runs and arrays.
  • stage 0 72 hours after reactor inoculation
  • the cells were moved into differentiation medium (Table 18) containing MCX and GDF8. Twenty-four hours after this media change significant alterations in gene expression patterns (Figures 49 and 50 fold expression versus undifferentiated control) were noted, such as a >10x increase in FOXA2, HAND2, PRDM1, PTH1R and SOX17 expression, >100x increase in CER1, FGF4, GATA4, GATA6, GSC, and MNX1 and a >1000x increase in EOMES, FGF17, MIXL1, and T expression. These increased expression levels indicated the cells were transitioning through a mesendodermal fate.
  • CDX2 levels were elevated at stage 1 day 1 versus undifferentiated cells (2700x increase in expression vs. control), however this was a transient increase in expression and CDX2 levels dropped 97% by stage 1 day 3 to levels comparable to those observed prior to induction of differentiation ( Figures 49 and 50 fold expression versus undifferentiated control).
  • stage 1 differentiation medium At 72 hours after exposure to the stage 1 differentiation medium, the cells expressed a profile consistent with specification to definitive endoderm, as CXCR4 levels peaked at ⁇ 400x over historical control, FOXA2 was expressed at 136x over control and SOX 17 was expressed at 470,000x over historical control. Consistent with definitive endoderm, it was also noted that gene expression of CER1, EOMES, FGF4, GSC, MIXL1, and T at the end of stage 1 (day 3) had dropped from the elevated levels observed at stage 1 day 1 ( Figures 49 and 50 fold expression versus undifferentiated control).
  • stage 2 Following the completion of definitive endoderm formation (stage 1) the medium was changed to one containing FGF7, a morphogen used to induce primitive foregut formation. Consistent with formation of primitive foregut, FTNF4a and GATA6 expression levels at stage 2 days 1 and 3 increased, while genes expressed at high levels on stage 1 day 3 (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX 17, and VWF) showed reduced expression by the end of stage 2 ( Figures 50 and 53 fold expression versus undifferentiated control). The expression of foregut genes (AFP, HHEX, PDX1, and PROX1) was increased ( Figure 53 fold expression versus undifferentiated control).
  • stage 3 medium (Table 18). Once in this medium the cells expressed markers consistent with an endodermal pancreatic lineage as measured by qRT-PCR assay for gene expression. Gene expression for PDX1 increased 60 fold from 12,000x over control at the end of stage 2 day 3 to 739,000x over control at the end of stage 3 day 3. These data indicated the cells were specifying to a pancreatic fate ( Figure 54).
  • the cells retained high levels of PDX1 (95.6% positive by FACS, -1,000,000 fold induction over control by qRT-PCR) and FOXA2 (99.5% positive by FACS) expression.
  • the cells showed an expression pattern consistent with pancreatic progenitor cells (39.2% positive for NKX6.1 by FACS) and a population of pancreatic endocrine cells (9.4% positive for PAX6, 12.4% positive for Chromogranin, 15.2% positive for NKX2.2; all by FACS).
  • This stage specific marker expression pattern indicated an efficient stage-wise differentiation from a pluripotent population to pancreatic precursor cells.
  • stage 1 basal medium or "Neat” medium for the purposes of this example.
  • Clusters were treated on the first day of differentiation with one of six conditions: (1) Neat, (2) 3 ⁇ MCX plus lOOng/ml GDF-8 (Catalog # 120-00, Peprotech), (3) 3 ⁇ MCX only, (4) lOOng/ml GDF-8 only, (5) 20ng/ml WNT-3A (Catalog # 1324-WN-002, R&D Systems, MN) plus lOOng/ml Activin A (Catalog # 338-AC, R&D Systems, MN), or (6) 20ng/ml W T-3A only.
  • This example demonstrates a process capable of both expanding and differentiating pluripotent stem cells (PSC) to achieve a scalable manufacturing process for generation of ectodermal or mesodermal tissues.
  • PSC pluripotent stem cells
  • the cells inoculated into the stirred tank bioreactor formed cell clusters in the continuously stirred tank, and were maintained in pluripotency medium (E8TM, supplemented with 0.5% BSA) in the reactor for three days total.
  • pluripotent cell differentiation was initiated by transferring cell clusters to plastic disposable Erlenmeyer flasks (PETG 125mL flask, Cat#4112, Thermo Scientific Rochester NY) in their respective differentiation medium (Table 19) to form mesoderm/cardiac tissue (1) or ectoderm/neural tissue (2).
  • the cells were maintained for ten (10) days at 100 rpm in a humidified, 5% CO 2 incubator on a shaker platform (MAXQ 416hp, Thermo Scientific, Rochester NY). At 1 day, 3 days, 5 days, and 7 days after the initiation of differentiation the medium in the flask was exchanged for fresh medium made as described in Table 19. qRT-PCR samples were taken prior to starting differentiation for reference and then 3, 5, 7, and 10 days after initiating differentiation.

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Abstract

La présente invention concerne des méthodes de préparation de groupes de cellules souches pluripotentes agrégés aux fins de différenciation. L'invention concerne en particulier des méthodes de différenciation de cellules souches pluripotentes en lignées de cellules béta, de cellules cardiaques et de cellules neuronales par regroupement en suspension. Les méthodes comprennent la préparation des groupes de cellules agrégés puis de la différenciation de ces agrégats.
PCT/US2014/038993 2013-11-01 2014-05-21 Mise en suspension et agrégation de cellules souches pluripotentes humaines aux fins de différenciation en cellules endocrines du pancréas WO2015065524A2 (fr)

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RU2016121409A RU2689710C2 (ru) 2013-11-01 2014-05-21 Суспендирование и кластеризация плюрипотентных стволовых клеток человека с целью их дифференцировки в панкреатические эндокринные клетки
CA2928639A CA2928639A1 (fr) 2013-11-01 2014-05-21 Mise en suspension et agregation de cellules souches pluripotentes humaines aux fins de differenciation en cellules endocrines du pancreas
CN201480059910.5A CN105793413A (zh) 2013-11-01 2014-05-21 用于分化成胰腺内分泌细胞的人多能干细胞的悬浮和群集
JP2016527338A JP2016534731A (ja) 2013-11-01 2014-05-21 膵内分泌細胞への分化のためのヒト多能性幹細胞の懸濁及び集団化
EP14857034.4A EP3063268A4 (fr) 2013-11-01 2014-05-21 Mise en suspension et agrégation de cellules souches pluripotentes humaines aux fins de différenciation en cellules endocrines du pancréas
AU2014342995A AU2014342995C1 (en) 2013-11-01 2014-05-21 Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells
KR1020167014409A KR20160079072A (ko) 2013-11-01 2014-05-21 췌장 내분비 세포로의 분화를 위한 인간 만능 줄기세포의 현탁 및 클러스터링
MX2016005657A MX2016005657A (es) 2013-11-01 2014-05-21 Suspensión y agrupamiento de células madre humanas pluripotentes para la diferenciación a células endocrinas pancreáticas.
KR1020187034291A KR20180128529A (ko) 2013-11-01 2014-05-21 췌장 내분비 세포로의 분화를 위한 인간 만능 줄기세포의 현탁 및 클러스터링
SG11201603045VA SG11201603045VA (en) 2013-11-01 2014-05-21 Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells
PH12016500782A PH12016500782A1 (en) 2013-11-01 2016-04-27 Suspension and clustering of human pluripotent stem cells for differentation into pancreatic endocrine cells
AU2018208707A AU2018208707A1 (en) 2013-11-01 2018-07-26 Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells

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US201361962158P 2013-11-01 2013-11-01
US61/962,158 2013-11-01
US13/998,974 2013-12-30
US13/998,974 US10377989B2 (en) 2012-12-31 2013-12-30 Methods for suspension cultures of human pluripotent stem cells

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PCT/US2014/042796 WO2015065537A1 (fr) 2013-11-01 2014-06-17 Mise en suspension et regroupement de cellules souches pluripotentes humaines pour la différenciation en cellules endocrines pancréatiques

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WO2020243663A1 (fr) 2019-05-31 2020-12-03 W. L. Gore & Associates, Inc. Composite à membrane biocompatible
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US11104918B2 (en) 2019-09-05 2021-08-31 Crispr Therapeutics Ag Universal donor cells
US11116798B2 (en) 2019-09-05 2021-09-14 Crispr Therapeutics Ag Universal donor cells
US11566230B2 (en) 2020-12-31 2023-01-31 Crispr Therapeutics Ag Universal donor cells
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EP3063269A4 (fr) * 2013-11-01 2017-07-12 Janssen Biotech, Inc. Mise en suspension et regroupement de cellules souches pluripotentes humaines pour la différenciation en cellules endocrines pancréatiques
US10391156B2 (en) 2017-07-12 2019-08-27 Viacyte, Inc. University donor cells and related methods
US11904001B2 (en) 2017-07-12 2024-02-20 Viacyte, Inc. Universal donor cells and related methods
JP2020527956A (ja) * 2017-07-21 2020-09-17 センマ・セラピューティクス・インコーポレーテッド 幹細胞由来の膵臓ベータ細胞の再凝集
US12104173B2 (en) 2018-01-18 2024-10-01 Agency For Science, Technology And Research Method for differentiation of human pluripotent stem cell lines in suspension culture
WO2019231111A1 (fr) * 2018-05-30 2019-12-05 주식회사 지스테믹스 Liquide de culture de cellules souches adultes et son procédé de préparation
US10865424B2 (en) 2018-09-07 2020-12-15 Crispr Therapeutics Ag Universal donor cells
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US11008586B2 (en) 2018-09-07 2021-05-18 Crispr Therapeutics Ag Universal donor cells
US11008587B2 (en) 2018-09-07 2021-05-18 Crispr Therapeutics Ag Universal donor cells
US10724052B2 (en) 2018-09-07 2020-07-28 Crispr Therapeutics Ag Universal donor cells
WO2020243665A1 (fr) 2019-05-31 2020-12-03 W. L. Gore & Associates, Inc. Composite à membrane biocompatible
WO2020243668A1 (fr) 2019-05-31 2020-12-03 W. L. Gore & Associates, Inc. Dispositifs d'encapsulation de cellules présentant des distances de diffusion d'oxygène régulées
WO2020243666A1 (fr) 2019-05-31 2020-12-03 W. L. Gore & Associates, Inc. Composite à membrane biocompatible
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US11104918B2 (en) 2019-09-05 2021-08-31 Crispr Therapeutics Ag Universal donor cells
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US11566230B2 (en) 2020-12-31 2023-01-31 Crispr Therapeutics Ag Universal donor cells
US11578309B2 (en) 2020-12-31 2023-02-14 Crispr Therapeutics Ag Universal donor cells

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MX2016005658A (es) 2017-01-05
AU2014342995B2 (en) 2018-04-26
AU2018208707A1 (en) 2018-08-16
CA2928741A1 (fr) 2015-05-07
JP2016534731A (ja) 2016-11-10
BR112016009393A8 (pt) 2020-03-24
EP3063268A4 (fr) 2017-11-29
AU2014343007C1 (en) 2018-08-09
JP2017500013A (ja) 2017-01-05
EP3063269A1 (fr) 2016-09-07
SG11201603047PA (en) 2016-05-30
CN105793413A (zh) 2016-07-20
AU2014342995C1 (en) 2018-08-09
AU2014342995A1 (en) 2016-05-05
EP3063269A4 (fr) 2017-07-12
PH12016500783A1 (en) 2016-06-13
EP3063268A2 (fr) 2016-09-07
KR20180130001A (ko) 2018-12-05
RU2016121409A3 (fr) 2018-03-29
CN105683362A (zh) 2016-06-15
CA2928639A1 (fr) 2015-05-07
KR20160079071A (ko) 2016-07-05
RU2016121409A (ru) 2017-12-04
KR20180128529A (ko) 2018-12-03
RU2689710C2 (ru) 2019-05-29
PH12016500782A1 (en) 2016-06-13
MX2016005657A (es) 2017-01-05
RU2016121404A (ru) 2017-12-04
RU2016121404A3 (fr) 2018-03-29
AU2014343007A1 (en) 2016-05-05
WO2015065537A1 (fr) 2015-05-07
AU2018208717A1 (en) 2018-08-16
SG11201603045VA (en) 2016-05-30
AU2014343007B2 (en) 2018-04-26
KR20160079072A (ko) 2016-07-05
WO2015065524A3 (fr) 2015-08-13

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