AU2015268690A1

AU2015268690A1 - Differentiation of human embryonic stem cells

Info

Publication number: AU2015268690A1
Application number: AU2015268690A
Authority: AU
Inventors: Janet Davis; Kevin Ditolvo; Christine Parmenter
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2009-12-23
Filing date: 2015-12-14
Publication date: 2016-01-07
Anticipated expiration: 2030-12-16
Also published as: AU2015268690B2

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to increase the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage.

Description

DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS FIELD OF THE INVENTION [0000] The present application is a divisional application of Australian Application No. 2010333840, which is incorporated in its entirety herein by reference. [0001] This application claims the benefit of U.S. Provisional Application No. 61/289,692, filed on December 23, 2009, which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION [0002] The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to increase the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage. BACKGROUND [0003] Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or s cells, appropriate for engraftment. One approach is the generation of functional P cells from pluripotent stem cells, such as, for example, embryonic stem cells. [0004] During vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF3 beta, GATA4, MIXL1, CXCR4 and SOX17. [0005] Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the - 1 pancreatic-duodenal homeobox gene, PDX1. In the absence of PDX1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, - laamong other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm. [00061 Cells bearing the features of islet cells have reportedly been derived in vitro from embryonic cells of the mouse. For example, Lumelsky et ai. (Science 29:2:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria ef at (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia when implanted in streptozotocin-induced diabetic mice. [00071 In one example, lori et a. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled P cells. [.00081 In another example, Blyszczuk ci al (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4, 100091 Micallef ef al reports that retinoic acid can regulate the commitment of embryonic stem cells to form PDX1 positive pancreatic endoderm. Retinoic acid is most effective at inducing PDX1 expression when added to cultures at day four of embryonic ;tei cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes S4:301, 2005), [00101 Miyazaki et aL reports a mouse embryonic stem cell line over-expressing Pdxl. Their results show that exogenous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurognin3, p48, Pa6, and -INF6 in the resulting differentiated cells (Diabetes 53: 1030, 2004). [0011.1 Skoudy et al-reports that activin A (a member of the TGF-l superfamily) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouse embryonic stem cells. The maximal effect was observed using I aM activin A. They also observed that the expression level of insulin and Pdxi mRNA was not affected by retinoic acid; however, 3nM FGF7 treatment resulted in an increased level of the transcript for Pdx 1 (Biochem. J. 379: 749, 2004). 2 [001.21 Shiraki et at studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into PDXI positive cells. They observed that TGF~32 reproducibly yielded a higher proportion of PDX1 positive cells (Genes Cells. 2005 Jun; 10(6): 503-16). 100131 Gordon et at demonstrated the induction of brachyury [positive], HNF3 beta [positive] endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A 1). [0014] Gordon et at (PN AS, Vol 103, page 16806. 2006) states "Wnt and TOF-beta/ nodal activin signaling simultaneously were required for the generation of the anterior primitive streak," [00151 However, the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans. 100161 Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonada tissue (Shamblott et aL, Proc. NatL Acad, Sci. USA 95:13726 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat, No. 6,200,806; WO 99120741; WO Oi/51616). 100171 D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endodenn in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005), Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of somc endodermal organs, Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX1 positive cells after addition of FGF-10 (US 2005/0266554AI). 100181 D'Amour et at (Nature Biotechnology - 24, 1392 - 1401 (2006)) states: "We have developed a differentiation process that converts human enibryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin., glucagon, 3 somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm. gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones". [0019] In another example, Fisk ei al, reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006M040387A LI. In this case, the differentiation pathway was divided into three stages. luman embryonic stem cells were first differemiated to endoderm using a combination of sodium butyrate and activin A. The cells were then cultured with TGF4 antagonists such as Noggin in combination with EGF or betacellulin to generate PDX 1 positive cells. The terminal difTrentiation was induced by nicotinamide. [0020] In one example, Benvenistry e t al states: "We conclude that over-expression of PDX I enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo" (Benvenistry et al, Stem Cells 2006; 24:1923-1930). [0021]. In another example, Grapin Botton el al stats: "Early activation of Ngn3 almost exclusively induced glucagon [positive] edls while depleting the pool of pancreas progenitors. As from El 15, PDX-1 progenitors became competent to differentiate into insulin [positive] and PP positive] calls" (Johansson KA et al, Developmental Cell 12, 457 - 465, March 2007). [0022] The expression of NON3 in cells expressing markers characteristic of the pancreatic endoderm lineage may reduce the ability of the cells to further differentiate into insulin expressing cells. Previou\ studies have showed that cells expressing markers characteristic of the pancreatic endoderm lineage that express NGN3 are more likely to produce glucagon expressing cells than insulin expressing cells, when subjected to further differentiation. However, NGN 3 expression is required to form pancreatic endocrine cells, or pancreatic endocrine precu\sor cells (cells that can form, for example glucag'on, or insulin expressing cells). Therefore, the temporal regulation of NGN3 is important in guiding the ultimate fate of pancreatic endocrine precursor cells toward insulin expressing cells. 4 [00231 Therefore, there still remains a significant need to develop conditions for establishing pharipotent stem cell lines that can be expanded to address the current clinical needs, while retaining the potential to differentiate into insulin expressing cells. The present invention takes an alternative approach to improve e teefficiency of differentiating human embry onic stem cells toward insulin expressing cellS, by providing a method to increase the expression of NGN3 and NKX6.1 in cells expressing markers characterliic of the pancreatic endocrine lineage. SUMMARY [00241 In one embodiment, the present invention provides a method to increase the expression of NGN3 and NKX6l in a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of: a) culturing pluripotent stern cells, b) differentiating the pluripotent stei cells into cells expressing markers characteristic of the definitive endoderm lineage, C) differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, supplementing the medium used to differentiate the cells expressing markers characteristic of the definitive endoderm lineage with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA 1004, PP2, PPI, LY 294002, Wortmannin, S1B-203580, SB-202190, Tyrphostin 25, Tyrphostin, AGl 478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7, and d) differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage. [00251 In one embodiment, the medium used to differentiate the cells expressing markers characteristic of the pancreatic endoderm lineage is supplemented with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA-1004, PP2, PP1, LY :294002, Wortmannin, SB-203580, S13-202190, Tyrphostin 25, Tyrphostin, AG 1478, Tyiphostin 46, GW 5074, Kenpaullone, HNMPA, AG490 Y2763 2, and ML-7 DETAILED DESCRIPTION [00261 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. Definitions 100271 Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderin aid ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts. 100281 Stem cells are classified by their developmental potential as: (I) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) m-ultipotent, meaning able to give rise to a subset of cell lineages but all, within a particular tissue, organ, or physiological system (for example, bematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors, and all cell types and clements (e,g platelets) that are normal components of the blood), (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e,,g, spermatogenic stem cells), 100291 Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell. The term "committed", when applied to the process of differentiation, refers to a cell 6 that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circurastances, differentiate into a different cell type or revert to a less differentiated cell type, De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can. give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted celI to the lineage of interest, 100301 "Cells expressing markers characteristic ofthe definitive endoderm lineage", or "Stage I cells", or "Stage 1", as used herein, refers to cells expressing at least one of the following markers: SOX 17, GATA4, HNF3 beta, OSC, CER 1, Nodal, FGFS, BrachyuryMix-like homeobox protein. FGF4 CD48, comesodermin (EIOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endodern cells. 100311 "Cells expressing markers characteristic of the pancreatic endoderm lineage", as used herein, refers to cells expressing at least one of the following markers: PDXI, HNFI beta, PTFI alpha, HNF6, NKX6.1, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells, primitive gut tube cells, and posterior foregut cells 100321 "Definitive endoderm", as used herein, refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives, Definitive endoderm cells express the following markers: HNF3 beta, GATA4, SeX17, Cerberus, OTX2, goosecoid, C-Kit. CD99, and MIXL L [0033] "Marken,", as used herein, ae nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently 7 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. [00341 "Pancreatic endocrine cell"., or"pancreatic hormone expressing cell", as used herein, refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin. and pancreatic polypeptide. Isolation, Expansion and Culture of Pluripotent Stem Cells Characterization af Puripotent Stem Cells 100351 Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SS EA) 3 and 4, and markers detectable using antibodies designated Tra-1 60 and Tria-1-8(rhomnson er alt Science 282: 1145, 1998). Differentiation of piuripotent stem cells in vitro results in the loss of SSEA-4, Tra 1-60, and Tra I -81 expression (if presem) and increased expression of SSEA- 1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldchyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories,. Burlingane Calif.). Undifferentiated pluripotent stem cells also typically express OCT4 and TERT. as detected by RT1 PCR, [00361 Another desirable phenotype ot propagated pluripotent stem cells is a potential to differentiate into cells of all three gerninal layers: endoden, .mesoderm, and ectoderm tissues, Pluripotency of plripotent stem cells can be confirmed, for example, by injecting cells into severe combined immnunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, piuripotency 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. 100371 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 8 that the cells are euploid, wherein all human chromosomes are present and not noticeably altered. Sowes of Pluripotent Stem Ce//s ,0038I The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-emrbryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-imiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and [19 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable arc cells taken from a plurnpotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, B00v (BresaGen, Athens, GA). [0039] In one embodiment, human embryonic stem cells are prepared as described by Thomson al, (U.S, Pat, No. 5,843,780; Science 282: 1145, 1998; Curr, Top, Dev. Bio. 38:133 ff., 1998; Proc. NatI, Acad. Sci. U.SA. 92:7844, 1995). Culture of P/uripotent Stem Ce/s 000] In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways. Alternatively, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation, The growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a mediun conditioned by culturing previously with another cell type. Alternatively, the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a clhenically defined medium. 9 [00411 For example, Reubinoff et al(Nature Biotechnology 18: 399 ~ 404 (2000)) and Thompson et ai (Science 6 November 1998: Vol. 282. no. 5391, pp. 1145 - 1147) disclose the culture of pluripotent stem cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer, [0042] Richards el al, (Stem Cells 21: 546-556. 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture. Richards ei al, states: "human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripolent", [00431 US200200721 17 discloses cell lines that produce r-iedia that support the growth of primate pluripotent stem cells in feeder-free culture, The cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stern cells. US20020072117 also discloses the use of the cell lines as a primary feeder cell layer. [00441 In another example, Wang et al (Stein Cells 23: 1221-1227, 2005) discloses methods for the long-term growth of human pluripotent stem cells on feeder cell layers derived from human embryonic stem cells, [00451 In another example, Stojkovic et al (Stem Cells 2005 23: 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic stem cells, 100461 In a further example, Miyamoto et al (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta. 100471 Amit ei al (BioL Reprod 68: 2150-2156, 2003) discloses a feeder cell layer derived from human foreskin. [0048] In another example, Inzunza et al (Stem Cells 23: 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts. 100491 US6642048 discloses media that support the growth of primate pluripotent stern (pPS) cells in feeder-free culture, and cell lines useful for production of such media. US6642048 states: "This invention includes mesenchymal and fibroblast-like cell 10 lines obtained from. embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure." [00501 In another example, W02005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. W02005014799 states; "The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells; in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte)" [0051] In another example, Xu et a? (Stem Cells 22: 972-980, 2004) discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase. 10052] In another example, US200700100 1I discloses a chemically defined culture medium for the maintenance of phiripotent stem cells. 100531 An alternative culture system employs serum-frec medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells. For example, Cheon et a? (BioReprod DOI:10,1095/biolreprod. 105.046870t October 19, 2005) disclose a feeder-free, serm-fee culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell Nelf-renewal. 100541 In another example, Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF. [00551 In another example, US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitam ins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth, factor signaling receptor,

II

wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium. [0056] In another example, US20050233446 discloses a defined medium useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the medium is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and art amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells. [00571 In another example, US6800480 states "In one embodiment. a cell culture medium for growing primate-detrived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stern cells. The basic medium is combined with a nutrient serum effective to support the growth of primate derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medim further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate sah," 100581 In another example, US20050244962 states: "In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures thet sten cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor." 100591 In a further example, W02005065354 discloses a defined, isotonic culture medium. that is essentially feeder-free and serum-free, comprising: a, a basal medium; b, an amount of bFGF sufficient. to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic 12 acid sufficient to support growth of substantially undifferentiated mammalian stem cells. [00601 In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method compinsing exposing a stem cell to a member of the transforming growth factor-beta (TGF ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result. [0061.1 The pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component. such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings, In one embodiment, the suitable culture substrate is MATIRIGEL* (Beeton Dickenson), MATRIGEL* is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane. [00621 Other extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations. 100631 The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics, All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art. [00641 Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibeo # 11965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; p-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256 029, 13 Formation of a Population of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage with Increased Expression of NGN3 and NKX6,1 [00651 In one embodiment, the present invention provides a method to increase the expression of NGN3 and NKX6, I in a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of: a) culturing pluripotent stem cells, b) differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, c) differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endodern lineage, supplementing the medium used to differentiate the cels exprtessing narkers characteristics of the definitive endodern lineage with a compound selected from the group consisting of 4-9, 1-89, G F 109203X, HA 1004, PP PI, LY 294002, Wortmannin, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, A0490, Y27632, and ML-7, and d) differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage. Dilkrentiation ofPiuripot ent Stem Cells into Cells Expressing Aarkers Ciaracteristic of the Definitive Endoderm Lineage [0066] Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers, Thus, differentiation of pluripotent cells is detected when cells begin to express them. [00671 Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention. 14 [00681 For example, phuripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Anour et al, Nature Biotechnology 23, 1534 - 1541 (2005). [00691 For example, pluripotent stem cells may be differentiated into cells expressing makers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651 - 1662 (2004), 100701 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endodern lineage according to the methods disclosed in McLean metal, Stem Cells 25, 29 - 38 (2007). [0071.1 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006). 100721 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration. An example of this method is disclosed in Nature Biotechnology 23, 1534 - 1541 (2005). [00731 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium contaiing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration. An example of this method is disclosed in D' Amour el al, Nature Biotechnology, 2005. [00741 For example, pluripoten stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent ster cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wit ligand and culturing the cells wsith acivin .A with serum. An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006). 15 [0075] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser No. 11/736,908, assigned to LifeScan, Inc. [0076] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 11/779,3 11, assigned to LifeScan, Inc. [00771 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 60/990,529, [00781 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Scr. No. 61/076,889. [00791 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61 "076,900. 10080] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,908. [0081]1 For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser, No. 61/076,915. 16 Ditereniiation of/Cells Expressing Arkers Characteristic of/the Definitive Endoderm Lineage into Cells Expres sing Alrkers Characieristic of the Pancreatic Endodern Lineage [00821 Cells expressing markers characteristic of the definitive endodermi lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention. [0083] For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnol 24:1392-1401, 2006. [00841 For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the .medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine, An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006). 100851 In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc. 100861 In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of timel, according to the methods disclosed in US patent application Ser, No. 11/779,311, assigned to LifeScan, Inc. 17 [00871 In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No. 60/990,529. 10088] The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage. [00891 Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These inchide quantitati ve reverse tramscriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e~g, Current Protocols in Molecular Biology (Ausubel et al eds. 2001 supplement)), and imtnmnoassays, such as immunolistochemical aMalysis of sectioned material., Westem blotting, and for markers that are accessible in intact cells, such as flow cytometric analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratoiy Press (1998)), [00901 Characteristics of pluripotcnt stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTFl, ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81. [00911 After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage. 100921 Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line -19 (NIH code: WA09), the human embryonic stem cell line El (NIH code: WAN1) the human embryonic stem cell line 1-7 (NIH code: WAO7.), and the human embryonic stem cell line SA002 (Cellartis, Sweden), i8 Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cclls: ABCO2, cripto, CD9, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. [0093] Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17. GATA4, RNF3 beta, GSC, CERI, Nodal, FGFSBrachyury, Mix-like homeobox protein, FGF4 CD48., comesodermin (EOMES), DKK4, FOF 17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present Invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell, In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cel. [00941 Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of PDX1, HNF1 beta, PTFl alpha, HNF6, HB9 and PROX. Suitable for use in the present ivention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endodern cell, [00951 In one embodiment, the cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cels expressing markers of the characteristic of the pancreatic endocrine lineage. The present invention provides methods to increase the expression of NGN3 and NKX6 i ni populations of cells expressing markers characteristic of the pancreatic endocrine lineage. [0096] Increasing the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage may be achieved by treating cells expressing markers expressing markers characteristic of the definitive endoderi lineage with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA-1004, PP2, PP I, LY 294002, Wortmannin, SB-203580, SB-202190, 19 Tyrphostin. 25, Tyrphostin, AC1.478, Tyrphostin 46., W 5074. Kenpaullone., HNMPA, AG490, Y27632, and ML-7. Alternatively, increasing the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage may be achieved by treating cells expression markers expressing markers characteristic of the pancreatic endoderm lineage with a compound selected from the group consisting of H -9, H-89, GF 109203X, HA- 1004, PP2, PP 1, LY 294002, Wortmannin, SB~203580, SB-202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7. [00971 In the case where cells expressing markers expressing markers characteristic of the definitive endoderm lineage are treated with a compound selected from the group consisting of X, Y, and Z, the cells are treated by supplementing the medium used to differentiate the cells to cells expressing markers characteristic of the pancreatic endoderm lineage with a compound selected from the group consisting of 1-9, 11-89, GF 109203X. HA-1 004, PP2. PP1. LY 94002, Wortmannin, SB-203580, SB 202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7. [00981 In the case where cells expressing markers expressing markers characteristic of the pancreatic endoderm lineage are treated with a compound selected from the group consisting of X, Y, and Z, the cells are treated by supplementing the medium used to differentiate tie cells to cells expressing markers characteristic of the pancreatic endocrine lineage with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA-Ik1004 PP2, PP1, LY 294002, Wortmannin, SB-203580, SB 202190, Tyrphostin 25, Tyrphostin, AG1478, Tyrpihostin 46, GW 5074, Kenpaullone, HNMPA, AC490, Y276,32, and ML-7. D~ern.ztiation ofCells Epressing Marksr Characteristic of she Pancreatic Endoder Lineage ink Cells IExpressing Markers Characteritic of the Pancreatic Endocrine Lineage wish an Increased Expression of NGN3 and NKX6. 1 [00991 Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method proposed in this invention, 20 [01001 For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, lGF~1 and HGF. An example of this method is disclosed in D' Amour er al, Nature Biotechnology, 2006, [0101] For example, cells expressing markers characteristic of the pancreatic endodemt lineage obtained according to the methods of the present invention are further ditTentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT (Sigma-Aldrich, MO) and exendin 4, An example of this method is disclosed in D' Amour et al. Nature Biotechnology, 2006. [01021 For example, cells expresing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further ditTerentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by cnlturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006. [01031 For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch. signaling pathway, according to the methods disclosed in US patent application Scr. No. 11/736,908, assigned to LifeScan, Inc, [01041 For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine 21 lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc. [01051 For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endodenm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 60/953,178, assigned to LifeScan, Inc. [0106] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No, 601990,529, assigned to LifeScan, Inc. 101071 Markers characteristic of the pancreatic endocrine lineage are selected fom the group consisting of NON3, NEUROD N IS , PD.X.1, NKX6,.1 PAX4, NGN3, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following horniones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for usc in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. in one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone-expressing cell Alternatively, the pancreatic endocrine cell may be a pancreatic hormone-secreting cell. [01,08 In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markets characteristic of the 0 cell lineage. A cell expressing markers characteristic of the p cell lineage expresses PDX I and at least one of the following 22 transcription factors: NGN3, NKX2,2, NKX6.I, NEUROD, ISLL, HNF3 beta, MAFA, PAX4, and PAX6. In one aspect of the present invention, a cell expressing markers characteristic of the Q cell lineage is a p cell. [0109] The present invention provides methods to increase the expression of NGN3 and NKX6,1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage. [01101 In one embodiment, increasing the expression of NGN3 and NKX6.1 in populations of cells expressing markers characteristic of the pancreatic endocrine lineage may be achieved by treating cells expressing markers expressing markers characteristic of the pancreatic endoderm lineage with a compound selected from the group consisting of H-9 1-89, GF 109203X, HA~ 1004. PP2, PP 1, LY 294002, Wortmannin, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin, AG 1478, Tytphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7, 101111 In the case where cells expressing markers expressing markers characteristic of the pancreatic endodennlineage are treated with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA- 1004 PP2, PP, LY 294002, Wortmannin, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin, AG 1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and ML-7, the cells are treated by supplementing the medium used to differentiate the cells to cells expressing markers characteristic of the pancreatic endocrine lineage with a compound selected from the group consisting of H-9, H-89, OF 109203X, HA-1004, PP2, PP1, LY 294002, Wortmannin, S3-203580, S3-202190, Tyrphostin 25, Tyrphostin, AG 1478, Tyrphostin 46, GW 5074, Kenpaullone, HNMPA, AG490, Y27632, and NL-7. [011.21 The present invention is further illustrated, but not limited by, the following examples. EXAMPLES Example I Screening for Small Molecule Analogues that Mediate NGN3 Expression 23 [0113] Expression of the transcription factor NGN3 is required during the progression of progenitor cells towards an endocrine cell fate. Enhancing the efTiciency of this process is a desirable outcome. A screen of small molecule compounds was performed on the assumption that enzymatic inhibitors may regulate cellular signals transmitted during differentiation and have direct or indirect effects on the gene expression of critical transcription factors such as NGN3, [0114] Preparation fcellsf1r asay: Stock cultures of human embryonic stem cells (H1 human embryonic stem cell line) were maintained in an undifferentiated, pluripotent state on reduced growth factor MATRIGEL (BD Biosciences: Cat # 35623 1)-coated dishes in MEF conditioned medium with passage on average every four days, Passage was performed by exposing cell cultures to a solution of I mg/ml dispase nvitrogen, Cat #: 17105-041) for 5 to 7 minutes at 3 7C followed by nasing the monolayer with MEF conditioned culture medium and gentle scraping to recover cell clusters Clusters were centrifuged at low sPeed to collect a cell pellet and remove residual dispase, Cell clusters were split at a 1:3 or 1:4 ratio for routine maintenance cult, All human embryonic stem cell lines were maintained at passage numbers less than 50 and routinely evaluated for normal karyotype and absence of mycoplasma, For screens in miniaturized assay format, clusters of H I human embryonic stem cells were harvested from culture with dispase treatment as described and plated with even dispersal at a ratio of 1:2 (surface area) on reduced growth factor MATRIGEL (BD Biosciences; Cat # 356231)-coated 96-well black plates (Packard ViewPlates: PerkinElmer; Cat #6005182) using volumes of 100 tl/well. Cells were allowed to attach and then recover log phase growth over a 1 to 3 day time period, feeding daily with MEF conditioned medium supplemented with 8ng/mI bFGF (R&D Systems; Cat # 233-FB). Plates were maintained at 37 0 C, 5% CO 2 in a humidified box throughout the duration of assay. [0115] Preparation ofcompounds: Screening was conducted using two commercial libraries of small molecule kinase inhibitors (BioMol Intl; Cat f 2832A(V2.2) and EMD Biosciences: Cat # 539745), Table I and Table 2 describe the compounds in these BioMol and EMD kinase inhibitor libraries, respectively, Compounds from these libraries were made available as 10 mM stocks in 96-well plate format, solubilized in 100% DMSO and stored at -80*C, The library compounds were further diluted to an 24 intermediate concentration of 2.5nM in 100% DMS( (Sigma: Cat # D2650), also stored at -80C until use. On the day of assay, compounds were diluted 1:12.5 into D.M1EM high glucose medi urm to yield a 200uM working stock in 8%:DMSO and then further dilted 1:80 into each assay test well for a final concentration of 2.5uM compound and 0.1% DMS0 [01161 Diferendation and Screening Assay: Step I of the differentiation protocol was conducted over three days, feeding daily by aspirating the medium from each well and replacing with a fresh aliquot (100 l). On the first day of assay, wells were fed using RPM[,- 1640 medium (lnvitrogen; Cat #: 22400) containing 2% Albumin Bovine Fraction V, Fatty Acid Free (FAF BSA) (Proliant Inc.; Cat 4: SKU 68700), 100ng/mi Activin A (PeproTech; Cat #,20-14), 20ng/ml WNta (R&D Systems; Cat # 1324 WN/CF), and 8ng/inl bFGF (R&D Systems; Cat # 233-FB), On the second and third day of assay, wells were fed with the same medium except that Wnt3a was removed, All wells were fed and treated identically. [01171 Step 2 of the differentiation protocol was conducted over two days. Cells were fed daily by aspirating the medium from each well and replacing with a fresh aliquot (1001) of DMEM:F 12 medium (Invitrogen; Cat # 11330-032) containing 2% FAF BSA, 50ng/mi FGF7 (PeproTech; Cat # 100-19), and 250nM KAAD-cyclopamine (Calbiochem; Cat # 239804). All wells were fed and treated identically. 10118] Step 3 of the differentiation protocol was conducted over four days. Cells were fed on alternating days by aspirating medium from each well and replacing with a fresh aliquot Qnl) of DME1-high glucose (Invitrogen; Cat 4 10569) supplemented with 0.1% Albumax (nvitrogen; Cat #: 11020-021), 0.5x Insulin Fransferrin-Selenium (ITS-X; Invitrogen; Cat # 51500056) 50 ng/mi FGF7, 100 ng/nil Noggin (R&D Systems; Cat # 3344-NG) 250 nM KAAD-cyclopaminc, 2 iM all-trans retinoic acid (RA) (Sigma-Aldrich; Cat # R2625), and 30ng/rl Activin A. During step 3, test samples of kinase inhibitors were added to single wells in two individual plates (Plates A and B-); a third plate (Plate C) was left untreated., in each plate, a total of 16 control wells were treated with an equivalent amount of 0,1% DMSO without any test compound. 25 [01191 Step 4 of the differentiation protocol was conducted over three days. Cells were fed on days 1 and 2, not day 3, by aspirating the medium from each well and replacing with a fresh aliquot (200ul) of DME-ihigh glucose supplemented with 0.1% Albuinax, 0,5x lnsulin-Transferrin-Seenium 100 uWnmi Noggin, and I pM Alk 5 inhibitor (Axxora; Cat t ALX-270-445). During step 4, test samples of kinase inhibitors were added to single wells in two individual plates (Plates B and C); a third plate was left untreated (Plate A). In each plate, a total of 16 control wells were treated with an equivalent amount of 0. 1% DMSO without any test compound. 101201 High Content Analykis: At the conclusion of step 4, medium from all assay plates was aspirated followed by fixing at room temperature for 20 mimintes with 4% paraformaldehyde (Sigma-Aldrich; Cat # 158127) diluted in PBS without divalent cautions (Invitrogen; Cat # 14190). then washing once with PBS. Sample wells were permeabilized with 0.5% Triton X-100 (VWR; Cat # VW3929-2) or 20 minutes at room temperature, washed two times with PBS, and blocked with 5% donkey serum (Jackson ImmunoResearch; Cat #017-000-121) in PBS for 30 minutes at room temperature. Primary antibody (sheep anti-NGN3; R&D Systems; AF3444) was diluted 1:300 in 5% donkey serum and added to each well for one hour at room temperature. After washing two times in PBS, Alexa Fluor 647 donkey anti-sheep secondary antibody (Invitrogen; Cat # A21448) was diluted 1: 100 and added to each sample well for 30 minutes at room temperature, followed by two washes in PBS. To counterstain nuclei, 4 g/mi Hoechst 33342 (Invitrogen; Cat #, R3570) was added for ten minutes at room temperature. Plates were washed once with PBS and left in 100 A/well PBS for imaging. [01211 Imaging was performed using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the MOM10bs dichroie for cells stained with Hoechst 33342 and Alexa Flour 647. Exposure times were optimized from positive control wells stained with secondary antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the bioassay and subsequent staining procedures. Measurements for total cell number and total NGN3 intensity were obtained from each well using IN Cell Developer Toolbox 1L7 (GE lealtheare) software. Segmentation for the nuclei was determined based on gray-scale levels (baseline range 100-300) and nuclear size. Total NGN3 protein expression was reported as total intensity or integrated intensity, 26 defined as total fluorescence of the cell multiplied by the area of the cell. Background was eliminated based on acceptance criteria of gray-scale ranges between 200 to 3500. Total Intensity data were normalized by dividing total intensities for each well by the average total intensity for the positive control. 101221 Screening results are shown in Table 3 from the combination of two kinase inhibitor libraries used to treat six assay plates in this single experiment Data shown are a representative ratio of the intensity of NGN3 staining for individual compound treated wells relative to staining in wells with DMSO vehicle alone. Intensity ratios as well as rank order comparisons are shown for individual compounds dosed during stage 3 alone or stage 4 alone or combined stages 3 and 4. Compounds with ratio intensities >1.4 relative to a vehicle treated control were tagged as hits for confirmation and additional evaluation. Of special interest, as summarized in Table 4, these compounds appear to target several cell signaling pathways that may be involved in the optimal expression pattern ofNNGN3 during endocrine differentiation. Example 2 Screening for Small Molecule Analogues that Mediate NKX6J and NGN3 Expression [01231 Expression of NKX6.1, along with NGN3 is required during the progression of progenitor cells towards an endocrine cell fate. A screen of kinase inhibitors was conducted to determine if any could up-regulate the. expression of one or both markers during differentiation. In this example, tIe HDAC inhibitor Trich-ostatin A was also included in the differentiation protocol to modulate chromatin remodeling and possibly enhance gene transcription. [01241 Preparation afeusfor sy: Stock cultures of human embryonic stem cells (H1 human embryonic stem cell line) were maintained in an undifferentiated, pluripotent state on reduced growth factor MATRIGEL (BD Biosciences; Cat # 35623 1 -coated dishes in MEF conditioned medium with passage on avegrae every four days, Passage was performed by exposing cell cultures to a solution of I mg/mi dispase (invitrogen, Cat #f: 17105-041) for S to 7 minutes at 37*C followed by rinsing the monolayer with MEF conditioned culture medium and gentle scraping to recover cell clusters. Clusters were centrifuged at low speed to collect a cell pellet and remove 27 residual dispase. Cell clusters were split at a 1:3 or 1:4 ratio for routine maintenance culture. All human embryonic stern cell lines were maintained at passage numbers less than 50 and routinely evaluated for normal karyotype and absence of mycoplasma. For screens in miniaturized assay format, cl busters of 111 human embryonic stem cells were harvested from culture with dispase treatment as described and plated with even dispersal at a natio of 1:2 (surface area) on reduced growth factor MATRIGEL (BD Biosciences; Cat # 56231)-coated 96well black plates (Packard View Plates; PerkinElmer; Cat #6005182) using volumes of 100 al/well. Cells were allowed to attach and then recover log phase growth over a I to 3 day time period, feeding daily with. MEF conditioned medium supplemented with 8ng/ml bFGF (R&D Systems; Cat i# 233-FB). Plates were maintained at 37*C, 5% CO 2 in a humidified box throughout the duration of assay. [01 25 Preparation af compounds: Screening was conducted using a single commercial library of small molecule kinase inhibitors (BioMol Inti; Cat # 2832A(V2:2) as defined in Table L Compounds from this library were made available as 10 mM stocks in 96-well plate fonna, solubilized in 100% DMSO and stored at -80*C. The library compounds were further diluted to an intermediate concentration of 2,5mM in 100% DMSO (Sigma; Cat # D2650). also stored at -804T until use. On the day of assay, compounds were diluted 1:12.5 into DMEM high glucose medium to yield a 200uM working stock in 8% DMSO and then further diluted 1:80 into each assay test well for a final concentration of 2.5pM compound and 0.,1 % DMSO, [01261 DIferendation and Screening Assay: Step I of the differentiation protocol was conducted over three days, feeding daily by aspirating the medium from each well and replacing with a fresh aliquot (l00pI), On the first day of assay, wells were fed using RPMI-1640 medium (itvitrogen; Cat #: 22400) containing 2% Albumin Bovine Fraction V, Fatty Acid Fret (FAF BSA) (Proliant Inc,; Cat #: SKU 68700), 1 00ng/ml Activin A (PeproTech; Cat #120~14), 2ing/ml W\nl3a (R&D Systems; Cat # 1324 WNICF), and 8ng/ml bFGF (R&D Systems; Cat # 233-1F) On the second and third day of assay, wells were fed with the same medium except that Wnt3a was removed. All wells were fed and treated identical. [0127] Step 2 of the differentiation protocol was conducted over two days. Cells were fed daily by aspirating the medium from each well and replacing with a fresh aliquot 28 (100pl) of DMEMI12 medium (lIwitrogen; Cat # 11330-032) containing 2% FAF BSA, MStng/ml FGF7 (PeproTech; Cat # 100-19), and 250aM KAAD-cyclopaminc (Calbiochem; Cat # 239804). All wells were fed and treated identically. 101281 Step 3 of the differentiation protocol was conducted over five days, Cells were fed on alternating days by aspirating medium from each well and replacing with a fresh aliquot (200p1) of DMEM-high glucose (Invitrogen; Cat -# 10569) supplemented with 01% Albumax Onvitrogen; Cat #: 11020 021). 0,5x Insulin-Transferrin-Selenium (ITS-X; Invitrogen; Cat f# 51500056), 50 ngi FGF7, 100 ng/ml Nogin (R&D Systems; Cat # 3344-NG) 250 riM KAAD-cyclopamine2 pM all-trans retinoic acid (RA) (Sigma-Aldrich; Cat # R2625), 30ng/ml Activin A, and IOOnM Trichostatin A (TsA; Sigma; Cat # T8552). During step 3, test samples of kinase inhibitors were added to single wells on days 2 and 4 hI each plate, a total of 16 control wells were treated with an equivalent amount of 0,1% DMSO without any test compound. 101291 Step 4 of the differentiation protocol was conducted over three days. Cells were fed daily by aspirating the medium from each well and replacing with a fresh aliquot

(

20 0 pl) of DMEM-high glucose supplemented with 0. 1% Albumax, 0.5x Insulin Transferrin-Selenium, 100 ng/ml Noggin, lPM Alk 5 inhibitor (Axxona; Cat f# ALX 270-445), and lug/mI DAPT (Sigma; Cat #D5942), During step 4, test samples of kinase inhibitors were added to single wells on the first day along with I 00nM Trichostatin A, then both test samples of kinase inhibitors and TsA were omitted during feeding on days 2 and 3. Ir each plate, a total of 16 control wells were treated with an equivalent amount of 0. 1 % DMSO without any test compound. [0130] [igh Contet Analsis: At the conclusion of step 4, medium from all wells was aspirated followed by fixing at room temperature for 20 minutes with 4% paraformaldehyde (Sigma-Aldrich; Cat # 158127) diluted in PBS without divalent nations (Invitrogen; Cat #Y 14190), then washing once with PBS. Sample wells were permeabilized with 0.5% Triton X- 00 (VWR; Cat # VW3929-2) for 20 minutes at room temperature, washed two times with PBS, and blocked with 5% donkey serum (Jackson inmunoResearch; Cat #017-000-121) in PBS for 30 minutes at room temperature. Primary antibodies (sheep anti-NGN3; R&D Systems; AF3444 or mouse anti-NKX6. 1 University of lowa; Cat # F55Al 2) were diluted (1:300 for anti 29 NGN3; 1:500 for anti-NKX6,1) in 5% donkey serum and added to each well for one hour at room temperature. After washing two times in PBS, Alexa Fluor 647 donkey anti-sheep secondary antibody (Invitrogen; Cat # A21448) and Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen; Cat #A21202) were diluted 1:100 (both secondary antibodies) and added to each sample well for 30 minutes at room temperature, followed by two washes in PBS, To counterstain nuclei, 4tginl Hoechst 33342 (Invitrogen; Cat # H3570) was added for ten minutes at room temperature. Plates were washed once with PBS and left in 100plt/well PBS for imaging, 10131] Imaging was performed using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 510081s dicbroic for cells stained with Hoechst 33342 and Alexa Fluor 488 and Alexa Flour 647. Exposure times were optimized from positive control wells stained with each secondary antibody alone, Images from 15 fields per well were acquired to compensate for any cell loss during the bioassay and subsequent staining procedures. Measurements for total cell number aid total NGN3 or NKX6.1 intensity were obtained from each well using IN Cell Developer Toolbox I7 (GE Healthcare) software. Segmentation for the nuclei was determined based on gray-scale levels (baseline range 100-300) and nuclear size. Total NGN3 or NKX6 1 protein expression was reported as total intensity or integrated intensity, dined as total fluorescence of the cell multiplied by the area of the cell. Background was eliminated based on acceptance criteria of gray-scale ranges between 200 to 3500. Total intensity data were nornralized by dividing total intensities for each well by the average total intensity for the positive control. 10132] Results from this screen are summarized in Table 5, Table 6, and Table 7. Data in Table 5 depict a representative ratio of NCN3 and NKX6.1 staining for each well treated with an individual compound relative to average staining in wells with DMSO alone, In addition, the rank order for each compound's effect on protein expression for either NGN3 or NKX6.1 is also shown. Table 6 lists ordered rankings for the top 16 hits having a positive effect on NGN3 and/or NKX6. I expression. Table 7 suimmarizes the targets and signal transduction pathways that correspond to these top hits. Pathways with multiple hits from this screen would appear to have greatest 30 validity for having an impact an expression an these two transcription factors critical for endocrine fate determination. Example 3 Confirmations for Small Molecule Analogues that Mediate NGN3 and NKX6.1 Expression 101331 Expression of a NKX6. 1, along with NGN3 is required during the progression of progenitor celLs towards an endocrine cell fate, A screen of kinase inhibitors was repeated to determine if any small molecule compounds could up-regulate expression of one or both markers during differentiation. In this example, the HDAC inhibitor Trichostatin .A was also included in the differentiation protocol to modulate chromatin remodeling and possibly enhance gene transcription. [01341 Preparation ofcellsfr ascay: Stock cultures of human embryonic stem cells (I human embryonic stem cell line) were maintained in an undifferentiated, pluripotent state on reduced growth factor MATRIGEL (BD Biosciences; Cat # 35623 1)-coated dishes in MEF conditioned medium with passage on average every four days Passage was performed by exposing cell cultures to a solution of I mg/ml dispase (Invitrogen, Cat #: 17105-041) for 5 to 7 minutes at 37*C followed by rinsing the monolayer with MEF conditioned culture medium and gentle scraping to recover cell clustens Clusters were centrifuged at low speed to collect a cell pellet and remove residual dispase, Cell clusters were split at a 1:3 or 1:4 ratio for routine maintenance culture, All Imman embryonic stem cell lines were maintained at passage numbers less than 50 and routinely evaluated for normal karyotype and absence of mycoplasma. For "creens in miniaturized assay format, clusters of HI human embryonic stem cells were harvested from culture with disease treatment as described and plated with even dispersal at a ratio of 1:2 (surface area) on, reduced growth factor MATRIGEL (BD Biosciences; Cat # 356231)-coated 96-well black plates (Packard ViewPlates; PerkinElmer; Cat #6005182) using volumes of 100 L/well. Cells were allowed to attach and then recover log phase growth over a 1 to 3 day time period, feeding daily with MEF conditioned medium supplemented with $ng/ml bFGF (R&D Systems; Cat # 233-FB). Plates were maintained at 37C, 5% CO2 in a humidified box throughout the duration of assay, 31 [01351 Prqparation of/compounfA: Confirmation screening was conducted using a single commercial library of small molecule kinase inhibitors (BioMol Intl Cat # 2832A(V2/2) as defined in Table 1. Compound hits of interest from this library were made available as 10 mM stocks in 96-well plate format, sol abilized in 100% DMSO and stored at -80"C. Individual library compounds of interest were further diluted to an intermediate concentration of 2,5mM in 100% DMSO (Sigrma; Cat # D2650), also stored at -80*C until use. On the day of assay, these individual compounds of interest were dil ted 1:12.5 into DMEM high glucose medium to yield a 200gM working stock in 8% DMSO and then further diluted 1:80 into each assay test well for a final concentration of 2.5pNI compound and 0. 1% DMSO 101361 DI/erentiation and Screening Assay: Step I of the differentiation protocol was conducted over three days, feeding daily by aspirating the medium from each well and replacing with a fresh aliquot (lOpl). On the first day of assay, wells were fed using RPMI- 1640 medium (Invitrogen; Cat #: 22400) containing 2% Albumin Bovine Fraction V, Fatty Acid Free (FAF BSA) (Proliant In, Cat #: SKU 68700), 100ng/mI Activin A (PeproTech; Cat #120-14), 20ng/mnl Wnt3a (R&D Systems; Cat # 1324 WNiCF), and 8ng/ml bFOF (R&D Systems; Cat # 233-FB). On the second and third day of assay, wells were fed with the same medium except that Wnt3a was removed. All wells were fed and treated identically. 101371 Step 2 of the differentiation protocol was conducted over two days, Cells were fed daily by aspirating the medium front each well and replacing with a fresh aliquot (100pl) of DMEM:F 12 medium (Invitrogen; Cat # 11330432) containing 2% FAF BSA, 50ngl/m FGF7 (PeproTech; Cat # 100- 19), and 250aMKAAD-cyclopamine (Calbiochem; Cat # 239804), All wells were ed and treated identically, [01381 Step 3 of the differentiation protocol was conducted over four days. Cells were fed on alternating days by aspirating medium from each well and replacing with a fresh aliquot ( 2 0 0 pL) of DMEM high glucose (Invitrogen; Cat # 10569) supplemented with 0,1% Albumax (Invitrogen; Cat #: 11020-021), 0,5x Insiinl ransferrin-Selenium (ITS-X; Invitrogen; Ca t # .1500056), 50 nig/nil FGF7, 100 ng/ml Noggin (R&D Systems; Cat # 3344-NG)t 250 nM KAAD-cyclopamine, 2 pM all-trans retinoic acid (RA) (Sigma-Aldrich; Cat # R2625), and 20ng/ml Activin A. During step 3, triplicate 32 test samples of kinase inhibitors were added to wells at the time offending on days I and 3, in each plate, a total of 16 control wells were treated with an equivalent amount of 0,1% DMSO without any test compound. [01391 Step 4 of the differentiation protocol was conducted over four days, Cells were fed on alternating days by aspirating the medium from each well and replacing with a fresh aliquot (200pl) of DMEM-high glucose supplemented with 0.1% Albumax, 0.5x Insulin-Transferrin-Seeniumn, 100 ng/mi Noggin, and 1pM Alk 5 inhibitor (Axxora; Cat # ALX-270-445). During step 4, triplicate test samples of kinase inhibitors were added to wells at the time of feeding on days 1, and 3. In each plate, a total of 16 control wells were treated with an equivalent amount of 0, 1% DMSO without any test compound, 101401 High Content Analysis: At the conclusion of step 4, medium from all wells was aspirated followed by fixing at room temperature for 20 minutes witl 4% paraformaldehyde (Sigma-Aldrich; Cat # 158127) diluted in PBS with out divalent cations (Invitrogen C at # 14190), then washing once with PBS. Sample wells were permeabilized with 0.5% Triton X-100 (VWR; Cat # VW3929-2) for 20 minutes at room temperature, washed two times with PBS, and blocked with 5% donkey serum (Jackson ImmunoResearch; Cat # 017-000-121) in PBS for 30 minutes at room temperature. Prinary antibodies (sheep anti-NGN3; R&D Systems; AF3444 or mouse anti-NKX6.1 ; University of Iowa; Cat # F55A1 2 wer diluted (1:300 for anti NGN3; 1:500 for anti-NKX6.1) in 5% donkey serum and added to cach vell for one hour at room temperature. After washing two times in PBS, Alexa Fluor 647 donkey anti-sheep secondary antibody (Invitrogen, Cat # A21.448) and Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen; Cat #A21202) were diluted 1:100 (both secondary antibodies) and added to each sample well for 30 minutes at room temperature, followed by two washes in PBS. To counterstain nuclei, 4ptg/ml Hoechst 33342 (Invitrogen; Cat # 3570) was added for ten minutes at room temperature. Plates were washed once with PBS and left in 100pl/well PBS for imaging. 101411 Imaging was performed using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008S dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488 and Alexa Flour 647. Exposure times were optimized from positive control wells stained 33 I with each secondary antibody alone, Images from 15 fields per well were acquired to compensate for any cell loss during the bioassay and subsequent staining procedures. Measurements for total cell number and total NGN3 or NKX6.1 intensity were obtained from each well using IN Cell Developer Toolbox 1 7 (GE Healthcare) software. Segmentation for the nuclei was determined based on gray-scale levels (baseline range 100-300) and nuclear size. Total NGN3 or NKX6 I protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell multiplied by the area of the cell. Background was eliminated based on acceptance criteria of gray-scale ranges between 200 to 3500. Total intensity data were normalized by dividing total intensities for each well by the average total intensity for the positive control. [0142] Results for these studies are shown in Table 8, Two compounds (Kenpaullon and BML-259) did not confirm and have no enhancing effects on either NGN3 or NKX6.I expression relative to a control treatment. 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nr N Tr0) U') Co C00 CV) CO to 0) N LO NCD 04 00 co co mD CC1 0) Nt N rr N 0 121 cco o CLC . c Co N.N r 04 0 00 r x ( N tCD COC CC 6C £ C 041 Table 5: NKX6.1 Intensity NGN3 Intensity Target Total WELL Activity COMPOUND Nuclei RATIO RANK RATIO RANK PKA, PKG, MLCK, and B-5 PKC. H-9 1,00 7.36 1 2.24 1 D-8 PKC Hypericin 1 07 2.15 18 2.22 2 D ~ 5 P1 3-K LY 294002 1-08 6.84 2 2.18 3 D-11 PKA H-89 1.09 5.39 3 2.13 4 F-4 Srcfamily PP2 1,01 4.07 7 209 5 B - 3 p38 MAPK SB-203580 1.07 505 4 1 95 6 Tyrphostin C-6 EGFRK AG 1478 1.10 241 14 1,89 7 F-8 cRAF GW 5074 1.06 3.48 10 1.78 8 D-6 P1 3-K Wortmannin 1.10 3.87 9 1 67 9 E- 1 PKA, PKG HA-1004 1.08 3.88 8 1 55 10 84 D-7 PKC GF 109203X 1,05 4,54 6 1,53 11 C ~ 12 Srofamiy PPI 099 1 90 22 1.43 12 F - 3 p38 MAPK SB-202190 1.08 1.90 23 1.40 13 G - 11 ROCK Y-27632 1.08 131 40 1,40 14 G - 12 GSK-3beta Kenpaullone 0-99 3.18 11 1.32 15 C-9 IRK HNMPA 1.01 2.31 15 1,32 16 PKA, PKG MLCK, and B-4 PKC. H-7 1,02 1.99 21 1 26 17 EGFRK, B-7 PDGFRK AG-494 1.10 1.57 30 1.26 18 Tyrosine Tyrphostln C-7 kinases AG 1295 1.08 1,43 36 1.26 19 BML-266 (Erlotinib H-7 EGFRK analog) 1.06 2.15 17 1,25 20 B-12 EGFRK Tyrphostin 25 102 4.68 5 1.24 21 E-2 PKA, PKG HA-1077 1.02 1.46 34 1.20 22 85 F-7 PDGFRK AG-1296 1.09 1,74 25 1,18 23 N9-sopropyl E-9 CDK olomoucine 1.05 1,42 37 118 24 B-11 EGFRK Tyrphostin 23 1 0 1.43 35 1.17 25 D- 12 PKA, PKG H-8 0,99 1,21 45 1.16 26 Tyrosine Tyrphostin C-5 kinases AG 1288 1,04 1,63 27 115 27 Negative control for iso E - 11 olomoucine. Olomoucine 1.04 1.51 31 1.14 28 F-6 Flk1 SU 4312 1.07 1,01 55 1,12 29 C-11 Syk Piceatannol 1.07 1 32 39 1.10 30 G-8 JNK SP 600125 1.04 1,67 26 1.09 31 H-11 DMSO 1.03 1.04 53 1.09 32 E-5 CaMK 11 KN-93 1.04 1.10 51 1.03 33 E - 12 CDK Roscovitine 1.03 0.67 75 1.03 34 F-11 Genistein 1.03 1.10 52 1.02 35 Tyrosine 86 Kinases B-8 HERI-2 AG-825 1.05 1.12 50 100 36 B-10 EGFRK RG-14620 1.02 1.26 41 100 37 Palmitoyi-DL F-9 PKC carniine C1 1.05 1.19 47 0.99 38 E - 10 CDK Olomoucine 1,04 122 44 0.99 39 D - 10 PKC Sphingosine 1.04 1.62 28 0.99 40 G-6 CK1 DRB 1.01 1.50 32 0.98 41 C -3 EGFRK Tyrphosfin 51 1,04 1 46 33 0.97 42 F -2 BTK LFM-Al3 0,97 1.82 24 0.96 43 p58 PITSLRE 2 E -8 betal Aminopurine 1.07 0.93 61 0.93 44 Lavendustin B-9 EGFRK A 1,02 0,98 57 0.92 45 PKC alpha, PKC G - 7 gamma HBDDE 1.07 0.92 63 0.91 46 87 D-2 IRAK AG-126 0.99 1,21 46 0,86 47 H - 12 DMSO 1.02 0 75 69 0.86 46 H-6 CK-I Apigenin 1.05 0.77 68 0.84 49 F-5 cRAF ZM 336372 1.06 136 38 0,83 50 Negative control for F - 12 Genistein. Daidzein 1.03 0.92 62 0.83 51 EGFRK, C - 1 PDGFRK Tyrphostin 46 1.05 254 13 0.82 52 H -9 DMSO 1.03 0.95 60 0.81 53 B - 1 MEK PD-98059 0.92 2.02 20 0.81 54 H - 1 BTK Terreic acid 1.02 1.24 42 0.81 55 H - 5 Cdk5/p25 BML-259 1 04 0.83 67 0.80 56 C-2 EGFRK Tyrphostin 47 1.00 1.13 49 0.79 57 Akt signaling H - 2 pathway Triciribine 0.73 2,06 19 0,78 58 88 D-3 PDGFRK AG-370 1.01 1.15 48 0.77 59 Indirubin~3~ G - 10 GSK-3beta monoxide 0.96 0,92 64 0,74 60 Negative control for tyrosine kinase C-4 inhibitors Tyrphostin 1 1.00 0.95 59 0.73 61 G -4 JAK-3 ZM 449829 1.01 1,23 43 0,73 62 D - 1 JAK-2 AG-490 1 04 2.26 16 0.72 63 -- --- -- ~~~-- -- -- -- -- -- -------- --- -- -- -- -- -- --- ----- i--- -- -- D -4 NGFRK AG-879 1 03 0.39 80 0.72 64 Quercetin G - 2 PI 3-K dihydrate 0.97 1.03 54 0.1 65 IKK G-5 pathway BAY 11-7082 0.93 1.00 56 0,68 66 H - 10 DMSO 099 0.88 65 0.67 67 GSK-3beta, G - 9 ODK5 Indirubin 1.01 0.59 77 0 66 68 E-6 MLCK ML-7 0.98 2.84 12 0,65 69 89 B -2 MEK U-0126 0,87 0.68 73 0,65 70 G - 3 FIkI SU1498 1 01 0.70 72 0.65 71 EGFRK, E -3 CaMK I1 HDBA 0.99 0.88 66 0.64 72 H -4 IKK2 SC-514 0.95 0,71 71 0.62 73 E-7 MLCK ML-9 1.06 1,60 29 0.59 74 F - 10 PKC delta Rottlerin 024 0.96 58 0.56 75 E -4 CaMK I KN-62 0,96 0.71 70 0.56 76 H - 3 Akt BML-257 0.98 0.68 74 0.55 77 H -8 mTOR Rapamycin 0.47 0.65 76 0.39 78 PKC C - 10 inhibitor PKC-412 0.17 0,45 79 0.39 79 Erbstatin G - 1 EGFRK analog 0.82 0.38 81 0.34 80 D -9 PKC Ro 31-8220 0.09 048 78 0.30 81 Pan B -6 specific Staurosporine 0.13 0.17 83 0.30 82 90 S- 8 PDGFRK Tyrphosfin 9 0,28 0,29 82 0,29 83 ERK2, adenosisie kinase, 5 F - I CKI, CK2, lodolubercidin 0.08 0.13 840.27 84 91 Table 6: RANK NKX6,1 RANK NGN3 RANK I H-9 H-9 2 LY 294002 Hypericin 3 H-89 LY 294002 4 SB-203580 H-89 5 Tyrphostin 25 PP2 6 GF 109203X SB-203580 7 PP2 Tyrphostin AG 1478 a HA-1004 GW 5074 9 Wortmannin Wortmannin 10 GW 5074 HA-1004 11 Kenpaullone GF 109203X 12 ML-7 PPI 13 Tyrphostin 46 SB-202190 92 14 Tyrphostin AG 1478 Y-27632 15 HNMPA Kenpauflone 16 AG490 HNMPA 93 Table 7: Pathway Compounds PKCIPKAPKG H-9, Hypericin H-89, GF 109203X, HA-1004 SRC kinase PP2, PPI P13 Kinase LY 294002, Wortmannin p38 MAP kinase SB-203580, SB-202190 Tyrphostin 25, Tyrphosdn AG1478, Tyrphostin EGF receptor kinase 46 --- -- -- -- -- -- -- -- -- -- ---- ---- ---- --- ---- ---- ---- ---- ---- ----- -- --- --- -------- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- - --- cRAF GW 5074 GSK3 beta Kenpaullone IRK HNMPA JAK2 AG490 ROCK Y27632 MLCK ML-7 94 Table 8: Total Nuclei NKX6.1 NGN3 Treatment Cell Count Intensity Cell Count Intensity Plate [conc] Ratio Ratio Ratio Ratio Ratio PD-98059 Plate 1 [2,SuM 0.96 2.08 2 37 2.21 2 41 SB-203580 Plate 1 (2uM] 105 2.93 2 58 5.26 4 74 Plate 1 H-7 [2.5uM] 1.09 2.02 188 2.75 244 Plate I H-9 [2.5uM] 1.13 1.93 1 76 2.85 2.47 AG-490 Plate I f2.5uM] 0.99 3.78 4 20 2.48 2 35 LY 294002 Plate 1 [2,SuMj 1.03 654 6.60 4.93 4,43 GF109203X Plate 1 [25uM] 0.82 5.20 3 157 4.17 3 33 H-89 Plate I [2.5uM] 1.08 2.41 2 30 4.00 3 74 Plate 1 1.02 0.69 061 0.81 077 KN-62 95 KN-93 Plate 1 [1uM] 1.05 0.59 0 55 0.84 0 79 Control Plate I Treatment 100 1.00 1 00 1.00 1 00 HA-1004 Plate 2 [2.5uM] 1.08 2.83 2 66 2.77 2 49

HA

Plate 2 1077[2 SuM] 1.07 1,48 1 31 257 2.27 SB-202190 Plate 2 12,5uM] 1.08 2.12 1 90 4,53 3,91 Plate 2 PP2 [2.5uMI 1.04 206 1.71 6.21 6,12 GW 5074 Plate 2 [2.5uM) 111 2.77 2.32 3,15 2,79 Kenpaulone Plate 2 [2,5uM] 1.02 0.53 0.45 1.52 1 40 t -- - - ---------- - ---- - - ---- - -t BML-259 Plate 2 12.SuM] 0.99 1.08 1.02 1.21 1 23 BML-265 Plate 2 [2,5uM] 0,97 6,12 i 6,34 4,50 4,65 Plate 2 1.03 0.71 0.67 0.72 0 71 KN-62 96 KN-93 Plate 2 [1uM] 1.04 0.75 0.76 0.93 0,93 Control Plate 2 Treatment 100 100 1.00 1.00 1 00 101431 Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly constned tinder principles of patent law. 97

Claims

2. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a compound selected from the group consisting of H-9, H-89, GF 109203X, HA-1004, PP2, PP1, SB-203580, SB-202190, Tyrphostin 25, Tyrphostin AG1478, Tyrphostin 46, and GW 5074.
3. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a compound selected from the group consisting of H-9, H-89, GF 109203X, and HA-1004.
4. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with PP2 or PP 1.
5. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with SB-203580 or SB-202190. - 98 -
6. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with Tyrphostin 25, Tyrphostin AG1478, or Tyrphostin 46.
7. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with GW 5074, hydroxy-2-naphthalenylmethylphosphonic acid or AG490.
8. The method of claim 13, wherein the method comprises culturing the cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with Y27632 or ML-7. - 99 -