WO2006138433A2

WO2006138433A2 - Induction of cell differentiation by class i bhlh polypeptides

Info

Publication number: WO2006138433A2
Application number: PCT/US2006/023263
Authority: WO
Inventors: Fred A. Levine; Pamela R. Itkin-Ansari
Original assignee: The Regents Of The University Of California
Priority date: 2005-06-14
Filing date: 2006-06-14
Publication date: 2006-12-28
Also published as: WO2006138433A3

Abstract

The present invention provides methods of inducing differentiation and modulation of the cell cycle of endocrine cells by expressing a recombinant polynucleotide encoding a class I basic helix-loop-helix (HLH) polypeptide in the cells, cells produced by such methods, and methods of treating diabetic subjects using such cells. Also provided by this invention are methods for identifying compounds that modulate differentiation and the cell cycle of endocrine cells by expressing a recombinant polynucleotide encoding a class I basic helix-loop-helix (HLH) polypeptide in the cells.

Description

INDUCTION OF CELL DIFFERENTIATION BY CLASS I BHLH

POLYPEPTIDES

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No.

60/690,607, filed June 14, 2005, the disclosure of which is hereby incorporated by reference herein in its entirety for all purposes.

STATEMENTAS TORIGHTS TOINVENTIONS MADEUNDER FEDERALLY SPONSOREDRESEARCH ORDEVELOPMENT

[0002] This invention was made with Government support under Grant No. 33368A, awarded by the NTH. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION [0003] Transplantation of cells exhibiting glucose-responsive insulin secretion has the potential to cure diabetes. However, this approach is limited by an inadequate supply of cells with that property, which is exhibited only by pancreatic β-cells. The development of expanded populations of human /3-cells that can be used for cell transplantation is therefore a major goal of diabetes research (D. R. W. Group, "Conquering diabetes: a strategic plan for the 21st century," NIH Publication No. 99-4398 (National Institutes of Health, 1999)). A number of alternative approaches are being pursued to achieve that goal, including using porcine tissue as a xenograft (Groth et ah, J. MoI. Med., 77:153-4 (1999)), expansion of primary human β -cells with growth factors and extracellular matrix (Beattie et ah, Diabetes, 48:1013-9 (1999)), and generation of immortalized cell lines that exhibit glucose-responsive insulin secretion (Levine, Diabetes/Metabolism Reviews, 1:209-46 (1997)).

[0004] Although there has been great interest in using porcine islets, they are difficult to manipulate in vitro and concerns have been raised about endogenous and exogenous xenobiotic viruses being transmitted to graft recipients (Weiss, Nature, 391:327-8 (1998)). With primary human /3-cells, entry into the cell cycle can be achieved using hepatocyte growth factor/scatter factor ("HGF/SF") plus extracellular matrix ("ECM") (Beattie et ah, Diabetes, 48:1013-9 (1999), Hayek et ah, Diabetes, 44:1458-1460 (1995)). However, this combination, while resulting in a 2-3xl0⁴-fold expansion in the number of cells, is limited by cellular senescence and loss of differentiated function, particularly pancreatic hormone expression (Beattie et al, Diabetes, 48:1013-9 (1999)).

[0005] Immortalized cell lines from the human endocrine pancreas have been created to develop j3-cell lines that exhibit glucose responsive insulin secretion (Wang et al, Cell

Transplantation, 6:59-67 (1997), Wang et al, Transplantation Proceedings, 29:2219 (1997), Halvorsen et al, Molecular and Cellular Biology, 19:1864-1870 (1999)). The cell lines are made by infecting primary cultures of cells from various sources including adult islets, fetal islets, and purified /3-cells, with viral vectors expressing the potent dominant oncogenes such as SV40 T antigen and H-ras^vaU2(Wang et al, Cell Transplantation, 6:59-67 (1997), Wang et al, Transplantation Proceedings, 29:2219 (1997), Halvorsen et al, Molecular and Cellular Biology, 19:1864-1870 (1999); see also U.S. Patent No. 5,723,333). The combined effect of those oncogenes is to trigger growth factor-independent and extracellular matrix (ECM)- independent entry into the cell cycle, as well as to prolong the lifespan of the cells from 10-15 population doublings or primary cells to approximately 150 doubling for the oncogene- expressing cells (Halvorsen et al, Molecular and Cellular Biology, 19:1864- 1870 (1999)). Further introduction of the gene encoding the hTRT component of telomerase results in immortalization, allowing the cells to be grown indefinitely (Halvorsen et al, Molecular and Cellular Biology, 19:1864-1870 (1999)). Although the cell lines grow indefinitely, they lose differentiated function, similar to growth-stimulated primary β-cells.

[0006] Another approach would be to use normal, non-transformed human jS-cells. However, this approach has been hampered by the low cell division rate observed in human /3-cells, even as compared with rodent β-cells. The studies of Tyrberg et al (Tyrberg, et al, Diabetes, 50:301-7 (2001)) demonstrated that cell division in human /3-cells was almost two orders of magnitude lower than in mouse /3-cells. Thus, the generation of large numbers of human /3-cells for treatments based on transplantation has proven to be difficult. Efforts by investigators to stimulate proliferation of human /3-cells in vitro has generally resulted in the rapid shut off of insulin gene expression. The down regulation of insulin expression in the resulting cells has defeated the purpose behind the generation of large numbers of cells. Thus, the apparent inverse relationship between growth and maintenance of a differentiated state must be overcome.

[0007] Ultimately, any therapy for diabetes will require an understanding of the relationship between cell growth and differentiation. Cell growth will be required for diabetes treatments based on transplantation of (3-cells, as well, as for treatments based on regeneration of cells within a diabetic pancreas. In both instances, a population of cells expanded after an appropriate degree of division, will then be required to display a differentiated phenotype of /3-cells, notably insulin secretion. [0008] Methods of stimulating both cell division and differentiation of cells (e.g. , into insulin-secreting /3-cells) are therefore desired. Such cells could then be transplanted in vivo as a treatment for diabetes. Alternatively, methods for stimulating cell growth and differentiation in situ in the pancreas would be desirable.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides methods for inducing differentiation in cultured cells by expressing class I basic helix-loop-helix (bHLH) proteins in the cells and cells produced by the methods described herein, hi some embodiments, the cultured cells are induced to differentiate into /3-cells, including insulin-producing /3-cells. The ability to grow unlimited quantities of functional human β-cells in vitro provides the means for a definitive cell transplantation therapy for treatment of diabetes.

[0010] In yet other embodiments, the invention provides methods for screening for compounds that are able to stimulate cell division and/or differentiation of human jS-cells. Such compounds can be applied to cells in vitro for transplantation therapy or can be administered to diabetic patients to promote the division and differentiation of b-cells in situ. [0011] Therefore, one embodiment of the present invention provides a method for inducing differentiation of cultured cells (e.g., endocrine cells or stem cells). A recombinant polypeptide encoding a class I bHLH polypeptide is expressed in the endocrine cells, thereby inducing differentiation of the cells. In some embodiments, the cells further express a recombinant polynucleotide encoding NeuroD/BETA2 and/or a recombinant polypeptide encoding PDX-I. The differentiated cells express glucokinase (GK), insulin, granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3, and/or ^lF^² at a level that is at least about 1 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide. In some embodiments, the cells express GK, SUR-I, MafA, insulin, chromogranin C, Synaptotamin like protein 3, and/or pSlF^² at a level that is at least about 2 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide. In some embodiments, the cells express chromogranin C and/or ^li^² at a level that is at least about 10 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide. In some embodiments, the cells express p57/^ffip2 at a level that is at least about 1000 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide. In some embodiments, the cells also express c-myc at a level that is at least about 1 fold lower than in the absence of the polynucleotide encoding a bHLH polypeptide. In some embodiments, the bHLH polypeptide is selected from E2A (e.g. , E47, E12 and/or E2-5), HEB/BETA1 , and E2- 2. In some embodiments, recombinant polynucleotide encoding the bHLH polypeptide is operably linked to a polynucleotide encoding an estrogen receptor. In some embodiments, the methods further comprise contacting the cells with an estrogen receptor antagonist (e.g., tamoxifen and/or keoxifene), thereby inducing differentiation of the cells. In some embodiments, the GLP-I receptor agonist is a GLP-I analog or has an amino acid sequence of a naturally occurring peptide. In some embodiments, the GLP-I receptor agonist is GLP- 1, exendin-3, or exendin-4.

[0012] In some embodiments, the endocrine cell is a pancreatic cell (e.g. , a j3-cell or a δ- cell). hi some embodiments, the pancreatic cell is a human cell. In some embodiments the δ- cell is a TRM-β cell.

[0013] In some embodiments, the cells express a recombinant oncogene, hi some embodiments, the cells express a recombinant telomerase gene.

[0014] In some embodiments, the cells are cultured as aggregates in suspension. In some embodiments, the cells are cultured under conditions such that the cells are in contact with other cells in the culture.

[0015] hi another aspect, the present invention provides a method of identifying a compound that modulates endocrine cell function, the method comprising the steps of contacting cells made by the method described above with the compound and determining the effect of the compound on endocrine cell function. [0016] hi another aspect, the present invention provides a method of treating a diabetic subject by providing to the subject an effective amount of cells that secrete insulin produced by the methods described above. In a further aspect, the present invention provides a method of treating a diabetic subject by providing to the subject an effective amount of cells that secrete insulin hi response to an estrogen receptor antagonist produced by the methods described above, hi various aspects, the subject has Type I insulin dependent diabetes or Type II insulin independent diabetes.

[0017] hi another aspect, the present invention provides a stable culture of endocrine cells produced by expressing a first recombinant polynucleotide encoding a class I basic helix- loop-helix (bHLH) polypeptide, thereby inducing differentiation of the cells, where the differentiated cells express glucokinase (GK), insulin, granuphilin, chromagranin A, chromogranin C, and Synaptotamin like protein 3, at a level that is at least about 1 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide. In some aspects, the bHLH polypeptide is E47. In yet other aspects, the polynucleotide encoding E47 is operably linked to a polynucleotide encoding an estrogen receptor. In some aspects, the endocrine cells are /3-cells. In further aspects, the /3-cells are human /3-cells. In various additional aspects, the endocrine cells are cultured under conditions such that the cells are in contact with other cells in the culture or as aggregates in suspension. [0018] In further embodiments, the present invention provides a method for identifying a compound that modulates endocrine cell function, the method by expressing a first recombinant polynucleotide encoding a class I bHLH polypeptide and a second recombinant polynucleotide encoding a reporter gene operably linked to a promoter responsive to the class I bHLH polypeptide in a cell, and contacting the cell with a compound and determining the effect of the compound on expression of the reporter gene. In some embodiments, the bHLH polypeptide is selected from E2A (e.g., E47, E12 and/or E2-5), HEB/BETA1, and E2-5. In some embodiments, recombinant polynucleotide encoding the bHLH polypeptide is operably linked to a polynucleotide encoding an estrogen receptor. In some embodiments, the methods further comprise contacting the cells with an estrogen receptor antagonist (e.g., tamoxifen and/or keoxifene). In additional embodiments, the promoter responsive to the class I bHLH polypeptide is the insulin promoter or p57/Kip2 promoter. In further embodiments, the reporter is GFP. hi one aspect, the endocrine cell is a /3-cell. hi a further aspect, the /3-cell can be a human /3-cell. hi further embodiments, the compound modulates the cell cycle of the cell by either inducing cell cycle progression or cell cycle arrest. [0019] In further embodiments, the present invention provides a method for identifying a compound that modulates the cell cycle in a cell by expressing a first recombinant polynucleotide encoding a class I bHLH polypeptide and a second recombinant polynucleotide encoding a reporter gene operably linked to a promoter responsive to the class I bHLH polypeptide in a cell, and contacting the cell with a compound and determining the effect of the compound on expression of the reporter gene, where expression of the reporter gene correlates with cell cycle progression or arrest. In some embodiments, the bHLH polypeptide is selected from E2A (e.g., E47, E12 and/or E2-5), HEB/BETA1, and E2-5. hi some embodiments, recombinant polynucleotide encoding the bHLH polypeptide is operably linked to a polynucleotide encoding an estrogen receptor. In some embodiments, the methods further comprise contacting the cells with an estrogen receptor antagonist (e.g., tamoxifen and/or keoxifene). In additional embodiments, the promoter responsive to the class I bHLH polypeptide is the insulin promoter or p57/Kiρ2 promoter. In further embodiments, the reporter is GFP. In one aspect, the endocrine cell is a /3-cell. In a further aspect, the /3-cell can be a human /3-cell. In further embodiments, the compound modulates the cell cycle of the cell by either inducing cell cycle progression or cell cycle arrest.

[0020] In yet additional embodiments, method for modulating the cell cycle in a cell by expressing a recombinant polynucleotide encoding a class I bHLH polypeptide in a cell, where expression of the class I bHLH polypeptide modulates cell cycle progression. In some embodiments, the bHLH polypeptide is selected from E2A (e.g., E47, E12 and/or E2-5), HEB/BETA1, and E2-5. hi some embodiments, recombinant polynucleotide encoding the bHLH polypeptide is operably linked to a polynucleotide encoding an estrogen receptor. In some embodiments, the methods further comprise contacting the cells with an estrogen receptor antagonist (e.g., tamoxifen and/or keoxifene). Li one aspect, the endocrine cell is a /3-cell. Li a further aspect, the /3-cell can be a human /3-cell. In further embodiments, the modulation of the cell cycle of the cell is by inducing cell cycle progression or cell cycle arrest.

[0021] In yet further embodiments, the present invention provides a method of treating a subject with a disorder characterized by an aberrant cell cycle by expressing in the cells of the subject a first recombinant polynucleotide encoding a class I basic helix-loop-helix (bHLH) polypeptide, wherein expression of the bHLH modulates the cell cycle in the cells. Li some embodiments, the bHLH polypeptide is selected from E2A (e.g., E47, E12 and/or E2-5), HEB/BETA1, and E2-5. Li some embodiments, recombinant polynucleotide encoding the bHLH polypeptide is operably linked to a polynucleotide encoding an estrogen receptor. Li some embodiments, the methods further comprise contacting the cells with an estrogen receptor antagonist (e.g., tamoxifen and/or keoxifene). In one aspect, the endocrine cell is a /3-cell. Li a further aspect, the /3-cell can be a human /3-cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 illustrates a model for the E-box as a central regulator of /3-cell growth and differentiation. Positive (e.g., E47 and NeuroDl) and negative (e.g., c-myc and Hesl) factors act on E-box elements within the promoters of genes that play important roles in the control of /3-cell growth (e.g., p57^Kip2 and p21^cipl) and differentiation (e.g., insulin, GK, and MafA). The balance between the positive and negative factors that act on the E-box is controlled by signaling pathways such as Notch and cadherins. [0023] Figure 2 illustrates CDKI expression in human islets and non-endrocrine cells. Hand-picked human islets and non-endocrine cell clusters we isolated as described (1). Labeled cRNA was used to probe Illumina BeadArray DNA microarrays and expression levels of the CDKIs was extracted. Only p21^Cipl and ffl^² in islets and ρ21^cipl in non- endocrine cells were expressed at a statistically significant level (p<0.05) (indicated by *). [0024] Figure 3 illustrates a construct used to express an E47 estrogen receptor fusion protein. The E47^MER fusion protein gene is inserted into a retroviral vector that also expresses the cell surface marker CD25, allowing for selection of infected cells. [0025] Figure 4 illustrates E47 induction of insulin gene expression in T6PNE cells. (A) Insulin mRNA is barely detectable by non-quantitative RT-PCR in the absence of tamoxifen in two biological replicates of T6PNE cells. With tamoxifen, insulin rnRNA is strongly induced. (B) A different set of two biological replicates of T6PNE cells were analyzed by quantitative real-time RT-PCR, again demonstrating a dramatic upregulation of insulin mRNA when E47 activity is induced by tamoxifen. The myosin heavy chain gene, which is cardiac specific and highly induced by E-boxes in its promoter, was not induced by tamoxifen (not shown).

[0026] Figure 5 illustrates that activation of E47 by tamoxifen results in upregulation of SUR-I, glucokinase, and MafA. RT-PCR was performed on three biological replicates of T6PNE cells with and without tamoxifen. In all three, GK, SUR-I, and MafA were highly induced when E47 was induced by tamoxifen.

[0027] Figure 6 illustrates growth arrest in T6PNE cells when E47 is activated. Cells cultured in the absence or presence of tamoxifen to activate E47 were counted on the indicated days. [0028] Figure 7 illustrates the induction of p57^Hp2 (CDKNlC) and p21^Cipl (CDKNlA) by E47 in T6PNE cells. Microarray analysis was used to determine genes in T6PNE cells that are downstream of E47. T6PN/E47-MER cells were treated with tamoxifen. RNA was isolated and assayed for the expression of 24,000 human genes using the Illumina BeadArray microarray technology. Each dot on the scatter plot (power function) represents a single gene. The values on the X and Y axes represent the level of hybridization to the oligonucleotide on the array, which is an indirect measure of the level of mRNA in the sample. p57^Kip2 is undetectable in the absence of tamoxifen and is expressed at an approximately 2,000 fold higher level in cells in which E47 is induced by tamoxifen. p21^Qpl was induced 2-fold. [0029] Figure 8 illustrates activation of the ρ57^Kp2 promoter by E47. A 6.3kb fragment from the 5' region of the p57^Kip2 gene was cloned upstream of a luciferase reporter gene. Consistent with the presence of an element that responds to E47, this promoter-reporter construct is activated in a dose responsive manner by tamoxifen in T6PNE cells. A p57^Kp2 minimal promoter did not respond to tamoxifen.

[0030] Figure 9 illustrates characterization of the p57^Hp2 promoter. Transient transfection reveals that the E47 response element lies between Sea and Sac sites in the 5' promoter region (A). Seven E-boxes were found in that region (B). ChIP analysis has found that E- box2 binding to E47MER is tamoxifen-responsive (C).

[0031] Figure 10 illustrates in vitro mutagenesis of E-boxes in the p57^Kip2 promoter. E- boxes El, E2, and E3 (Figure 9B) were mutated to eliminate bHLH binding activity. Transient transfections of the wild-type Sac4.0 promoter fragment and the mutated Sac4.0 fragments in the presence and absence of tamoxifen revealed that mutating E2 (mE2) caused loss of responsiveness to activation of E47 by tamoxifen. mEl and mE3 retained E47 responsiveness. [0032] Figure 11 illustrates that induction of p57^Kip2 expression by E47 is not dependent on protein synthesis. T6PNE cells were treated with cycloheximide to inhibit protein synthesis and tested for their ability to increase pSδ^² mRNA levels in response to E47 activation by tamoxifen. p57^Kip2 mRNA levels were measured by quantitative RT-PCR. Cycloheximide had no effect on the induction of p57^Kip2. However, induction of GFP protein from an insulin promoter-GFP transgene was completely inhibited by cycloheximide. [0033] Figure 12 illustrates a lentiviral vector that expresses eGFP under the control of the human insulin promoter.

[0034] Figure 13 illustrates the results from a secondary screen by RT-PCR for insulin and GFP mRNA. 3 compounds that repressed that repressed (1, 2, 3) and 3 that increased (7, 10, 11) GFP fluorescence were tested. Cells were exposed to 5 μM compound and 0.5 μM tamoxifen. Controls had 0.5 μM tamoxifen. DMSO control had no tamoxifen. The 4 μM tamoxifen control represents the maximum induction of insulin mRNA by tamoxifen. GFP and insulin mRNA levels are normalized for GAPDH mRNA to rule out nonspecific effects.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0035] The present invention provides methods of inducing differentiation of cultured cells by expressing class I bHLH polypeptides in the cells. Human endocrine cells were transfected with a nucleic acid encoding a bHLH protein under inducible control. Expression of the bHLH protein in the endocrine cells induced differentiation of the cells into insulin producing /3-cells.

[0036] The differentiated endocrine cells exhibit characteristics of /3-cells, including, e.g., insulin production. The differentiated endocrine cells express insulin, glucokinase (GK), granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3, and/or ρ57/^Kip2at a level that is at least about 1 fold higher than an undifferentiated endocrine cell; GK, SUR-I, MafA, insulin, chromogranin C, Synaptotamin like protein 3, and/or p57/^Hp2 at a level that is at least about 2 fold higher than an undifferentiated endocrine cell; chromogranin C and/or p57/^κip2 at a level that is at least about 10 fold higher than an undifferentiated endocrine cell; p₅₇/^Ki _P ² _{at a} j_evei tø_at -_{s at} 2_{east a}b_out 1000 fold higher than an undifferentiated endocrine cell; and/or c-myc at a level that is at least about 1 fold lower than an undifferentiated endocrine cell. The differentiated endocrine cells are also highly stable cell cultures that can replicate through many generations. Thus, the invention provides methods of creating cells that produce a high level of insulin over many generations.

[0037] One striking effect of expressing the E47 bHLH in endocrine cells was cell cycle arrest. The cell cycle arrest is mediated, at least in part, by the induction of cyclin dependent kinase inhibitor (CDKI) expression upon activation of E47. The CDKI mostly highly upregulated by E47 is p57^Kip2. Further investigation revealed that the regulation of p57^Kip2 by E47 is effected by the direct interaction of E47 with the -pSl^² promoter. Additional studies demonstrated that E47 also activated the insulin promoter. Thus, another aspect of this invention provides cells that express a bHLH protein as well a reporter gene under the control of a promoter responsive to the bHLH. Cells in which the reporter gene is easily detectable and measured can be used for high-throughput screening to identify compounds that modulate the cell cycle and cell differentiation of /3-cells, as well as, other cell types. Such agents can be used to control the proliferation and differentiation of human /3-cells for therapy of diabetes. Other agents, for instance, those that arrest the cell cycle in other cell types, may be useful for the treatment of diseases of aberrant cell growth, such as cancer.

[0038] The availability of an unlimited source of functional human β-cells has important implications for diabetes treatment. One straightforward application is in exploring aspects of β-cell biology that would benefit from an unlimited, homogeneous source of cells. Additionally, the present invention provides a means to expand and differentiate populations of human b-cells for transplantation into diabetic patients. Cells expressing a recombinant polynucleotide encoding a class I bHLH protein is transplanted into a suitable mammalian host, preferably a human.

[0039] High-throughput screening for new diabetes drugs is another application of this invention. The cells of the invention can be used, e.g., to screen for small molecules or other compounds that can induce endocrine cell division and differentiation. Such compounds can be applied to b-cells (or their precursors) in vitro to stimulate proliferation and differentiation prior to transplantation into a diabetic patient. Alternatively, such compounds can be administered to patients directly to stimulate proliferation of jS-cells in situ. The compounds of the invention, by modulating cell cycle progression will find application as well to the treatment of disorders of aberrant cell cycle such as cancer.

II. Definitions

[0040] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0041] "Class I basic helix-loop-helix protein" or "class I bHLH protein" refers to a protein or polypeptide that is a member of a group of widely expressed eukaryotic transcription factors characterized by a basic region adjacent to a HLH motif and which bind to DNA as homodimers, heterodimers, or both {see, e.g., Roberts et al, PNAS USA 90:7583-7587 (1993) and Massari et al, MoI Cell Biol. 18(6):3130-3139 (1998). The HLH motif binds to the E- box, a DNA sequence with the following consensus sequence: 5'-CANNTG-3', in which N is any nucleotide (see, e.g., Itkin-Ansari et al., Endocrinology, 137(8):3540-3543 (1996);

Massari et al., Molecular Cell, 4:63-73 (1999) and Robinson and Lopes, Nucleic Acids Res., 28(7): 1499-1505 (2000)). Class I bHLH proteins are also known as E proteins and include, e.g., E2A (e.g., E 12, E47, and E2-5) as described in Itkin-Ansari et al, Endocrinology, 137(8):3540-3543 (1996) and Roberts et al, PNAS USA, 90:7583-7587 (1993). E47, E12 and E2-5 are each encoded by the E2A gene and are produced by differential exon splicing (see, Itkin-Ansari et al, Endocrinology, 137(8):3540-3543 (1996) and Roberts et al, PNAS USA, 90:7583-7587 (1993)). E2A nucleotide sequences are set forth in Genbank Accession Nos. NM_003200; BCOl 1665; BC005166; BC014680; and M65214. Additional class I bHLH proteins include, e.g.; HEB/BETA1 and E2-2 (see, e.g., Murre et al, Biochim Biophys. Acta, 1218: 129-135 (1994); Hu et al, MoI Cell Biol, 12(3):1031-42 (1992)). HEB/BETAl sequences are set forth in Genbank Accession Nos. BC050556; NM_207038; NM_207037; NMJ207036; NM_003205; NM_207040; and M80627. E2-2 sequences are set forth in Genbank Accession Nos. NM_003199 and BC031056.

[0042] "Stem cells" are undifferentiated cells that have the potential to become a wide variety of specialized cell types including, e.g., endocrine cells and /3-cells). Stem cells include, e.g., embryonic stem cells, adult stem cells, and pancreas-derived multipotent precursor cells as described in, e.g., Seaberg et ah, Nat. Biotechnoh, 22(9): 1115-24 (2004). [0043] "Endocrine cell" refers to a cell originally derived from an adult or fetal endocrine gland {e.g., pancreas and islets of langerhans). "Endocrine pancreas cell" refers to a cell originally derived from an adult or fetal pancreas, preferably islet cells. "Cultured" endocrine pancreas cells refers to primary cultures as well as cells that have been transformed with recombinant polynucleotides encoding class I bHLH polypeptides {e.g., E2A, HEB/BETA1, and/or E2-2). Cultured endocrine pancreas cells also refer to cells that have been transformed with oncogenes, e.g., SV40 T antigen, ras, or a telomerase gene {e.g., hTRT). [0044] "Inducing endocrine cell differentiation" refers to inducing differentiation of an endocrine cell such that the cell expresses genes and cell surface proteins typically expressed by insulin producing /3-cells. For example, a "differentiated" endocrine cell typically expresses insulin, glucokinase (GK), granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3, and/or p57/Kip2 at a level that is at least about 1 fold higher than an undifferentiated endocrine cell; GK, SUR-I, MafA, insulin, chromogranin C, Synaptotamin like protein 3, and/or p57/Kip2 at a level that is at least about 2 fold higher than an undifferentiated endocrine cell; chromogranin C and/or p57/Kip2 at a level that is at least about 10 fold higher than an undifferentiated endocrine cell; ρ57/Kiρ2 at a level that is at least about 1000 fold higher than an undifferentiated endocrine cell; and/or c-myc at a level that is at least about 1 fold lower than an undifferentiated endocrine cell. Differentiated endocrine cell gene expression can be measured by methods known to those of skill in the art, e.g., by measuring RNA expression, polypeptide production, or cell surface protein expression, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, and immunoassays. [0045] Cells that "secrete insulin in response to glucose" are cells or a cell culture that, in comparison to a negative control (either non-insulin responsive cells or insulin responsive cells that are not exposed to glucose), have increased insulin secretion in response to glucose of at least about 10%, preferably 25%, 50%, 100%, 500%, 1000%, 5000%, or higher than the control cells (measured as described above). [0046] "Culturing cells" so that the cells are "in contact with other cells in the culture" refers to culture conditions that allow cell to cell contact. Under such conditions, many but not all, cells are in contact with one or more other cells of the culture. Such conditions include culturing the cells on a solid surface, such as a plate or a bead, or culturing the cells in suspension such that the cell-to-cell contact is greater than in cells grown in monolayer culture. Examples of such conditions include growth of cells in three-dimensional aggregates.

[0047] "Estrogen receptor" or "ER" as used herein refers to a ligand activated transcription factor that is classified as a Class I nuclear receptor that interacts with estrogen and estrogen like molecules {e.g. tamoxifen and keioxifene) to form a DNA binding complex to regulate expression of downstream genes. Estrogen receptors include ER-a and ER-b. ER-a (i.e., ESRl) is mapped to the long arm of chromosome 6 and comprises 595 amino acids with a N- terminal modulating domain, central DNA-binding domain (DBD) and a C-terminal ligand- binding domain (LBD). ER-b (i.e., ESR2) is mapped to band q22-24 of chromosome 14 and comprises 530 amino acids. ER-b contains all the same functional domain except that ER-b lacks a portion of the C-terminal domain (see, e.g., Klinge, Steroids, 65(5):227-51 (2000); Enmark and Gustafsson, J. Intern. Med., 246(2):133-8 (1999); and Osborne et al, J. Clin. Oncol, 2000 Sep;18(17):3172-86 (2000). ER-a sequences are set forth in Genbank Accession Nos. NM_000125; AY891064; AY888432; and AY893286. ER-b sequences are set forth in Genbank Accession Nos. NM_001437; AY785359; and BC024181.

[0048] A "GLP-I receptor agonist" refers to GLP-I, a GLP-I analog, or a naturally occurring peptide that binds to the GLP-I receptor (e.g., exendin -3 or -4), thereby activating signal transduction from the receptor.

[0049] "Culturing" refers to growing cells ex vivo or in vitro. Cultured cells can be non- naturally occurring cells, e.g., cells that have been transduced with an exogenous gene such as an oncogene or a transcription factor such as NeuroD/BETA2 and/or PDX-I . Cultured cells can also be naturally occurring isolates or primary cultures.

[0050] "Pancreatic hormones" refer to hormones synthesized by the pancreas and include, e.g., insulin and glucagon. [0051] A "stable" cell line or culture is one that can grow in vitro for an extended period of time, such as for at least about 50 cell divisions, or for about 6 months, more preferably for at least about 150 cell divisions, or at least about ten months, and more preferably at least about a year. [0052] "Modulating β-cell function" refers to a compound that increases (activates) or decreases (inhibits) glucose responsive insulin secretion of an endocrine pancreas cell. Glucose responsive insulin secretion can be measured by a number of methods, including analysis of insulin mRNA expression, preproinsulin production, proinsulin production, insulin production, and c-peptide production, using standard methods known to of skill in the art. To examine the extent of modulation, cells are treated with a potential activator or inhibitor and are compared to control samples without the activator or inhibitor. Control samples (untreated with inhibitors or activators) are assigned a relative insulin value of 100%. Inhibition is achieved when the insulin value relative to the control is about 90%, preferably 75%, 50%, and more preferably 25-0%. Activation is achieved when the insulin value relative to the control is 110%, more preferably 125%, 150%, and most preferably at least 200-500% higher or 1000% or higher.

[0053] "Modulating cell cycle progression" refers to a compound that either increases the fraction of cells undergoing cell cycle progression or which increases the fraction of cells to arrest. "Modulating cell cycle progression" can also refer to a compound that alters the transition time of cells through the cell cycle, e.g., by altering the length of the phases of the cell cycle, such as Go, Gl, S, or M phase. A number of assays can be used for the measurement of cell cycle progression and cell proliferation. Cell proliferation may be assayed, among other means, via bromodeoxyuridine (BrdU) or ³H-thymidine incorporation. Progression of cells through the various phases of the cell cycle may be determined by flow cytometry using labels such as propidium iodide, Hoechst 33342, and DAPI, as well as, other reagents known to those of skill in the art.

[0054] "Transduction" refers to any method of delivering an exogenous nucleic acid, e.g., an expression vector, to a cell, including transfection, lipofection, electroporation, viral transduction, microinjection, particle bombardment, receptor mediated endocytosis, and the like.

[0055] A diabetic subject is a mammalian subject, often a human subject, that has any type of diabetes, including primary and secondary diabetes, type 1 non-insulin dependent diabetes mellitus (NIDDM)-transient, type 1 insulin-dependent diabetes mellitus (IDDM), type 2 IDDM-transient, type 2 NIDDM, and type 2 maturity onset diabetes of the young (MODY), as described in Harrison 's Internal Medicine, 14th ed., 1998.

[0056] "A disorder characterized by an aberrant cell cycle" is a disorder characterized by cell growth that is not appropriate for proper cell, organ, or organism functioning. Examples of such disorders include cancer, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders. Cancers affecting most major organs and blood cells are well known. (For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia.) An example of a hypoproliferative disorders is hypoproliferative anemia. [0057] A cancer subject is a mammalian subject, often a human subject, that has any type of cancer. Cancers can occur in many organs and cells of the body and include, but are not limited to, cancer of the head, neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovary, testicle, kidney, liver, pancreas, brain, intestine, heart or adrenals, and blood and immune cells. (For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). [0058] "Expressing" a gene or a nucleic acid refers to expression of a recombinant or endogenous gene or nucleic acid, e.g., resulting in mRNA or protein production from the gene or nucleic acid. A recombinant gene or nucleic acid can be integrated into the genome or in an extrachromosomal element. [0059] The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen, e.g., ELISA, Western blotting, RIA, immunoprecipitation, fluorescence activated cell sorting, and the like. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen (e.g., insulin), hi some embodiments, immunoassays are used to quantify the amount of insulin produced by the differentiated endocrine cells of the invention. In some embodiments, immunoassays are used to assess the markers expressed by the differentiated endocrine cells of the invention, hi other embodiments, any marker of interest, such as markers for cell growth or tumor antigens may be subjected to immunoassay. [0060] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0061] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res., 19:5081 (1991); Ohtsuka et ah, J. Biol. Chein., 260:2605-2608 (1985); Rossolini et al., MoI. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0062] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0063] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, 7- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0064] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0065] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. [0066] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0067] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Argmine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) {see, e.g., Creighton, Proteins (1984)).

[0068] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, an estrogen receptor, or array of transcription factor binding sites) and a second nucleic acid sequence (e.g., a class I bHLH sequence), wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. [0069] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantry produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). [0070] An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter or another expression control sequence such as an estrogen receptor sequence.

III. Cells of the Invention

[0071] The present invention provides methods of inducing differentiation of cells (e.g. , embryonic stem cells, adult stem cells, pancreas-derived multipotent cells, and endocrine cells) by expressing nucleic acids encoding class I bHLH polypeptides in the cells. The cells of the invention may be primary cells or may be cells maintained in culture. Techniques and methods for establishing a primary culture of cells for use in the methods of the invention are known to those of skill in the art. See e.g., Humason, ANIMAL TISSUE TECHNIQUES, 4^m ed., W. H. Freeman and Company (1979), and Ricciardelli et al, (1989) In Vitro CellDev. Biol, 25:1016. Suitable cells include, for example, endocrine cells and stem cells (e.g., embryonic stem cells, adult stem cells, and pancreas-derived multipotent precursor cells). Suitable endocrine cells include, for example, pancreatic cells, islet cells (e.g., /3-cells and δ-cells). Islet cells may be derived from, for example, adult pancreatic tissue, fetal pancreatic tissue and islet-like cell clusters (ICCs) that contain a heterogenous population of cells. [0072] The cells may be derived from any suitable mammal or embryo thereof. For example the cells may be obtained from a rodents such as, for example, mice, rats, guinea pigs, and rabbits; non-rodent mammals such as, for example, dogs, cats, pigs, sheep, horses, cows, and goats; primates such as, for example, chimpanzees and humans. [0073] Expression of a class I bHLH polypeptide in a cell (e.g. , an endocrine cell) leads to differentiation of the cell into, e.g., an insulin-producing 0-cell. The differentiated cells of the invention typically express insulin, glucokinase (GK), granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3, and/or p57/Kip2at a level that is at least about

1 fold higher than an undifferentiated endocrine cell; GK, SUR-I, MafA, insulin, chromogranin C, Synaptotamin like protein 3, and/or p57/Kip2 at a level that is at least about

2 fold higher than an undifferentiated endocrine cell; chromogranin C and/or p57/Kip2 at a level that is at least about 10 fold higher than an undifferentiated endocrine cell; p57/Kip2 at a level that is at least about 1000 fold higher than an undifferentiated endocrine cell; and/or c- myc at a level that is at least about 1 fold lower than an undifferentiated endocrine cell. In some embodiments, the differentiated endocrine cells divide at least twice and preferably at least about 5 or at least about 10 times while producing insulin or retaining the ability to produce insulin. [0074] Suitable class I bHLH polypeptides include, e.g. , E2A, HEB/BETA1 , and E2-2. In a preferred embodiment, nucleic acids encoding E47 are expressed in endocrine cells. [0075] In some embodiments, the nucleic acid encoding E47 is operably linked to a nucleic acid encoding an estrogen receptor and the endocrine cells are contacted with an estrogen receptor ligand (i.e., tamoxifen, keioxifene, or estrogen) to induce their differentiation into insulin-producing /3-cells.

[0076] In some embodiments, the cells of the invention also express a construct comprising either the p57Kip57 promoter or the insulin promoter directing the expression of a reporter gene.

[0077] hi some embodiments, the differentiated cells of the invention also express either endogenous or recombinant NeuroD/BETA2 having NeuroD/BETA2 activity , e.g. , alleles, polymorphic variants, and orthologs (see, e.g., U.S. Patent No. 5,795,723; Miyachi, T., et al. MoI Brain Res., 69, 223-231 (1999); Lee, et al. Science, 268:836-844 (1995); Wilson et al, Nature, 368, 32-38 (1994); Naya et al, Genes Dev., 9:1009-1019 (1995)). Human NeuroD/BETA2 alleles and variants are particularly desirable. Recombinant PDX-I is delivered to the cells using expression vectors, e.g., viral vectors such as retroviral vectors, as described above.

[0078] Li some embodiments, the differentiated cells of the invention also express either endogenous or recombinant PDX-I having PDX-I activity, e.g., alleles, polymorphic variants, and orthologs (see, e.g., Sander et al, J. MoI. Med., 71:327-340 (1997)). Endogenous expression of PDX-I can be induced using transcription factors such as hepatocyte nuclear factor 3 beta, which is involved in pancreatic /3-cell expression of the PDX-I gene (see, e.g., Wu et al, Molecular and Cellular Biology, 17:6002-6013 (1997)). Recombinant PDX-I is delivered to the cells using expression vectors, e.g., viral vectors such as retroviral vectors, as described in U.S. Patent No. 6,448,045.

[0079] In some embodiments, the cells are contacted with a GLP-I receptor agonist to further induce insulin expression. Suitable GLP-I receptor agonists include, e.g., naturally occurring peptides such as GLP-I, exendin-3, and exendin-4 (see, e.g., U.S. Patent No. 5,424,286; U.S. Patent No. 5,705,483, U.S. Patent No. 5,977,071; U.S. Patent No. 5,670,360; U.S. Patent No. 5,614,492), GLP-I analogs (see, e.g., U.S. Patent No. 5,545,618 and U.S. Patent No. 5,981,488), and small molecule analogs. GLP-I receptor agonists may be tested for activity as described in U.S. Patent No. 5,981,488. Cells are contacted with a GLP-I receptor agonist in a time and amount effective to induce insulin mRNA expression, as described in U.S. Patent Publication No. 20030077259. Typically, the cells are contacted with the GLP-I receptor agonists for a discrete time period, as the GLP-I receptor agonist is believed to act as a switch for insulin gene expression. Continuous administration of the GLP-I receptor agonist is therefore not required.

[0080] In some embodiments, the cells of the invention are contacted with a Myc antagonist to further induce endocrine cell differentiation. Exemplary Myc antagonists include antisense molecules or nucleic acid catalysts (e.g., ribozymes) that target Myc transcripts. Expression cassettes containing promoters (e.g., constitutive or inducible) can be operably linked to polynucleotides coding for antisense RNA. Expression of such constructs results in decreased translation of the c-Myc gene product. In another example, polypeptides that antagonize Myc function, such as MADl (Cultraro, et al. Curr. Topics Micro. Immunol, 224:149-158 (1997)), can be expressed in cells. In a third example, small molecules which antagonize c-myc are contacted with the cells of the invention.

[0081] In one embodiment, cells useful in the practice of the invention express one or more oncogenes, such as SV40 T antigen and Hras^va112, which minimally transform the cells but stimulate growth and bypass cellular senescence. Other suitable oncogenes include, e.g., HPV E7, HPV E6, c-myc, and CDK4 (see also U.S. Patent No. 5,723,333). In addition, the cells can be transduced with an oncogene encoding mammalian telomerase, such as hTRT, to facilitate immortalization. Suitable oncogenes can be identified by those of skill in the art, and partial lists of oncogenes are provided in Bishop et al., RNA Tumor Viruses, vol. 1, pp. 1004-1005 (Weiss et ah, eds, 1984), and Watson et al, Molecular Biology of the Gene (4^th ed. 1987). In some cases the oncogenes provide growth factor-independent and ECM- independent entry into the cell cycle. Often the oncogenes are dominant oncogenes. The cells can be analyzed for recombinant oncogene expression by analysis of oncogene RNA or protein expression. Integration of an oncogene into the genome can be confirmed, e.g., by Southern blot analysis. Often, the oncogenes are delivered to the cells using a viral vector, preferably a retroviral vector, although any suitable expression vector can be used to transduce the cells (see, e.g., U.S. Patent No. 5,723,333, which describes construction of vectors encoding one or more oncogenes and transduction of pancreas endocrine cells, see also Halvorsen et al, Molecular and Cellular Biology, 19:1864-1870 (1999)). [0082] The vectors used to transduce the cells can be any suitable vector including vectors that integrate into the host genome and transient vectors. Suitable vectors include viral vectors such as retroviral vectors or adeno-associated viral vectors. Examples of retroviruses, from which viral vectors of the invention can be derived, include avian retroviruses such as avian erythroblastosis virus (AMV), avian leukosis virus (ALV), avian myeloblastosis virus (ABV), avian sarcoma virus (ACV), spleen necrosis virus (SNV), and Rous sarcoma virus (RSV); non-avian retroviruses such as bovine leukemia virus (BLV); feline retroviruses such as feline leukemia virus (FeLV) or feline sarcoma virus (FeSV); murine retroviruses such as murine leukemia virus (MuLV), mouse mammary tumor virus (MMTV), murine sarcoma virus (MSV), and Moloney murine sarcoma virus (MoMSV); rat sarcoma virus (RaSV); and primate retroviruses such as human T-cell lymphotropic viruses 1 and 2 (HTLV-1, 2) and simian sarcoma virus (SSV). Many other suitable retroviruses are know to those of skill in the art. Often the viruses are replication deficient, i.e., capable of integration into the host genome but not capable of replication to provide infective virus. [0083] In another embodiment of the invention, the vector is a transient vector such as an adenoviral vector, e.g. , for transducing the cells with a recombinase to delete the integrated oncogenes.

[0084] In some embodiments, the vectors incorporate recombinase sites, such as lox sites, so that the oncogenes can be deleted by expression of a recombinase, such as the ere recombinase, in the cells following expansion (Halvorsen et ah, Molecular and Cellular Biology, 19:1864-1870 (1999)). Deletion of the oncogenes is useful for cells that are to be transplanted in to a mammalian subject. Other recombinase systems include Saccharomyces cerevisiae FLP/FRT, lambda att/uit, R recombinase of Zygosaccharomyces rouxii. In addition, transposable elements and transposases could be used. Deletion of the oncogene can be confirmed, e.g., by analysis of oncogene RNA or protein expression, or by Southern blot analysis.

[0085] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).

IV. Cell Culture

[0086] This invention relies upon routine techniques in the field of cell culture, and suitable methods can be determined by those of skill in the art using known methodology {see, e.g., Freshney et al, Culture of Animal Cells (3^rd ed. 1994)). In general, the cell culture environment includes consideration of such factors as the substrate for cell growth, cell density and cell contract, the gas phase, the medium, and temperature. [0087] In some embodiments, the cells of the invention are grown under conditions that provide for maximal cell to cell contact. For instance, in some embodiments, the cell-to-cell contact occurs to a greater degree than found in monolayer cell cultures. In a preferred embodiment, the cells are grown in suspension as three dimensional aggregates. Suspension cultures can be achieved by using, e.g., a flask with a magnetic stirrer or a large surface area paddle, or on a plate that has been coated to prevent the cells from adhering to the bottom of the dish, hi a preferred embodiment, the cells are grown in Costar dishes that have been coated with a hydrogel to prevent them from adhering to the bottom of the dish.

[0088] For the cells of the invention that are cultured under adherent conditions, plastic dishes, flasks, roller bottles, or microcarriers in suspension are used. Other artificial substrates can be used such as glass and metals. The substrate is often treated by etching, or by coating with substances such as collagen, chondronectin, fibronectin, and laminin. The type of culture vessel depends on the culture conditions, e.g., multi-well plates, petri dishes, tissue culture tubes, flasks, roller bottles, and the like. [0089] Cells are grown at optimal densities that are determined empirically based on the cell type. For example, a typical cell density for TRM-6 and/or T6PBTE47 cultures varies from IxIO³ to IxIO⁷ cells per ml. Cells are passaged when the cell density is above optimal. [0090] Cultured cells are normally grown in an incubator that provides a suitable temperature, e.g. , the body temperature of the animal from which is the cells were obtained, accounting for regional variations in temperature. Generally, 37°C is the preferred temperature for cell culture. Most incubators are humidified to approximately atmospheric conditions. [0091] Important constituents of the gas phase are oxygen and carbon dioxide. Typically, atmospheric oxygen tensions are used for cell cultures. Culture vessels are usually vented into the incubator atmosphere to allow gas exchange by using gas permeable caps or by preventing sealing of the culture vessels. Carbon dioxide plays a role in pH stabilization, along with buffer in the cell media and is typically present at a concentration of 1-10% in the incubator. The preferred CO₂ concentration typically is 5%. [0092] Defined cell media are available as packaged, premixed powders or presterilized solutions. Examples of commonly used media include DME, RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium (see, e.g., GibcoBRL/Life Tech-nologies Catalogue and Reference Guide; Sigma Catalogue). Typically, low glucose DME or RPMI 1640 are used in the methods of the invention. Defined cell culture media are often supplemented with 5-20% serum, typically heat inactivated, e.g., human horse, calf, and fetal bovine serum. Typically, 10% fetal bovine serum is used in the methods of the invention. The culture medium is usually buffered to maintain the cells at a pH preferably from 1.2-1 A. Other supplements to the media include, e.g., antibiotics, amino acids, sugars, and growth factors such as hepatocyte growth factor/scatter factor. [0093] The growth media of cells can contain appropriate concentrations of the compounds of the invention. Appropriate concentrations for use in cell culture can be routinely determined by persons of ordinary skill in the art by varying the concentration of compounds in the growth media and assaying for characteristics of interest such as growth rate or a phenotype associated with the differentiated state. V. Methods of Treating Diabetic Subj ects

[0094] The cells of the invention can be used to treat diabetic subjects. For example, differentiated endocrine cells produced by the methods described herein are administered to a patient, e.g., a human with type II insulin-dependent diabetes. [0095] In determining the effective amount of the cells to be administered in the treatment or prophylaxis of conditions owing to diminished or aberrant insulin expression, the physician evaluates cell toxicity, transplantation reactions, progression of the disease, and the production of anti-cell antibodies. For administration, cells of the present invention can be administered in an amount effective to provide normalized glucose responsive-insulin production and normalized glucose levels to the subject, taking into account the side-effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. [0096] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular cells employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient. [0097] Immune rejection of grafted cells has previously been a major obstacle to successful islet transplantation. Any universal human donor cell will be recognized by the immune system as an allograft. However, recent advances in therapy for allograft rejection may make this less of a concern (see, e.g., Kenyon et at, PNAS USA, 96:8132-7 (1999)). An advantage of using an immortalized cell line as described herein as a source of transplantable cells is that they can be engineered to exhibit desirable qualities, including avoidance or suppression of host immune responses.

VI. Methods of Treating Cancer Subjects

[0098] Cancers or neoplastic diseases and related disorders that can be treated or prevented by administration of a compound of the invention include, but are not limited to, cancers of the head, neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovary, testicle, kidney, liver, pancreas, brain, intestine, heart, adrenals, and blood and immune cells. (For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia.)

[0099] In determining the effective amount of the compounds of the invention to be administered in the treatment of a cancer, the physician evaluates among other factors, the type of cancer and the progression of the disease. For administration, the compounds of the present invention can be administered with or without other chemotherapeutic agents in an amount effective to stop the proliferation of cancer cells, taking into account the side-effects of the compound at various concentrations, as determined by the compound's effect on the overall health of the patient. Administration can be accomplished via single or divided doses. Examples of chemotherapeutic agents that may be combined with the compounds of the present invention are well known in the art. {See, e.g., Goodman and Gilman's Pharmacological Basis of Therapeutics (11th ed. 2005)).

[0100] Pharmaceutically acceptable carriers for the above are determined in part by the particular composition being administered (e.g., a cell or small molecule), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17^th ed., 1989).

[0101] Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by direct surgical transplantation under the kidney, intraportal administration, intravenous infusion, or intraperitoneal infusion. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

VII. Assays for Modulators of Endocrine Cell Function and Modulation of Cell Cycle

[0102] In some embodiments, the differentiated cells produced by the methods described herein are used to identify additional modulators of endocrine cell function. A. Assays

[0103] Assays using the differentiated cells of the invention can be used to test for modulators (e.g., inhibitors and activators) of endocrine cell function (e.g., /3-cell function or δ-cell function), e.g., by expression of GK, SUR-I, MafA, insulin, granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3; by GK, SUR-I, MafA, insulin (including, e.g., glucose responsive insulin production), granuphilin, chromagranin A, chromogranin C, Synaptotamin like protein 3 production. Such modulators are useful for treating various disorders involving glucose metabolism, such as diabetes and hypoglycemia. Treatment of dysfunctions include, e.g., diabetes mellitus (all types); hyperinsulinism caused by insulinoma, drug-related, e.g., sulfonylureas or excessive insulin, immune disease with insulin or insulin receptor antibodies, etc. (see, e.g., Harrison 's Internal Medicine (14^th ed.

1998)).

[0104] Modulation is tested using the cultures of the invention by measuring GK, SUR-I,

MafA, insulin, granuphilin, chromagranin A, chromogranin C, and/or Synaptotamin like protein 3 gene expression, optionally with administration of glucose, e.g., analysis of GK, SUR-I, MafA, insulin, granuphilin, chromagranin A, chromogranin C, and/or Synaptotamin like protein 3 mRNA expression using northern blot, dot blot, PCR, oligonucleotide arrays, and the like; and analysis of GK, SUR-I, MafA, insulin (i.e., preproinsulin, proinsulin, insulin, or c-peptide), granuphilin, chromagranin A, chromogranin C, and/or Synaptotamin like protein 3 using, e.g., Western blotting, radioimmunoassays, ELISAs, and the like. Downstream effects of GK, SUR-I, MafA, insulin, granuphilin, chromagranin A, chromogranin C, and/or Synaptotamin like protein 3 modulation can also be examined. Physical or chemical changes can be measured to determine the functional effect of the compound on endocrine cell function. Samples or assays that are treated with a potential inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation.

[0105] In some embodiments, the cells are transfected with a reporter gene operably linked to an appropriate promoter prior to contacting the cells with a potential modulator. Expression of the reporter gene indicates that the test compound is a modulator of /3-cell function. Suitable reporter genes include, e.g., green fluorescent protein (GFP), chloramphenicol acetyltransferase gene, a firefly luciferase gene, a bacterial luciferase gene, a /3-galactosidase gene, or an alkaline phosphatase gene. Suitable promoters include, e.g., the insulin promoter (see, e.g., Pino et at, MoI, Endocrinol, 19(5):1343-60 (2005) and Odagiri et al, JBC, 271(4):1909-1915 (1996), the PDX-I promoter (see, e.g., Sander et al, J. MoI Med., 71:327-340 (1997) and Wu et al, Molecular and Cellular Biology, 17:6002-6013 (1997)), and the Neuro D/Beta-2 promoter (see, e.g., U.S. Patent No. 5,795,723; Miyachi et al., MoI. Brain Res., 69, 223-231 (1999); Lee et al, Science, 268:836-844 (1995); Wilson et al, Nature, 368, 32-38 (1994); Naya et al, Genes Dev., 9:1009-1019 (1995)).

In the case of hBLH proteins, suitable promoters include promoters responsive to bHLH protein binding. In one embodiment, the promoter can be the p57^Kp2 or insulin promoters. [0106] When these promoters are used to direct the expression of an easily detected reporter gene such as GFP, the assay can be adapted to high through put screening as described below.

B. Modulators

[0107] The compounds tested as modulators of endocrine cell function (e.g., /?-cell function or δ-cell function) or as cell cycle modulators can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika- Biochemica Analytika (Buchs Switzerland) and the like.

C. Methods of Identifying Modulators

[0108] A variety of methods may be used to identify modulators of endocrine cell function or cell cycle progression. Typically, an assay that provides a readily measured parameter is adapted to be performed in the wells of multi-well plates in order to facilitate the screening of members of a library of test compounds as described herein. Thus, in one embodiment, an appropriate number of cells can be plated into the cells of a multi-well plate, and the effect of a test compound on a detectable parameter of cell function can be determined. The effect of a test compound on endocrine cell function may be determined by measuring any of a number of parameters, such as the expression of a marker associated with endocrine cell differentiation. Such markers, e.g., insulin, glucokinase, etc., are described herein. Alternatively, the expression of a reporter gene, e.g., GFP, which is under the control of a promoter of interest, such as an endocrine specific promoter, can be measured. Similarly, to determine the effect of a test compound on cell cycle progression, markers characteristic of cell division can be measured or the expression of reporter genes under the control of cell cycle promoters can be monitored. Additionally, cell cycle progression can also be monitored by measuring the DNA content of cells using any of a number of reagents that label DNA as described herein. In one embodiment, the effect of a test compound can be tested using cells expressing an inducible transcription factor that directs the expression of a reporter gene under the control of a promoter responsive to the transcription factor. [0109] One of skill in the art will readily recognize that among the methods that can be used to detect the effect of a compound in a particular assay format will include the measurement of fluorescence. Such fluorescence measurement can be performed using high throughput microscopy which allows, for instance, DNA content on a per cell basis to be determined.

[0110] To optimize the assays used, the toxicity of compounds over a range of concentrations can be determined to optimize the concentration of compounds to be tested. Additionally, control plates can be interspersed within a screening run to control for plate-to- plate variability. Background subtraction methods can be used to eliminate false positives that may arise due to artifacts such as autofluorescence.

[0111] Candidate compounds that have an effect on endocrine cell function or cell cycle progression in an initial screen can be subjected to further confirmatory testing. For example, dose response curves and EC50 values can be generated for candidate compounds to confirm that a test compound is not having a non-specific effect in the assay used for screening. Compounds that fulfill the requirements of a primary confirmation test can be subjected to additional confirmatory tests such as the determination of endogenous mRNA levels using methods such as RT-PCR or RNase protection assays in the case of screening assays in which the read-out is expression of a reporter gene. After confirmatory testing, candidate compounds that remain as viable candidates can be subjected to further biological testing, such as testing of the compounds on primary human /3-cells to determine the effect of candidate compounds on endocrine cell function. Candidate compounds that appear to be modulators of cell cycle progression can be tested for their effect on the growth of cells, both normal and cancerous, in culture. The testing of candidate compounds can also be performed by administering compounds to animals and monitoring effects on parameters such as endocrine cell proliferation and differentiation or inhibition of tumor cell growth.

D. High Throughput Screening

[0112] In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0113] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i. e. , the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [0114] Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton et al, Nature, 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids {e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, PNAS USA, 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc, 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc, 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc, 116:2661 (1994)), oligocarbamates (Cho et al, Science, 261 : 1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem., 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506,337; benzodiazepines, 5,288,514, and the like). Libraries of chemical compounds include the CheniBridge DiverSet Library and libraries prepared by the Molecular Libraries Screening Centers. [0115] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[0116] The assays can be solid phase or solution phase assays. In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 or more different compounds is possible using the integrated systems of the invention. [0117] In some embodiments, the assay read out is detection by measurement of fluorescence. One such example of a fluorescent read out is the fluorescence resulting from the expression of GFP. Other examples include fluorescence from agents such as propidium iodide, DAPI or Hoechst 33342, which are used to monitor DNA content. In other embodiments, the assay read out is detectable in the visible spectrum. Examples of such assays include assays for β-galactosidase, among others known in the art. Such assays in which the read out is detectable by the absorption or emission of light are readily adaptable to high through put screening of compounds. While assays based on detection of light have been discussed above, it will be readily apparent to those of skill in the art that other measurable parameters may be used for high through put screening. [0118] Alternatively, high through put screening can be performed using microscopic detection. Plate readers which are capable of high through put microscopy are available through sources such as GE/Amersham.

EXAMPLES

[0119] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Example 1: Generation of an endocrine cell line comprising an inducible class I bHLH polypeptide

[0120] A nucleic acid encoding the class I bHLH polypeptide E47 was fused to a mutated estrogen receptor to generate an inducible expression construct that could be induced by ligands for the estrogen receptor including, e.g., tamoxifen. The inducible construct was expressed in TRM-6 cells expressing PDX-I and Neuro Dl to produce T6PBTE47 cells. Gene expression (i.e., mRNA expression) of the cells following addition of tamoxifen was profiled using Illumina Gene Chips. Expression of multiple /3-cell markers and cell cycle markers was affected following tamoxifen-induced E47 expression. For example, insulin expression was increased 3.4 fold; granuphilin expression was increased 1.4 fold; chromagranin A expression was increased 12.2 fold; chromogranin C expression was increased 1.7 fold; and Synaptotamin like protein 3 expression was increased 2.7 fold. In addition, c-myc expression was decreased 1.6 fold and p57/Kip2 expression was increased 1700 fold.

Example 2: The class I bHLH protein E47 induces insulin expression in a human cell line [0121] T6PBTE47 cells described in Example 1 were cultured in the presence and absence of tamoxifen to induce E47 expression and endogenous insulin gene expression was measured. A 100 fold increase in the level of insulin mRNA produced by the T6PBTE47cells expressing E47 was observed. In addition, expression of multiple components of the /3-cell glucose sensing pathway (e.g., glucokinase ("GK") and the sulfonylurea receptor ("SUR-I") and musculoaponeurotic fibrosarcoma oncogene homolog A ("MafA")) was also observed. Expression levels of some genes in T6PBTE47cells induced to express E47 are similar to those of normal islet cells. Gene expression levels of GK and SUR-I are about 10% of the levels in normal islet cells.

Example 3: E47 induces cell cycle arrest and the CDKIs p57^Kp2 and ρ21^cipl in T6PNE cells [0122] One pronounced effect of inducing E47 in T6PNE cells, as described in Example 1, was growth arrest, as demonstrated by inhibition of growth rate and BrdU incorporation (Figure 6). To investigate the mechanism by which E47 effected cell cycle arrest, a microarray study was performed to identify cell cycle regulatory genes that are affected by tamoxifen induction of E47 (Figure 7). Of the known cell cycle regulatory genes on the array, the cyclin dependent kinase inhibitor (CDKI), p57^Kip2, showed a dramatic induction. While the expression of p57^Kip2 was initially undetectable in untreated cells, the mRNA for p57^Kip2 was induced about 2,000-fold when the inducing agent tamoxifen was added to T6PNE cells (Figure 7). Also induced by E47 is ρ21CIPl, another CDKI (Figure 7). The induction levels as determined in the microarray experiments were confirmed by RT-PCR experiments. Further confirmation of the induction of pSl^² by tamoxifen in T6PNE cells was performed using immunohistochemical analyses.

[0123] Consistent with the hypothesis that pδl^² is playing an important and specific role in regulating cell cycle entry in the human islet cells expressing E47^MBR, E47 was shown to not have an effect on growth rate or induction of p57^Kp2 in the human cardiomyocyte cell line hfc-08.

Example 4: Expression of CDKIs in human pancreas cells

[0124] It was of interest to determine the distribution of CDKIs in primary tissues and cells to establish the validity of the T6PNE cell system. Accordingly,

expression was examined in the human fetal pancreas by immunohistochemistry. When such a study was performed, insulin-positive cells were found to express p57^Kip2 in the human fetal pancreas. Most, but not all, non-b-cells were ρ57^Hp2 negative.

[0125] To determine in greater detail which CDKIs are differentially expressed in the human pancreas, DNA microarrays were probed using cRNA from highly purified, hand- picked preparations of human islets and non-endocrine cells, including ducts and acini (1). As shown in Figure 7, when the expression of all the known CDKIs was analyzed, it was determined that only two, p21^Qpl and p57^Kip2, were expressed in human islet cells. p21^Cipl was expressed at high levels in both islet and non-endocrine cells, while p57^Kip2 expression was islet restricted, consistent with previous reports (7) (Figure 2). No other CDKI was expressed at a statistically significant level. This correlation provided support for the validity of using T6PNE cells as a model for islet cell proliferation and differentiation. [0126] Moreover, the demonstration that the only two CDKIs are expressed in human islet cells suggested that a strategy directed toward down regulation of these CDKIs might be effective at inducing /3-cells to enter the cell cycle.

Example 5: E47 directly activates the p57^Kip2 promoter

[0127] Transient transfection of cells with an inducible-E47 expression vector and p57^Kip2 promoter-reporter constructs was used to address the issue of whether p57^Kip2 induction by E47 was direct (i.e., whether E47 acts directly to activate the p57^Kip2 promoter) or indirect (i.e., whether E47 acts through a transcriptional cascade involving other, as yet unknown, transcription factors). The p57^Kip2 promoter has been isolated and shown to respond appropriately to glucocorticoids (27). The full length p57^Kip2 promoter and a series of nested deletions driving the luciferase reporter gene were obtained from Dr. Okret of the Karolinska Institute (27). Analysis of the full-length and a minimal promoter by co-transfection of those constructs along with an E47^MER expression vector revealed dose-responsive activation of the p57^Kip2 promoter by tamoxifen, while the Tpδl^² minimal promoter was completely inactive (Figure 8). Analysis of promoter deletion constructs revealed that the E47 response element resided in a 900bp region beginning 3. lkb upstream of the TATA box, bounded by Sea and Sac sites (Figure 9 A, B) and contained 7 E-boxes (Figure 9 B). To further define the locations of critical regulatory elements, ChEP analysis was performed with the finding that only one E box sequence, designated E2, consistently gave a stronger signal in the presence versus the absence of tamoxifen (Figure 9C). Site-directed mutagenesis of the E-boxes in that region confirmed that E2 is critical for E47 responsiveness (Figure 10). Induction of pβl^² mRNA by E47 did not depend upon protein synthesis (Figure 11), providing confirmation that the effect of E47 on the p57^Kp2 promoter is direct and not through secondary induction of another transcription factor. Thus, these studies show that E47 acts directly on the p57^Kip2 promoter.

Example 6: p57^Kip2 is sufficient to induce growth arrest in T6PN cells

[0128] While it has been shown in Example 3 that E47 induces growth arrest, as well as, p57^Kip2, this does not prove that p57^Kip2 expression is sufficient to cause growth arrest in T6PNE cells. Thus, a p57^Kip2 expression vector was transfected into T6PN cells (cells without E47MER), and it was shown by BrdU labeling that expression of p57^Kip2 induced growth arrest. Thus, p57^Kip2 is sufficient to mediate the growth arrest induced by activation of E47.

Example 7: Development of a reporter system for use in assays of small molecules that regulate the insulin promoter

[0129] A reporter construct containing an insulin promoter-eGFP transgene was generated and expressed in T6PNE cells for use in high-through-put screening for small-molecule compounds that modulate insulin promoter activity. [0130] The insulin promoter is regulated by a complex of transcription factors that bind to multiple sequence elements, but of central importance is an E-box approximately 100 bp 5' of the transcription start site (22). This E-box binds to a heterodimeric complex consisting of a class I (widely expressed) and class II (tissue specific) bHLH transcription factor. The products of the E2A gene, E12 and E47, are the major class I bHLH factors in the /3-cell. [0131] A lentiviral vector pRRL.SIN-18.cPPT.hINS-EGFP.WPRE expressing the enhanced green fluorescent protein (eGFP) reporter gene driven by the human insulin gene promoter was generated by inserting a 1.4-kb Sall-Hindlll fragment containing the human insulin promoter (28), into pRRL.SIN-18.cPPT.hPGK-EGFP.WPRE (29) (Figure 12). RRL.SIN- 18.cPPT.hINS-EGFP.WPRE virus was used to infect the mouse insulinoma cell line MIN6 and the cervical carcinoma cell line HeLa. High level GFP expression was observed in MIN6 but not in HeLa, consistent with the endogenous insulin promoter activity in MIN6 but not HeLa. The /3-cell specific expression of the insulin promoter construct was also observed in studies in which primary islet cells were infected.

[0132] The insulin promoter-eGFP lentiviral vector was then used to infect T6PNE cells. After infection, the resulting cells were tested for induction of eGFP by tamoxifen, which causes E47^MER to translocate to the nucleus and thus active the insulin promoter. High levels of eGFP were observed in response to tamoxifen by fluorescence microscopy.

Example 8: Assay for small molecule compounds that regulate the insulin promoter [0133] T6PNE cells that had been infected with the insulin promoter-eGFP virus were tested at different cell plating densities and tamoxifen concentrations to optimize conditions for a high-throughput assay. Under the optimal conditions in which a baseline of 0.5 μM tamoxifen was added to each well of a 384 well plate in which 2,000 cells were plated into each well, the calculated z' was 0.6. Having optimized the assay and validated it to a high z' value, a small-scale screen of a subset of compounds from the ChemBridge DiverSet library was commenced. This screen is in preparation for a larger scale screen of compounds available through the Molecular Libraries Screening Center.

[0134] A) Initial screening of 8,000 compounds from the ChemBridge DiverSet library yields candidate compounds that regulate the insulin promoter both positively and negatively. [0135] Cells were plated into 384 well plates, and a submaximal dose of tamoxifen was added to induce an intermediate level of GFP fluorescence (indicative of insulin promoter activation). 8,000 compounds from the ChemBridge DiverSet library were added, one compound per well (at 5 μM concentration per compound). After two days, the plates were fixed in paraformaldehyde and the GFP fluorescence was measured by high-throughput microscopy. The integrated eGFP and DAPI fluorescence data per well for each of the 8,000 screened compounds was measured. To calculate integrated fluorescence intensity, objects were extracted from the images using an intensity- and size-thresholded image mask to exclude background and this value was used to select initial hits. Of the 8,000 compounds screened, 52 profoundly repressed eGFP fluorescence and approximately 11 increased eGFP fluorescence.

[0136] B) Primary confirmatory assay yields a preliminary true positive rate of approximately 50%.

[0137] For primary confirmation, a dose response curve on a small number of active compounds was performed in triplicate. Three of four compounds that repressed GFP fluorescence and three of seven compounds that increased GFP fluorescence were confirmed, for a true positive rate of approximately 50% of the initial hits. The EC50 values were in the low micromolar range. [0138] C) Secondary Confirmatory Assay [0139] Compounds that passed the primary confirmatory assay were subjected to further testing in a secondary confirmatory assay. This consisted of determination of GFP and endogenous insulin mPJSfA levels by RT-PCR (Figure 13). The levels of both GFP and endogenous insulin mRNAs were determined because of the possibility that a compound may affect the 1.4 kb insulin promoter transgene but not the endogenous insulin promoter, which may be under more complex control. Three compounds had potent repressive effects on both GFP and endogenous insulin mRNA. Three activating compounds were less potent in modulating insulin mRNA than the repressing compounds, as might be expected since activators are generally more difficult to screen for than are inhibitors. Further characterization of these candidates is underway. Example 9: Assay for small molecule compounds that affect cell cycle entry and p57^Ep2 promoter activity in human pancreatic endocrine cell lines

[0140] Since pδl^² is a major CDKI in human /3-cells, and there is evidence from focal PHHI that pδl^² deletion induces β-cell proliferation, a screen for molecules that modulate pS?*^² promoter activity can be undertaken to understand and manipulate the signaling pathways that control /3-cell replication. Because Tpδl¹^² is a potent inducer of cell cycle exit, a first step is to develop a cell line from the human endocrine pancreas in which the endogenous pδl^² promoter is tightly and inducibly regulated. Such a cell line can be used to discover pathways that control pδl^² expression and function. [0141] A) Assay development: The 6.3 kb p57^Kp2 promoter fragment that was shown to be responsive to E47 (Figure 8-11) will be inserted 5' of the eGFP gene and then subcloned into a lentiviral vector in place of the insulin promoter that was used previously in Example 8 (Figure 12). To characterize the ipδl^² promoter-eGFP vector, it will be tested in T6PNE cells in the presence and absence of tamoxifen and in primary human islets infected in monolayer culture. Simultaneous visualization of eGFP fluorescence and hormone expression by immunohistochemistry will be used to ensure that /3-cell specific expression is maintained. [0142] Upon infection with the lentiviral vector, T6PNE clones that exhibit high level tamoxifen-inducible upregulation of eGFP with low background expression in the absence of tamoxifen will be isolated. Assay optimization will be done by systematically varying the cell plating density and the tamoxifen dose to induce p57^Kip2. Optimized assay conditions that yield a z' >0.5 will be used for the screen. This is the same approach that was successful in developing an assay for molecules that modulate insulin promoter activity in T6PNE cells as shown in Example 8. [0143] B) High-throughput, high-content screening: To validate the assay and to test its robustness, the Chembridge DiverSet library, comprising a total of 50,000 compounds, will be screened. To perform the screen for small molecules that affect p57^Kip2 expression and/or function, cells will be plated into 1536 well plates in the presence of a dose of tamoxifen titrated to induce an intermediate level of p57^Kip2 expression. The rationale for using this dose of tamoxifen is that it allows for the discovery of compounds that both induce and repress p57^Kp2 promoter activity. In addition, it maintains the assay in a dynamic range that is most likely to be responsive to regulation by an added compound.

[0144] A single compound from among the 50,000 in the DiverSet library (30-32) will be added to each well. To determine the optimal concentration of compound to add, the toxicity over a range of concentration and compounds was examined and 5 μM was found to be optimal. DMSO effects were also examined, and DMSO does not affect assay performance. To control for plate-to-plate variability, control plates will be interspersed throughout the screen with one at the beginning, middle, and end of the run. Each control plate will consist of an extensive tamoxifen dose response, with 8-16 replicate wells at each dose. This will permit calculation of a z' at different times during the screen, ensuring that technical characteristics of assay performance are within the desirable range. [0145] Forty-eight hours after compound addition, a time found to be optimal for ^Sl^² induction by tamoxifen, the cells will be fixed with paraformaldehyde and stained with DAPI to allow DNA content on a per cell basis (a surrogate for cell cycle status) to be measured. The plates will then be read on a GE/Amersham InCeIl 1000 high throughput microscopy system. Three channels of data (green, red, and blue) will be acquired. The green channel reflects GFP fluorescence while the blue channel reflects DAPI fluorescence. The red channel is used for background subtraction. [0146] C) Background subtraction: Background subtraction methods are an important aspect of this screening method. Artifacts such as autofluorescence must be eliminated from each image to remove false positives. A number of background subtraction algorithms have been tested. An advantageous method is based upon a pixel-by-pixel subtraction of the red from the green channel. After filtering out any well data flagged for errors (e.g. debris or pipetting errors), plate distributions will be normalized by the mode value of each plate; the validity of this normalization is checked by evaluating a set of negative (untreated) controls and positive (high tamoxifen dose) wells. Numerical and meta data will be collated in pre- configured excel-type spreadsheets (ActivityBase) and linked to image data maintained in a relational database. [0147] One advantage of using a high-content microscope-based system, rather than a plate reader, is that one can rule out many sources of artifactual hits by examining the digital images collected from each well. For example, compounds that fluoresce and stain the cells can easily be ruled out. Compounds that are toxic so that cells have very low DAPI channel signal and/or induce cellular autofluorescence can also be detected by virtue of the fact that such cells autofluoresce in the red channel. On the other hand, compounds that stimulate replication will cause an increase in overall DAPI intensity/well (integrated pixel intensity in the DAPI channel), an increase in the number of nuclei, or an increase in the incidence of cells in G2/S/M phase (elevated DAPI/nucleus). The latter two parameters are apparent after segmentation of the images. [0148] D) Selection of "hits": Although every assay has unique properties, the inventors have determined that setting the "hit rate" at 0.1% provides a good threshold value. However, the "hit rate" may be adjusted depending on the distribution of compounds that result from initial screening using the pδl^² promoter assay. The "hit rate" can be modified as needed on the positive and negative ends.

[0149] E) Primary Confirmatory Screen: For primary confirmation of hits, a dose response curve will be performed, with each dose being done in triplicate. In this screen, in addition to compounds that modulate GFP activity, compounds that affect cell cycle entry as measured by DAPI fluorescence will also be identified. [0150] F) Secondary screen. Once obvious artifacts and false positives revealed by the primary confirmatory screen are eliminated, the remaining compounds will be subjected to a secondary screen for GFP mRNA levels by quantitative RT-PCR. This will eliminate any compounds that alter GFP stability or translation and enrich for compounds that regulate p_romotø. _activity. An advantage of high-content screening is that DAPI fluorescence is measured both on a per-well and per-cell basis, allowing the effect of each compound on cell cycle status to be determined. Thus, direct effects of each compound on the cell cycle status of the target cells will be determine independently of the pδl^² promoter-reporter. The secondary screens will serve to detect compounds that reproducibly affect pSl^² promoter activity and/or a corresponding effect on DAPI fluorescence. Secondary screens will be done in 384-well plates, as opposed to the 1536-well plates for the primary screens. This will increase the number of cells per well, providing the statistical advantage of increasing the number of events detected by the microscope per well. Each compound in the secondary screen will be assayed at a range of doses to give a dose-response curve, and each dose will be done in triplicate. Thus, highly statistically significant data on the effect of each compound on the cell will be obtained from this screen.

[0151] G) Tertiary screen. For the tertiary screen, compounds that decrease Tpδl^² promoter activity or that have a direct effect on cell cycle status in the cell line will be tested on primary human /3-cells, both in suspension and in monolayer culture. Islets will be obtained from the NCRR Islet Cell Resource Centers. In suspension culture, islets will be cultured under standard conditions including 10% FBS. In monolayer culture, the cells will be cultured on HTB9 matrix in the presence and absence of HGF, as those conditions have been reported to promote /3-cell replication (10). Cell cycle entry will be measured by Ki67 immunohistochemistry and BrdU incorporation. [0152] H) Possible classes of compounds: This screen is designed to discover compounds that affect endogenous pS?^² expression and cell cycle entry in the human /3-cell. In theory, compounds that pass the secondary screen, i.e. have effects on the endogenous pβl^² promoter or that have reproducible effects on DAPI fluorescence, thus indicating effects on cell cycle entry, could fall into the following classes:

1. Compounds that induce a decrease in φδl^² promoter activity and promote cell cycle entry as measured by an increase in the number of cells (reflected as increased integrated pixel intensity in the DAPI channel, indicating increased DNA content per nucleus, i.e., cells in S, G2, and M phase). Such compounds would be valuable candidates for inducing /3-cell regeneration.

2. Compounds that do not affect eGFP fluorescence but that induce entry into the cell cycle as determined by DAPI fluorescence. Compounds in this class might target post- transcriptional functioning of pδl^².

3. Compounds that induce a decrease in pδl^² promoter activity without affecting cell division. Compounds in this class would dissociate pβl^² levels from cell cycle control and might target potential redundant, compensatory and/or independent downstream regulators of cell cycle.

[0153] A large number of studies will be required to follow up on the compounds that are identified in this screen. Especially interesting will be to determine the molecular targets of the compounds. The molecular targets affected by the compounds identified in the screens described above can be determined using a variety of methods. Such methods include: microarray studies to identify candidate signaling pathways that are being altered by a compound, proteomic approaches to determine direct effects of compounds on activation or repression of a pathway, e.g., changes in protein phosphorylation patterns, and direct affinity binding approaches which require derivatization of the compound to attach it to a solid support.

[0154] REFERENCES

1. E Hao, B Tyrberg, P Itkin-Ansari, J Lakey, E Monosov, M Barcova, M Mercola, F Levine: Beta-cell differentiation from non-endocrine epithelial cells of the adult human pancreas. Nat Med 2006, hi Press.

2. AE Butler, J Janson, S Bonner-Weir, R Ritzel, RA Rizza, PC Butler: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003, 52:102-10. 3. B Tyrberg, J Ustinov, T Otonkoski, A Andersson: Stimulated endocrine cell proliferation and differentiation in transplanted human pancreatic islets: effects of the ob gene and compensatory growth of the implantation organ. Diabetes 2001, 50:301-7. 4. DT Finegood, L Scaglia, S Bonner-Weir: Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes 1995, 44:249-56.

5. GM Beattie, P Itkin-Ansari, V Cirulli, G Leibowitz, AD Lopez, S Bossie, MI MaUy, F Levine, A Hayek: Sustained proliferation of PDX-1+ cells derived from human islets. Diabetes 1999, 48:1013-9.

6. P Itkin-Ansari, F Levine: Sources of beta-Cells for Human Cell-Based Therapies for Diabetes. Cell Biochem Biophys 2004, 40:103-12. 7. SA Kassem, I Ariel, PS Thornton, K Hussain, V Smith, KJ Lindley, A Aynsley- Green, B Glaser: p57(KIP2) expression in normal islet cells and in hyperinsulinism of infancy. Diabetes 2001, 50:2763-9.

8. T Uchida, T Nakamura, N Hashimoto, T Matsuda, K Kotani, H Sakaue, Y Kido, Y Hayashi, KI Nakayama, MF White, et al: Deletion of Cdknlb ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice. Nat Med 2005, 11:175-82.

9. B Tyrberg, A Andersson, LAH Borg: Species differences in susceptibility of transplanted and cultured pancreatic islets to the b-cell toxin alloxan. Gen Comp Endocrinol 2001, 122:238-251.

10. A Hayek, GM Beattie, V Cirulli, AD Lopez, C Ricordi, JS Rubin: Growth factor/matrix-induced proliferation of human adult beta-cells. Diabetes 1995, 44:1458-1460. 11. VH Lefebvre, T Otonkoski, J Ustinov, MA Huotari, DG Pipeleers, L Bouwens:

Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells. Diabetes 1998, 47:134-7.

12. CJ Sherr, JM Roberts: CDK inhibitors: positive and negative regulators of Gl -phase progression. Genes Dev 1999, 13:1501-12.

13. P Zhang, NJ Liegeois, C Wong, M Finegold, H Hou, JC Thompson, A Silverman, JW Harper, RA DePinho, SJ Elledge: Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith- Wiedemann syndrome. Nature 1997, 387:151- 8.

14. P Zhang, C Wong, RA DePinho, JW Harper, SJ Elledge: Cooperation between the Cdk inhibitors p27(KJJPl) and p57(KIP2) in the control of tissue growth and development. Genes Dev 1998, 12:3162-7.

15. B Joseph, A Wallen-Mackenzie, G Benoit, T Murata, E Joodmardi, S Okret, T Perlmann: p57(Kip2) cooperates with Nurrl in developing dopamine cells. Proc Natl Acad Sci U S A 2003, 100:15619-24. 16. EG Reynaud, MP Leibovitch, LA Tintignac, K Pelpel, M Guillier, SA Leibovitch: Stabilization of MyoD by direct binding to ρ57(Kip2). J Biol Chem 2000, 275:18767-76.

17. P Itkin-Ansari, I Geron, E Hao, C Demeterco, B Tyrberg, F Levine: Cell-based therapies for diabetes: progress towards a transplantable human beta cell line. Ann N Y Acad Sci 2003, 1005:138-47.

18. D Dufayet de Ia Tour, T Halvorsen, C Demeterco, B Tyrberg, P Itkin-Ansari, M Loy, S-J Yoo, E Hao, S Bossie, F Levine: b-cell differentiation from a human pancreatic cell line in vitro and in vivo. MoI Endocrinol 2001, 15:476-483.

19. P Itkin-Ansari, C Demeterco, S Bossie, DD de Ia Tour, GM Beattie, J Movassat, MI Mally, A Hayek, F Levine: PDX-I and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. MoI Endocrinol 2000, 14:814-22.

20. S Wang, GM Beattie, MI Mally, AD Lopez, A Hayek, F Levine: Analysis of a human fetal pancreatic islet cell line. Transplant Proc 1997, 29:2219. 21. P Itkin-Ansari, E Marcora, I Geron, B Tyrberg, C Demeterco, E Hao, C Padilla, C Ratineau, A Leiter, JE Lee, et al: NeuroDl in the endocrine pancreas: Localization and dual function as an activator and repressor. Dev Dyn 2005, 233:946-953.

22. MS German, MA Blanar, C Nelson, LG Moss, WJ Rutter: Two related helix-loop- helix proteins participate in separate cell- specific complexes that bind the insulin enhancer.

MoI Endocrinol 1991, 5:292-9.

23. G Bain, ECR Maandag, DJ Izon, D Amsen, AM Kruisbeek, BC Weintraub, I Krop, MS Schlissel, AJ Feeney, M van Roon, et al: E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 1994, 79:885-892.

24. P Itkin-Ansari, G Bain, GM Beattie_5l C Murre, A Hayek, F Levine: E2A gene products are not required for insulin gene expression. Endocrinology 1996, 137:3540-3. 25. M Peyton, LG Moss, M-J Tsai: Two distinct class A helix-loop-helix transcription factors, E2A and BETAl, form separate DNA binding complexes on the insulin gene E box. J Biol Chem 1994, 269:25936-25941.

26. CE Sayegh, MW Quong, Y Agata, C Murre: E-proteins directly regulate expression of activation-induced deaminase in mature B cells. Nat Immunol 2003, 4:586-93.

27. K Alheim, J Corness, MK Samuelsson, LG Bladh, T Murata, T Nilsson, S Okret: Identification of a functional glucocorticoid response element in the promoter of the cyclin- dependent kinase inhibitor p57Kip2. J MoI Endocrinol 2003, 30:359-68.

28. H Odagiri, J Wang, MS German: Function of the human insulin promoter in primary cultured islet cells. J Biol Chem 1996, 271:1909-1915. 29. A Follenzi, LE Ailles, S Bakovic, M Geuna, L Naldini: Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-I pol sequences. Nat Genet 2000, 25:217-22. 30. JL Jenkins, RY Kao, R Shapiro: Virtual screening to enrich hit lists from high- throughput screening: a case study on small-molecule inhibitors of angiogenin. Proteins 2003, 50:81-93.

31. RY Kao, JL Jenkins, KA Olson, ME Key, JW Fett, R Shapiro: A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity. Proc Natl Acad Sci U S A 2002, 99:10066-71.

32. TR Webb: Current directions in the evolution of compound libraries. Curr Opin Drug Discov Devel 2005, 8:303-8.

[0155] All publications, patents, patent publications, and Genbank Accession Nos. cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for inducing differentiation of cultured cells, the method comprising the steps of: expressing a recombinant polynucleotide encoding a class I basic helix-loop- helix (bHLH) polypeptide in the cells, thereby inducing differentiation of the cells, wherein the differentiated cells express glucokinase (GK), insulin, granuphilin, chromagranin A, chromogranin C, and Synaptotamin like protein 3, at a level that is at least about 1 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

2. The method of claim 1, wherein the bHLH polypeptide is selected from the group consisting of: E2A, HEB/BETA1, and E2-2.

3. The method of claim 1 , wherein the bHLH polypeptide is E2A.

4. The method of claim 3, wherein the E2A is selected from the group consisting of: E47, E12, and E2-5.

5. The method of claim 3 , wherein the E2 A is E47.

6. The method of claim 5, wherein the recombinant polynucleotide encoding E47 is operably linked to a polynucleotide encoding an estrogen receptor.

7. The method of claim 6, further comprising contacting the cells with an estrogen receptor antagonist, thereby inducing differentiation of the cells

8. The method of claim 7, wherein the estrogen receptor antagonist is selected from the group consisting of: tamoxifen and keoxifene.

9. The method of claim 1, wherein the cell is a member selected from the group consisting of: embryonic stem cells, adult stem cells, pancreas-derived multipotent precursor cells, and endocrine cells.

10. The method of claim 1, wherein the cell is an endocrine cell.

11. The method of claim 10, wherein the endocrine cell is a /3-cell.

12. The method of claim 11, wherein the β-cells are human β-cells.

13. The method of claim 1, wherein the differentiated cells further express SUR-I, MafA, and p57/Kip2, at a level that is at least about 1 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

14. The method of claim 1, wherein the differentiated cells express GK, SUR-I, MafA, insulin, chromagranin A, Synaptotamin like protein 3, and p57/Kip2, at a level that is at least about 2 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

15. The method of claim 1, wherein the differentiated endocrine cells express chromagranin A and p57/BCip2, at a level that is at least about 10 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

16. The method of claim 1, wherein the differentiated cells express p57/Kip2 at a level that is at least about 1000 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

17. The method of claim 1, wherein the differentiated endocrine cells express c-myc at a level that is at least about 1 fold lower than in the absence of the polynucleotide encoding a bHLH polypeptide.

18. The method of claim 1, wherein the cells further comprise a recombinant polynucleotide encoding NeuroD/BETA2.

19. The method of claim 18, wherein the cells further comprise a recombinant polynucleotide encoding PDX- 1.

20. The method of claim 1, wherein the cells are cultured under conditions such that the cells are in contact with other cells in the culture.

21. The method of claim 1, wherein the cells are cultured as aggregates in suspension.

22. A method of identifying a compound that modulates endocrine cell function, the method comprising the steps of contacting cells made by the method of claim 1 with the compound and determining the effect of the compound on endocrine cell function.

23. A method of treating a diabetic subj ect by providing to the subj ect cells that secrete insulin, the method comprising the step of administering to the subject an effective amount of cells according to claim 1.

24. The method of claim 23, wherein the subject has Type I insulin dependent diabetes.

25. The method of claim 23, wherein the subject has Type II insulin independent diabetes.

26. A method of treating a diabetic subject by providing to the subject cells that secrete insulin in response to an estrogen receptor antagonist, the method comprising the step of administering to the subject an effective amount of cells according to claim 6.

27. A stable culture of endocrine cells, wherein said cells express a first recombinant polynucleotide encoding a class I basic helix-loop-helix (bHLH) polypeptide, thereby inducing differentiation of the cells, wherein the differentiated cells express glucokinase (GK), insulin, granuphilin, chromagranin A, chromogranin C, and Synaptotamin like protein 3, at a level that is at least about 1 fold higher than in the absence of the polynucleotide encoding a bHLH polypeptide.

28. The culture of claim 27 further comprising a second recombinant polynucleotide encoding a reporter gene operably linked to a promoter responsive to the class I bHLH polypeptide.

29. The culture of claim 27, wherein the bHLH polypeptide is E47.

30. The culture of claim 29, wherein the first recombinant polynucleotide encoding E47 is operably linked to a polynucleotide encoding an estrogen receptor.

31. The culture of claim 27, wherein the endocrine cells are /3-cells.

32. The culture of claim 31 , wherein the β-cells are human β-cells.

33. The culture of claim 27, wherein the endocrine cells are cultured under conditions such that the cells are in contact with other cells in the culture.

34. The culture of claim 27, wherein the endocrine cells are cultured as aggregates in suspension.

35. A method for identifying a compound that modulates endocrine cell function, the method comprising the steps of: expressing a first recombinant polynucleotide encoding a class I bHLH polypeptide and a second recombinant polynucleotide encoding a reporter gene operably linked to a promoter responsive to the class I bHLH polypeptide in a cell; and contacting said cell with a compound and determining the effect of the compound on expression of the reporter gene.

36. The method of claim 35, wherein the bHLH polypeptide is E2A.

37. The method of claim 36, wherein the E2 A is selected from the group consisting of E47, E12, and E2-5.

38. The method of claim 37, wherein the E2A is E47.

39. The method of claim 38, wherein the recombinant polynucleotide encoding E47 is operably linked to a polynucleotide encoding an estrogen receptor.

40. The method of claim 39 further comprising contacting the cells with an estrogen receptor antagonist, thereby activating the polynucleotide encoded E47.

41. The method of claim 40, wherein the estrogen receptor antagonist is selected from the group consisting of: tamoxifen and keoxifene.

42. The method of claim 41, wherein the promoter responsive to the class I bHLH polypeptide is selected from the group consisting of: insulin promoter and p57/Kip2 promoter.

43. The method of claim 42, wherein the promoter responsive to the class I bHLH polypeptide is the insulin promoter.

44. The method of claim 42, wherein the promoter responsive to the class I bHLH polypeptide is the p57/Kip2 promoter.

45. The method of claim 35, wherein the reporter is GFP.

46. The method of claim 35, wherein the endocrine cell is a /3-cell.

47. The method of claim 46, wherein the /3-cells are human /3-cells.

48. The method of claim 35, wherein said compound modulates the cell cycle of the cell.

49. The method of claim 48, wherein said compound modulates the cell cycle of the cell by inducing cell cycle progression.

50. The method of claim 48, wherein said compound modulates the cell cycle of the cell by inducing cell cycle arrest. ₍

51. A method for identifying a compound that modulates the cell cycle in a cell, the method comprising the steps of: expressing a first recombinant polynucleotide encoding a class I bHLH polypeptide and a second recombinant polynucleotide encoding a reporter gene operably linked to a promoter responsive to the class I bHLH polypeptide in a cell; and contacting said cell with a compound and determining the effect of the compound on expression of the reporter gene, wherein expression of the reporter gene correlates with cell cycle progression or arrest.

52. A method for modulating the cell cycle in a cell, the method comprising the steps of: expressing a recombinant polynucleotide encoding a class I bHLH polypeptide in a cell, wherein expression of the class I bHLH polypeptide modulates cell cycle progression.

53. A method of treating a subj ect with a disorder characterized by an aberrant cell cycle comprising expressing in the cells of the subject a first recombinant polynucleotide encoding a class I basic helix-loop-helix (bHLH) polypeptide, wherein expression of the bHLH modulates the cell cycle in the cells.