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Induction of a beta cell differentiation in human cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripInduction of a beta cell differentiation in human cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060210963, Induction of a beta cell differentiation in human cells. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE OF RELATED PATENT APPLICATIONS [0001] The present application is a continuation of U.S. patent application Ser. No. 11/125,716, filed May 9, 2005, which is a divisional of U.S. patent application Ser. No. 10/041,845, filed Oct. 18, 2001, now U.S. Pat. No. 6,911,324, the disclosures of which are hereby incorporated by reference in their entirety. 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, with is exhibited only by pancreatic .beta.-cells. The development of expanded populations of human .beta.-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 al., J Mol Med 77:153-4 (1999)), expansion of primary human .beta.-cells with growth factors and extracellular matrix (Beattie et al., 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 .beta.-cells, entry into the cell cycle can be achieved using hepatocyte growth factor/scatter factor ("HGF/SF") plus extracellular matrix ("ECM"") (Beattie et al., Diabetes 48:1013-9 (1999), Hayek et al., Diabetes 44:1458-1460 (1995)). However, this combination, while resulting in a 2-3.times.10.sup.4-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 .beta.-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 .beta.-cells, with viral vectors expressing the potent dominant oncogenes such as SV40 T antigen and H-ras.sup.val12 (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. Pat. 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)). [0006] Although the cell lines grow indefinitely, they lose differentiated function, similar to growth-stimulated primary .beta.-cells. Methods of stimulating differentiation of the cell lines into insulin-secreting .beta.-cells are therefore desired. Such cells could then be transplanted in vivo as a treatment for diabetes. SUMMARY OF THE INVENTION [0007] Induction of .beta.-cell differentiation in cultured human .beta.-cells was achieved by stimulating multiple signaling pathways, including those downstream of the homeodomain transcription factors NeuroD/BETA2 and PDX-1, cell-cell contact, and the glucagon-like peptide-1 (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation of the cultured human .beta.-cells, which initially express no detectable pancreatic hormones, into fully functional .beta.-cells that exhibit glucose-responsive insulin secretion. Furthermore, these cells can be transplanted in vivo and demonstrate glucose-responsive expression of insulin. The ability to grow unlimited quantities of functional human .beta.-cells in vitro provides the means for a definitive cell transplantation therapy for treatment of diabetes. [0008] The expression of the transcription factor NeuroD/BETA2 in human .beta.-cells that express PDX-1, are in cell to cell contact, and are contacted with a GLP-1 receptor agonist resulted in certain desirable characteristics. For example, surprisingly, the resulting cells produce high levels of insulin compared to cells that do not express NeuroD/BETA2. Moreover, the cells expressing NeuroD/BETA2 are highly stable in cell culture and can be grown in culture for multiple generations. Thus, in some embodiments of the invention, the present invention provides a method for inducing insulin gene expression in cultured endocrine pancreas cells, the method comprising the steps of (i) expressing a recombinant NeuroD/BETA2 polynucleotide and a recombinant PDX-1 gene in cells that have been cultured under conditions such that the cells are in contact with other cells in the culture; and (ii) contacting the cells with a GLP-1 receptor agonist, thereby inducing insulin gene expression in the cells. In some embodiments, the cells do not initially produce any detectable pancreatic hormones such as insulin and glucagon. [0009] In another aspect, the present invention provides a method of identifying a compound that modulates .beta.-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 .beta.-cell function. [0010] In another aspect, the present invention provides a stable culture of endocrine pancreas cells, wherein the cells are in contact with other cells in the culture, wherein the cells express a recombinant NeuroD/BETA2 gene and a recombinant PDX-1 gene, and wherein insulin gene expression is stimulated in the cells when exposed to an effective amount of a GLP-1 receptor agonist. [0011] In another aspect, the present invention provides a method of treating a diabetic subject by providing to the subject cells that secrete insulin in response to glucose, the method comprising the steps of: (i) contacting a culture of endocrine pancreas cells expressing a NeuroD/BETA2 gene and a PDX-1 gene with a GLP-1 receptor agonist, wherein the cells have been cultured under conditions such that the cells are in contact with other cells in the culture; and (ii) administering the cells to the subject, thereby providing to the subject cells that secrete insulin in response to glucose. [0012] In some embodiments, the GLP-1 receptor agonist is a GLP-1 analog or has an amino acid sequence of a naturally occurring peptide. In some embodiments, the GLP-1 receptor agonist is GLP-1, exendin-3, or exendin-4. [0013] In some embodiments, the cells are cultured as aggregates in suspension. [0014] In one embodiment, the cells are human cells. In another embodiment, the cells are .beta.lox5 cells. A deposit of the .beta.lox5 cells, which are human pancreatic cells, was made on Jul. 19, 2001 under accession number PTA-3532 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. [0015] In some embodiments, the cells express a recombinant oncogene. In some embodiments, the cells express a recombinant oncogene. In some embodiments, the cells express a recombinant oncogene. In some embodiments, the cells express a recombinant telomerase gene. [0016] In some embodiments, the diabetic subject is a human. In some embodiments, the subject has Type I insulin dependent diabetes. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1. Flow cytometric analysis of FAD autofluorescence in a single cell suspension of human islets used to develop the .beta.lox5 cell line. [0018] FIG. 2: RT-PCR analysis of pancreatic hormone gene expression in .beta.lox5. The conditions tested were .beta.lox5 cells grown in monolayer culture, .beta.lox5 cells infected with a retroviral vector expressing PDX-1, .beta.lox5 cells grown as three-dimensional aggregates, and .beta.lox5 cells treated with exendin-4. FIG. 2(A) insulin; FIG. 2(B) quantitative RT-PCR analysis of insulin gene expression; FIG. 2(C) other pancreatic hormones. Exendin-4 (Sigma) was used at a concentration of 10 nM. RT-PCR for insulin, somatostatin, glucagon, and IAPP have been described previously (Itkin-Ansari et al., submitted). Quantitative RT-PCR was done by interpolation from a standard curve constructed using a plasmid containing the human insulin cDNA. [0019] FIG. 3: Analysis of transcription factors expressed in .beta.lox5 cells. FIG. 3(A) electrophoretic mobility shift assay (EMSA) of PDX-1. EMSA for PDX-1 was performed using a probe derived from the human insulin promoter A5 element. FIGS. 3(B&C) RT-PCR analysis of BETA2 and Pax6. FIG. 3(D) Western blot analysis of CREB. FIG. 3(E) EMSA of RIPE3b. [0020] FIG. 4. Insulin protein in induced .beta.lox5 cells. FIG. 4(A-C) Insulin immunohistochemistry; FIG. 4(D) Insulin western blot analysis of conditioned medium. [0021] FIG. 5. Analysis of glucose-responsive insulin secretion. FIG. 5(A) RT-PCR for glucokinase. FIG. 5(B) Radioimmunoassay for insulin secreted from induced .beta.lox5 cells grown in culture medium containing increasing concentrations of glucose. Induced .beta.lox5 cells were cultured in DME containing a single concentration of added glucose for one hour. Medium was harvested and assayed for insulin by RIA. Continue reading about Induction of a beta cell differentiation in human cells... 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