Human glucose-dependent insulin-secreting cell line

#Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express β-cell differentiation factor IDX-1. Cells so transfected respond to glucagon-like peptide-1 by differentiating into insulin-secreting β cells.

#Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express β-cell differentiation factor IDX-1. Cells so transfected respond to glucogon-like/peptide-1 by differentiating into insulin-secreting βcells.

#Insulin is essential for proper metabolism in humans: in addition to its familiar role as the chief regulator of blood sugar levels in humans, it is essential for carbohydrate, lipid, and protein metabolism, as well. Pancreatic beta (β) cells of the islets of Langerhans, epithelial cells dispersed throughout the pancreas, secrete insulin. When β cells are destroyed or their function impaired, insulin production declines, and diabetes results.

#The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Office upon request and payment of the necessary fee.

#FIGS. 1A-F are photographs showing the morphological changes of rat pancreatic tumor duct cells (ARIP) and human pancreatic tumor duct cells (PANC-1) cells treated with GLP-1. ARIP and PANC-1 cells (transfected with human IDX-1 or solely with a neomycin-resistant gene) were cultured in serum-free medium with or without GLP-1 (10 nM) for 72 h, and stained with hematoxylin and eosin. FIGS. 1A and 1B show ARIP cells cultured with vehicle (FIG. 1A) or with medium containing GLP-1 (FIG. 1B). FIGS. 1E and F show PANC-1 cells cultured in the absence (FIG. 1C) or in the presence (FIG. 1D) of GLP-1.

#FIGS. 2A-H are photographs showing the immunocytochemistry for insulin of ARIP and PANC-1 cells treated with or without GLP-1. ARIP and PANC-1 cells (transfected with human IDX-1 or solely with a neomycin-resistant gene) were cultured with or without GLP-1 (10 nM) for 72 h and subjected to immunostaining with an anti-human insulin antibody. FIGS. 2A and 2B show ARIP cells cultured in serum-free medium in the absence (FIG. 2A) or presence (FIG. 2B) of GLP-1. FIGS. 2C and 2D show non-transfected PANC-1 cells cultured in the absence (FIG. 2C) or presence (FIG. 2D) of GLP-1. FIGS. 2E and 2F show PANC-1/IDX-1 cells cultured in the absence (FIG. 2E) or presence (FIG. 2F) of GLP-1. PANC-1/IDX-1 cells, treated with GLP-1 (10 nM for 72 h), and stained solely with secondary antibody, were used as a negative control (FIG. 2G). Rat pancreas was used as a positive control (FIG. 2H). All photographs in FIG. 2 depict their subject magnified 200 times.

#The present invention is based on the inventor's discovery that the treatment of pancreatic cells with the gastrointestinal incretin hormone glucagon-like peptide-1 (GLP-1) promotes the differentiation of such cells into pancreatic M-like cells; and that, before this can happen, GLP-1 requires the gene expression of the islet differentiation factor IDX-1 to exert its differentiation-promoting activity.

#The method of the invention comprises the steps of providing pancreatic cells, transfecting the cells with DNA or mRNA that leads to the expression of IDX-1, and then culturing the cells in human GLP-1.

#Any pancreatic cell may be used in the method of the invention, including cells of the islet of Langerhans (such as A, B, D, and F islet cells), epithelial cells of intralobular ducts, acinar cells, centroacinar cells, basket cells, or any other pancreatic exocrine cells—whether they secrete insulin or digestive enzymes—and their associated ducts. In a preferred embodiment of the invention, the cells are obtained from a cell line. As used herein, “cell line” has the usual meaning: a cell culture of a uniform population of cells. Any cell line of pancreatic cells may be used in the method of the invention, including, for example, AsPc-1, AR42J, BxPc-3, Capan-1, Capan-2, Colo357, HPAC, HPAF, HPAF-II, Hs766T, human insulinoma cell line, MiaPaCa, MIAPaCa-2, PANC-1, Panc89, QGP-1, S2CP9, T3M4, or any other pancreatic cell line whether derived from human, rat, or other animal. Use of cells from PANC-1 (human pancreas tumor cell line) is especially preferred.