| Budget Amount *help |
¥3,650,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Pancreatic beta-cells of the islets of Langerhans are the only insulin producing cells but they have a limited capacity for regeneration. Recently, the use for pancreatic or embryonic stem cells has been proposed as a source of cells for the beta-cell replacement. Furthermore, recent studies have suggested that several tissues such as bone marrow, adipose tissue, placenta, liver, and small intestine are candidates for source of insulin-producing cells. However, molecular mechanisms of transdifferentiation into the insulin-producing cells in those stem/progenitor cells have not been clear yet.
The AR42J cells that were derived from a chemically induced rat pancreatic carcinoma have been used as a model for pancreatic transdifferentiation. The cells convert into insulin-producing cells after treatments with a combination of activin-A and betacellulin or HGF.
In this study, transcription factors such as PDX-1, Regl, Reg3 α, and Foxa2 that have been known to be involved in the pancreatic dev
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elopment and differentiation were analyzed using AR42J cells. Increase in Reg3 α mRNA expression and decrease in Foxa2 mRNA expression were detected after the treatment of AR42J cells with activin-A and HGF. The results suggest that induction of Reg3 α and the suppression of Foxa2 is the prerequisite for the induction of differentiation of AR42J cells into insulin producing cells.
After growing in vitro for a certain culture period, mesenchymal stem cells (MSC) form spheroids with the similar morphological features and the marker expressions to the embryoid bodies. Long-term cultures and increased passage numbers of MSC resulted in the morphological and phenotypic change and caused also cell senescence and loss of differentiation capacity.
In this study, human bone marrow mesenchymal stem cells (hMSC) were cultured on gelatin coated dish containing serum-free medium supplemented with bFGF according to the method described by Battula. The culture condition maintains the spindle-shaped fibroblastic morphology and immature phenotype of hMSC. To induce the insulin-expressing cells from hMSC, the cells were treated with serum-free DMEM/F12 medium containing tamine (2 mM) , nicotinamide (10 mM) , glucose (25 mM) , activin-A (2 nM) , betacellulin (4 nM) , N2-supplement and G27. In the medium condition, some fraction of the spindle-shaped hMSC formed spheroids and clusters. The size of the dusters increased during the culture. Expression of Insulin and Glut-2, the islet markers, were confirmed by using immunofluorescence method. The cells in the clusters were strongly positive with these markers. In the non-treated control group, a very small fraction of cells were found also positive to insulin, however, the positive cells were much fewer than that of the cells cultured in the induction medium and fluorescence intensity was very low. We have demonstrated that hMSC differentiated into the insulin-expressing cells by the treatment of a combination with activin-A, betacellulin, and glucose concentrations without serum. Less
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