Ideniification and isolation of pancreatic stem cells

朗格汉斯胰岛的胰岛β细胞是唯一的产生胰岛素的细胞，但它们的再生能力有限。最近，胰腺或胚胎干细胞被提议作为β细胞替代的细胞来源。此外，最近的研究表明，骨髓、脂肪组织、胎盘、肝脏和小肠等几种组织是胰岛素产生细胞的候选来源。然而，这些干细胞/祖细胞向胰岛素分泌细胞转分化的分子机制尚不清楚。AR42J细胞来源于化学诱导的大鼠胰腺癌，已被用作胰腺转分化的模型。在激活素-A和β细胞蛋白或HGF的联合处理后，细胞转化为胰岛素产生细胞。在这项研究中，转录因子，如pdx-1，reg1，reg3α和foxa2，已知与胰腺发育…有关。用AR42J细胞分析更多的发育和分化。激活素-A和肝细胞生长因子联合作用后，AR42J细胞Reg3Foxa2mRNA表达增加，α表达降低。结果提示，诱导分化为胰岛素分泌细胞是诱导分化为胰岛素分泌细胞的前提。间充质干细胞在体外培养一段时间后，可形成形态特征与类胚体相似的球体，其标志物表达与类胚体相似。人骨髓间充质干细胞长期培养和传代次数增加会引起细胞形态和表型的改变，并导致细胞衰老和分化能力丧失。该培养条件保持了人骨髓间充质干细胞的纺锤形成纤维细胞形态和未成熟表型。用含2 mM他胺、10 mM烟酰胺、25 mM葡萄糖、2 nM激活素A、4 nMβ细胞球蛋白的DMEM/F12无血清培养基加G27诱导hMSC分化为胰岛素表达细胞。在培养条件下，部分纺锤形的hMSC形成球体和簇状结构。在培养过程中，除尘器的大小增加了。免疫荧光法检测胰岛标志物胰岛素和GLUT-2的表达。簇中的细胞对这些标记呈强阳性。在未经处理的对照组中，极少数细胞也呈胰岛素阳性，但阳性细胞比在诱导培养液中培养的细胞少得多，而且荧光强度很低。我们已经证明，在没有血清的情况下，hMSC在激活素-A、β-细胞蛋白和葡萄糖浓度的共同作用下分化为胰岛素表达细胞。较少

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 … More 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