Ideniification and isolation of pancreatic stem cells

朗格汉斯胰岛的胰腺β细胞是唯一产生胰岛素的细胞，但它们的再生能力有限。最近，胰脏或胚胎干细胞被提议作为替代β细胞的细胞来源。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都是胰岛素产生细胞的候选来源。然而，这些干细胞/祖细胞转分化为胰岛素生成细胞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细胞素或HGF联合治疗后，这些细胞转化为产生胰岛素的细胞。本研究利用AR42J细胞分析了参与胰腺发育的转录因子，如PDX-1、Regl、Reg3 α和Foxa2等。激活素- a和HGF处理AR42J细胞后，Reg3 α mRNA表达升高，Foxa2 mRNA表达降低。结果提示，Reg3 α的诱导和Foxa2的抑制是诱导AR42J细胞向胰岛素生成细胞分化的前提。间充质干细胞（mesenchymal stem cells， MSC）在体外培养一定时间后形成球状体，其形态特征和标志物表达与胚状体相似。长期培养和增加MSC传代数导致细胞形态和表型变化，也导致细胞衰老和分化能力丧失。本研究采用batula方法，在明胶包被培养皿中添加bFGF的无血清培养基上培养人骨髓间充质干细胞（hMSC）。培养条件维持hMSC纺锤形成纤维细胞形态和未成熟表型。为了诱导hMSC中表达胰岛素的细胞，将细胞用含胺（2 mM）、烟酰胺（10 mM）、葡萄糖（25 mM）、激活素- a （2 nM）、β细胞素（4 nM）、n2 -补充剂和G27的无血清DMEM/F12培养基处理。在中等条件下，部分纺锤形hMSC形成球状和团簇。在培养过程中，粉尘的大小增加了。免疫荧光法检测胰岛标志物胰岛素和Glut-2的表达。这些标记物在细胞群中呈强阳性。在未处理的对照组中，发现有极少数细胞对胰岛素也呈阳性，但阳性细胞比在诱导培养基中培养的细胞少得多，荧光强度也很低。我们已经证明，hMSC通过联合激活素- a、β细胞素和葡萄糖浓度处理，在不使用血清的情况下分化为表达胰岛素的细胞。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