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Insulin releasing cells derived from embryonic stem cells and new semipermeable membranes for bioartificial pancreas.

来自胚胎干细胞的胰岛素释放细胞和用于生物人工胰腺的新型半透膜。

基本信息

批准号：
14207048
负责人：
IWATA Hiroo
金额：
$ 34.36万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2005
项目状态：
已结题

项目摘要

Dopaminergic Neurons Derived from ES Cells : Neural inducing factors (NIFs) produced by washing PA6 cells were collected. ES cells enclosed in semipermeable membranes could be efficiently differentiated to dopaminergic neurons using medium containing NIFs. β-tubulin type III positive cells and tyrosine hydroxylase positive cells were clearly observed in ES cell progenies after 16 days culture, and dopamine release were seen when cells were exposed to 56 mM KCl for 15 mm through deporization of neurons. Differentiation of ES cells in semipermeable membranes is one of crucially important procedures when cell therapy of Parkinson's disease will be realized.Insulin Releasing Cells Derived from ES Cells : Insulin releasing ES cell progeny was derived in culture media with/without fetal calf serum by the 4 step procedure. Cells of which cytoplasm was stained by the anti-insulin antibody and by anti-C-peptide antibodies were obtained. Those cells can be subculture 9 times. The results suggest that a sufficient number of insulin releasing cells could be obtained from ES cells for the cell transplantation therapy of diabetic patients.Membrane for Immunoisolation : An amino groups-terminated poly (ethylene glycol)-conjugated phospholipids (PEG-lipids, Mw : 5000) spontaneously formed a thin layer on cells, when they were added to cells suspension. The layer-by-layer membrane could be further formed on the PEG-lipid layer through the polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly-L-lysine. Cells could be microencapsulated by this method without volume increase.

ES细胞衍生多巴胺能神经元：收集PA6细胞洗涤后产生的神经诱导因子（Neural inducing factors, nif）。在含有nif的培养基中，半透膜包裹的胚胎干细胞可以有效地分化为多巴胺能神经元。培养16天后，胚胎干细胞子代中明显可见β-微管蛋白III型阳性细胞和酪氨酸羟化酶阳性细胞，当细胞暴露于56 mM KCl中15 mM时，通过神经元脱porization可以观察到多巴胺的释放。胚胎干细胞在半透膜中的分化是实现帕金森病细胞治疗的关键步骤之一。从胚胎干细胞中获得胰岛素释放细胞：通过4步程序在含/不含胎牛血清的培养基中获得释放胰岛素的胚胎干细胞后代。获得细胞质经抗胰岛素抗体和抗c肽抗体染色的细胞。这些细胞可以传代培养9次。提示胚胎干细胞可获得足够数量的胰岛素释放细胞，用于糖尿病患者的细胞移植治疗。免疫隔离膜：端氨基聚乙二醇偶联磷脂（PEG-lipids，分子量：5000）加入细胞悬浮液时，在细胞上自发形成薄层。通过聚乙二醇链末端的氨基与海藻酸钠和聚l -赖氨酸形成多离子络合物，进一步在聚乙二醇脂质层上形成一层一层的膜。该方法可以在不增加细胞体积的情况下实现细胞微囊化。