Novel methods for the induction on the differentiation of human amniotic stem cell to the pancreatic islet cells.

采用流式细胞仪从足月胎盘羊膜中分离人羊膜干细胞，成功建立了人羊膜干细胞系HAM-R1、HAM-R2和HAM-R3。用胚胎营养因子（ETFs）悬滴法从上述细胞系（HAM-R1）中分化出拟胚体。然后，通过灌流装置，从用添加ETFs的培养基培养的胚状体发育胚胎怪物。从拟胚体发育成胚状体的平均比例约为2%。胰岛原基从胚胎怪物中取出。用0.1%胰蛋白酶-0.02%EDTA/PBS（-）从去内脏原基中分离获得单细胞，并在添加15%FBS的DMEM/F12培养基中培养。选择分泌胰岛素的原代培养物，并用上述生长培养基培养。对于开放剂，胰岛素分泌量的增加与培养基中葡萄糖的量呈剂量依赖性。但到第6代，胰岛素分泌量逐渐减少。传12代时，未观察到胰岛素分泌。用注射器将细胞植入胶原海绵中进行胰岛重建。重组胰岛在40天内胰岛素分泌量与葡萄糖浓度呈剂量依赖性，随后胰岛素分泌量下降。

The HAM-R1,HAM-R2 and HAM-R3 cell lines were successfully established from human amniotic stem cell isolated by FACS from amnion of fullterm placenta. Embryoid body was differentiated from above cell line (HAM-R1) by hanging drop method using embryotrophic factors (ETFs). Then, embryonic monster was developed from embryoid body cultured with ETFs-supplemented medium by circumfusion apparatus. Average of the development of embryonic monster from the embryoid body was about 2%. The anlage of the pancreatic islets was eviscerated from the embryonic monster. The single cells were obtained from the eviscerated anlage by dissociation with 0.1%trypsin-0.02%EDTA/PBS(-) and were cultured in DMEM/F12 medium supplemented 15%FBS. Insulin secreted primary cultures were selected and cultured with above growth medium. For openers, the amount of insulin secretion was increased dose dependently on the amount of glucose in the medium. However the amount of insulin secretion was decreased gradually by the passage at 6th. At 12 passages, the insulin secretion was not observed. Whereas, the cells were put into the collagen sponge by syringe for the reconstruction of islets. The reconstructed islet was secreted insulin dose dependently to the glucose during 40 days, then insulin secretion was decreased.