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

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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.