Differentiation Of Stem Cells Into Islet-Like Clusters In Vitro

Objective To study and to optimize culture conditions of islet-like cells induced in vitro by fetus bone marrow mesenchymal stem cells (MSCs), fetus pancreatic stem cells (PSCs) and human embryonic stem cells(hESs) from somatic cell nuclear transfer (SCNT), and to provide suitable "seed" cells for replacement therapy of diabetes mellitus.Methods (1) Bone marrow(BM) was obtained from the femurs and tibias of human spontaneous abortion fetus. BM cells (1×10~6/ml) were cultured (37℃, 5% CO2) in plastic flasks with DMEM medium containing 10% FBS(fetal bovine serum). 48h later, total media in the culture was changed. Following three to four passages, the cells were used to the differentiation into islet-like clusters in DMEM containing 2-mercaptoethanol, EGF, bFGF and nicotinamide through three stages. (2) In the same time, pancreatic stem cells were obtained from the fetal pancreas by the digestion, and then cultured in plates with DMEM containing 10% FBS and 1XB27. 48h later, total media in the culture was changed with DMEM-F12 medium containing 2%FBS and 1XB27. Two weeks later, adherent cells gaining 80% confluence were passaged every five days. Surprisingly, PSCs generated both neural and pancreatic phenotype in the serum -free media conditions supplement with ITS-A and EGF. In addition, PSCs were used to the orient differentiation into islet-like cells with EGF, bFGF, B27 and nicotinamide through two stages. (3) SCNT-hESs were obtained from Shandong research center of stem cell engineering and induced to differentiate into islet-like clusters according to the six stages: stage 1: undifferentiated hES grown on MEF; stage 2: generation of EBs supplemented with15%FBS in suspension(6d); stage 3: plating EBs in ITS-A and fibronectin medium (6d); stage 4: expansion of pancreatic progenitor cells in N2 and B27 supplemented with bFGF; stage 5: withdrawal of bFGF and addition of nicotinamide; stage 6 maturation in suspension. Then to identificate all the cells with dithizone staining, immunofluorescence together with the specific insulin secretion from extracellular or intracellular.Results (1)BM cells attached to the surface of plastic flasks with spindle-shaped or fibrocyte-like morpho -logy within 24h. After 8-9d, adherent cells gaining 80% confluence were passaged every three days.The doubling time of hMSCs was about 30h. During the course of differentiation , MSCs changed rapidly into...