Purification And Differentiation Of Human Mesenchymal Stem Cells In Vitro And Analysis Of Their Biological Characteristics

Objective To purify,expand hMSCs and investigate the process of differentiating them into cardiomyocytes in vitro directionally and indentify their characteristics, investigate differentiation and apoptosis during the process of differentiating hMSCs,detect electrophysiological properties during the process of differentiating,and provide experimental data for clinical transplant therapy of hMSCs.Methods (1)hMSCs were isolated and expanded as undifferentiated cells in culture until 4th passage,then were treated with 5μmol.l^-15-azacytidine for 24 hours and then cultured for 8 weeks,The differentiation of hMSCs was observed by Phase-Contrast microscope,Flow Cytometry, Immunohistochemistry. Membrane potential was observed in the whole-cell patch-clamp mode. (2)After being isolated and expanded, hMSCs were divided into 3 groups and treated with 0,2.5,5.0μmol.l^-15-azacytidine respectively for 24 h and then cultured for 8 weeks.The differentiation of hMSCs was observed by Flow Cytometry and RT-PCR technique.Results hMSCs had a spindle morphology,but formed a ball-like or strick-like morphology at 2 days after induction,1week later,approximately 30% of the cells gradually increased in size and granuies were seen concentrating in cells. ANP and CX43 began to express at 2nd week after induction and increased gradually,about 60% of the resulting myogenic cells were positive at 4th week after induction ,they were negative for uninduced cells.hMSCs'surface antigen profiles obtained by Flow Cytometry were negative for CD31\CD34\CD45 before and after induction,but CD90 expressed higher after induction while was weak positive before induction(P<0.05). Apotosis index was correlated with the cultural time after induction,The apoptosis rate of induced hMSCs was remarkably higher than control group(P<0.01),and the variation between groups was notable(P<0.05),the cell cycle analysis showed that the percentages of G₀/G₁phases were reduced significantly after induction. The expresstion levels ofβ-MHC and CTNT mRNA were undetectable before induction,began to increase at 1st,4th week after induction,reached the peak at 6th week and decreased after that,the expression of Bcl-2 and Bax mRNA varied regularly after treated with 5-azacytidine. hMSCs'resting membrane potential(RMP),range and rate of depolarization were heightened gradually after being induced.Conclusions hMSCs can express biological characteristics of cardiomyocytes phenotype cells after being purified,expanded,induced in vitro,but apoptosis is observed in the process of differentiation.Compared with nomal density of 5-azacytidine ,the lower one could reduce the ratio of apoptosis.hMSCs can provide reliable cells for clinical transplant therapy,but they may be the sources of unanticipated arrythmias after cell transplantation.