Study on biological characteristics of adult adipose-derived mesenchymal stem cells and its differentiation into cardiomyocytes in vitro

Objective To isolate and culture adult adipose-derived mesenchymal stem cells(ADMSCs) in vitro,observe their biological characteristics,and to induce them differentiate into cardiomyocytes,so as to provide a good source of stem cells for myocardial regeneration.Methods ADMSCs were isolated with collagenase digestion and cultured from adult adipose tissue in vitro.The morphological characteristics of cells were observed by inverted phase contrast microscope.The expressions of CD29,CD31,CD34,CD44 and cell cycles were detected by flow cytometry.MTT colorimetry was used to investigate cell growth curve.The third-generation AMDSCs were induced and examined for outcomes of differentiation with various concentrations of 5-azacytidine(5-Aza,1,3,5,10,15 and 20 μmol/L) and various duration(12,24,48 and 72 h).The optimal concentration(10 μmol/L) and duration(24 h) were used in another experiment.At 7,14,21 and 28 days after induction,expressions of α-Sarcomeric Actin,myosin heavy chain(MHC) and Troponin I(cTn1) in rabbit cardiomyocytes were detected by immunocytochemistry.The expression of ANP was detected by RT-PCR.Results The ADMSCs were fibroblast-like spindle cells,with round or oval nuclei and occasional dual-nuclei.The morphology of passage cells were similar to that of primary cells,arranged a certain of direction.Expression of cell surface antigens,CD29 and CD44,were negative for weak five positively in the 1st,3rd and 5th passage cells,but CD31 expression were negative.The expression of CD34 was weakly positive in the 1st and 3rd passage cells,and were examined with colorimetry.The cell growth curve showed that a latency from days 1 to 3,logarithmic phase on day 4 and a peak on day 10.The cell cycle studies revealed that there were 85.93% of the cells in G0/G1,7.24% in S and 6.83 % in G2/M.α-Sarcomeric Actin,myosin heavy chain and Troponin I were positively expressed 28 d after induction by immunocytochemistry.The 14th days NKX2.5 were positive by RT-PCR.Conclusion ADMSCs can be isolated from adult adipose tissue and cultured in vitro,and induced by 5-Aza to differentiate into cardiomyocytes.ADMSCs might be a potential source of cell transplantation for myocardial and may offer ideal seed cells for tissue engineering.