Characteristics Of Adult ADMSCs Versus EPCs From Adult Peripheral Blood Differentiating Into Cardiomyocyte-like Cells

Objective：To explore the method for isolation and cultivation of adipose tissue-derivedmesenchymal stem cells (ADMSCs) and endothelial progenitor cells (EPCs) from adult peripheralblood in vitro respectively. This study prove that ADMSCs can be differentiated intocardiomyocyte-like cells induced by5-azacytidine (5-aza) and explore the possibilities that EPCscan be differentiated into cardiomyocyte-like cells induced by5-aza. We observe the changes ofthe cardiac-specific transcription factor GATA-4and Nkx2.5expression during the study. Thecardiomyocyte differentiation potential of ADMSCs were compared with EPCs．We hope to finda new suitable source of seed stem cells and provide experimental foundation for stem cellstransplantation for treating coronary heart disease.Methods：(1)①Isolating and culturing ADMSCs：ADMSCs were separated from adultabdominal hypodermic adipose tissue with type I collagenase and cultured with adherent filtrationmethod.The cells in better growth state of3rd passage were used to detemine CD29，CD44，factorⅧ and HLA-DR with indirect immunocytochemistry， with the blank control of phosphatebuffer solution (PBS).②Inducing ADMSCs in vitro：The cells in better growth state of3rdpassage were used to be induced by5-aza，and then we identify the cardiac-specific surfacemarkers Desmin and troponin T (cTnT) in the induced cells with indirect immunocytochemistry.(2)①Isolating and culturing EPCs：The EPCs from adult peripheral blood were separated andcultured by the method of density gradient centrifugation and adherent filtration. Primary cellscultured for7days were taken to determine FITC-UEA-Ⅰand Dil-ac-LDL with double-immunofluorescence and CD133，CD34，KDR with immunocytochemistry.②Inducing EPCsin vitro：Primary cells cultured for7days were used to be induced by5-aza，and the inducedcells were taken to determine the cardiac-specific surface markers Desmin and cTnT with indirectimmunocytochemistry.(3) On the7th，14th，21th day，ADMSCs and EPCs induced weredetected the cardiac-specific transcription factor GATA-4and Nkx2.5expression during theinduction by PCR respectively. And both were compared. Result：(1) A large amount of long spindle mesenchymal stem cells were found in adultadipose tissue. And they were isolated and cultured in vitro easily. Indirect immunocytochemistryshows that most cells were CD29，CD44positive，factor Ⅷ，HLA-DR negative. ADMSCsinduced by5-aza became longer and bigger and changed in the long spindle for rod-like orpolygonal.The arrangement was the same direction gradually. During the late period，the cellsbecame spherical or irregular in shape.Desmin and cTnT were positive in these cells.(2) Theperipheral blood of adult contains EPCs. FITC-UEA-Ⅰ，Dil-ac-LDL，CD133，CD34and KDRin EPCs were all positive. EPCs induced by5-aza became longer and bigger.The arrangementwas the same direction gradually. Desmin and cTnT were positive in these cells.(3) The band ofPCR product showed positive expression of the gene of GATA-4and Nkx2.5in ADMSCs andEPCs induced groups. And the GATA-4and Nkx2.5relative expression levels increased alongwith time of cells being induced extension gradually.In the same induction period，the relativeexpression of myocardial specific gene in ADMSCs induced groups is higher than in the EPCsinduced groupsConclusion：(1). ADMSCs may differentiate into cardiomyocyte-like cells after beinginduced by5-aza.(2)EPCs may differentiate into cardiomyocyte-like cells after being induced by5-aza，but it’s very difficult.(3) ADMSCs induced by5-aza are much easier than EPCs.ADMSCs is a new suitable source of seed cells for stem cells transplantation in the treatment ofcoronary heart disease.