Isolation and differentiation of c-kit+ cardiac stem cells from adult mice

WEN Dezhong; ZHOU Min; XUE Song; JIANG Aixia; HUANG Ritai

doi:10.3969/j.issn.1002-266X.2017.07.005

Abstract：Objective To isolate the c-kit + stem cells from the hearts of adult mice,and to explore the induced differentiation of cardiac stem cells (CSC).Methods The freshly isolated hearts were obtained from 3 male C57BL/6 mice,and the hearts were dissected into single cells.The c-kit + stem cells were isolated from the CD45-cells by magnetic microbeads.Flow cytometric analysis and Immunofluorescence were used to measure the purity and the phenotype of c-kit + cells.The cell purity was determined and cell phenotype was observed.The c-kit + cells were cultured in suspension medi um to form the CardioStem sphere.RT-PCR was used to analyze the expression of the smooth muscle cells marker (α-SMA and sm-22α),endothelial cells marker (vWF),myocardial cells marker (cTnT) and cardiac stem cells marker (c-kit) after differentiation.Immunofluorescence was used to measure the phenotypes after the differentiation.Results The purity of c-kit + reached 89.3％,and the cardiac stem cells expressed the c-kit antibody.The primary cells grew slowly,but faster after being passaged.When cultured in suspension,the cells can form the CardioStem sphere.The expression of α-SMA,sm-22α,vWF,and cTnT mRNA was significantly increased,but the expression of c-kit mRNA was significantly decreased on day 14 as compared with that on day 0 after differentiation (all P ＜ 0.05).cTnT-positive cells were observed in cardiomyocyte-induced differentiation medium for about 14 days in cardiac c-kit + stem cells.vWF positive cells and α-SMA positive cells were observed in the medium without LIF on day 14.Conclusion Highly purified c-kit + CSCs can be isolated and cultured from the hearts of adult C57BL mice,c-kit + CSCs can be differentiated into cardiomyocytes,smooth muscle cells,and endothelial cells.