Gata4, Tbx5and Baf60c Genes Modified CD73Positive Subpopulation Of Mouse ADMSCs For Differentiating Into Beating Cardiomyocytes

Cardiomyocyte transplantation and cardiac tissue engineering are emerging as an attractive new therapy for myocardial infarction and heart failure because of the limited regeneration capacity of the heart. Because of its unique advantages, the adipose tissue-derived mesenchymal stem cells (ADMSCs) has been one of the most promising stem cells in cardiac tissue engineering, regenerative medicine and cell therapy. Over the recent years, there have been many researches about ADMSCs. However, basic and clinical researches are still required. For example, the cultured ADMSCs in vitro is short of specific and unique markers; the relationship between cell senescence and culturing time of these cells(ADMSCs) remains elusive; there has been few researches about ultrastructure of ADMSCs; ADMSCs are a heterogeneous cell population and different subpopulations share similar but not identical biological features and differentiation potential, which limits the application effect of ADMSCs. Therefore, the isolation and research of the different subpopoulations of ADMSCs would have an important significance for clinical and academic study; ADMSCs can differentiate into cardiomyocyte, and it has been shown that overexpression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes would reprogram beating cardiomyocyte-like cells from ADMSCs has been unknown.The morphology characters, biological behaviour and differentiation potency of mice ADMSCs were cultured in vitro and detected; The cardiac differentiated potency about subpopulations of CD73+/-ADMSCs were evaluated; The key factors for cardiac function were probed by transfecting Gata4、Baf60cand Tbx5genes. These insights aim to provide an easily applicable quality control for cell model of heart tissue engineering and regenerative medicine, to probe the theoretical basis of studying the genetics of cardiomyocyte development and to increase effectiveness and standardization in the emerging field of cell-based therapy and regenerative medicine.The cultured ADMSCs were consistent with international accepted standard for MSCs. Morphous of mice ADMSCs which were cultured in vitro was diverse. ADMSCs expressed surface markers of MSCs such as CD73, CD90and CD105, and had pluripotent capacity to differentiate into osteocytes, adipocytes and cardiomyocytes in vitro.The cultured ADMSCs was consistent with international accepted standard for MSCs and could be used to experimental study.Index of cell senescence of ADMSCs was positively correlated with the number of passages and culturing time in vitro. Techniques for tracing cell growth curve, aging test, sorting cell generation cycle and electronic microscopy were used to definite optimal time and cultured stage of utilizing ADMSCs. The growth curve of P3ADMSCs was "S"-shaped. Index of cell senescence was positive correlation with culturing time in vitro. With increasing of passages, the percentage of ADMSCs in G0/G1raised also; in contrast, the percentage of ADMSCs in G2/M decreased, that means the speed of division growth was descent. TEM result showed that morphous of passage8cells was irregular, and there were few big protrusions and many microspikes on cell membrane; nucleus was located in the center or side of the cell and had little heterochromatin, and there were many lipid droplets, Golgi apparatus and autophagosomes in cytoplasm. The strongest stage of ADMSCs was at before passage8.The specific CD73+ADMSCs are an undifferentiated subpopulation and have high potential for differentiation toward cardiomyocytes. CD73+subset were mainly small cells, containing RS cells and SS cells, and were steadily positive for all three surface markers. In contrast, CD73-cells showed a homogeneous morphologic pattern with FC shapes, dubbed big cells and lost CD73expression and the expression of CD105and CD90were partially positive. Furthermore, compared to CD73"ADMSCs, nanog expression at the transcriptive and proteomic levels in CD73+ADMSCs was significantly higher. Two subpopulations can both be differentiated into cardiomyocytes, adipocytes and osteoblasts in vitro. Quantitative analysis revealed there were no differences about osteogenic and adipogenic differentiation between CD73+cells and CD73-cells. In contrast, the percentage of cardiomyocyte transdifferentiation in CD73+cells was obviously higher than that in CD73-subset in vitro. After transplanted into mice heart, the differentiation potency of two subpopulations were as same as in vitro. All results demonstrated that CD73+cell possessed preferable therapy effect for heart disease.Gata4. Tbx5and Baf60c modified CD73+ADMSCs for the expressions of cTnT and cardio-genes. Gata4, Baf60c and Tbx5over expression lentivirus were constructed successfully. Experiment groups:CD73+ADMSCs (Con); GFP-LV infected CD73+ADMSCs (NC); Tbx5-GFP-LV infected CD73+ADMSCs(OE1); Gata4-LV and Baf60c-LV infected CD73+ADMSCs(OE2); Gata4-LV, Baf60c-LV and Tbx5-GFP-LV infected CD73+ADMSCs (OE3). The techniques of cytochemistry and Real-time PCR showed the expression of Gata4and Baf60c ADMSCs were higher which proved that genes transfection were done well.After GTB was transfected with CD73+ADMSCs for2weeks, cTnT expressions in Con, NC and OE1were negative, but few fraction cells of OE2and OE3displayed cTnT. Fluorescence labeling of cells expressing cTnT displayed the morphology of myofilament. FCM result showed the expression rate of cTnT in OE3was5.71%. After transfecting GTB with CD73+ADMSCs, the expressions of cardio-genes and stem cell marker were as following:Nanog, stem cell marker, was positive in Con and NC, but negative in OE1, OE2and OE3, which means the ADMSCs has the differentiation tendency after transfecting the GTB. Nkx2.5, which is the pristine cardiac differentiation marker, was continually expressed in OE2and OE3only after GTB transduction7d. cTnT, Myh6and Actcl are terminal cardiac differentiation markers, which were expressed in OE3with lentivirus transduction14d, meanwhile the expressions of cTnT and Myh6were detected in OE2. Gata4, Tbx5and Baf60c genes modified CD73+ADMSCs for differentiating into beating cardiomyocytes. Beating cardiomyocytes appeared from day8post-transduction in OE3which always expressed GFP, while other groups didn’t display beating cells. The beating rate was from80to120per minute.Conclusions:ADMSCs possess pluripotent capacity to differentiate into osteocytes, adipocytes and cardiomyocytes in vitro. The specific CD73+ADMSCs are an undifferentiated subpopulation and have high potential for differentiation toward cardiomyocytes. Combination of Gata4, Tbx5and Baf60c produces the regulating mechanism of the adult stem cells differentiating toward cardiomyocyte. A forced expression of GTB in ADMSCs is sufficient to induce cardiac specification that leads to the formation of beating cardiomyocytes with very similar gene-expression and phenotypical characteristics. Gata4and Baf60c were key gene to myocardial differentiation and Tbx5was a kind of function gene determined cardiac cell beating.