Evaluation About Differentiation Ability Of CD73 Positive Subpopulation Of Adipose Tissue-derived Mesenchymal Stem Cells

BackgroundCardiomyocyte 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. 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.ObjectivesThe two subpopulations, CD73+ADMSC and CD73-ADMSC, were sorted by flow cytometer. Then systematic and extensive parallel comparison between the two subpopulations about differentiation into cardiomyocytes, adipocytes and osteoblasts had been done in vitro to select advantage subpopulation of myocardial differentiation and provide experimental reference for cell-based therapy.Methods1. Using flow cytometry to separate CD73+/CD73 ADMSCs subgroups, their shape were detected by HE dyeing. Then the expression of mesenchymal stem cell surface markers of CD73, CD90 and CD 105 were detected by immunofluorescence chemical method.2. After ADMSCs subgroups induced by 5-aza, the expression of c-TnT (myocardial specificity protein myocardial troponin) was detected by immunofluorescent chemical method, and using real-time PCR to check c-TnT and Gata-4 gene level changes.3. After fat inducers induced CD73 +/- ADMSCs subgroup, adipocyte differentiation ability of subgroups was evaluated by oil red O detection and spectrophotometer testing.4. After osteogenesis inducers induced CD73 +/- ADMSCs subgroup, osteoblast differentiation was tested by alizarin red, and using real-time PCR to confirm OST and RUNX2 gene level changes.Results1. CD73+subset were mainly small cells, possessed stellate cells, triangle cells, slender and spindle cells, and expressed CD73, CD 105 and CD90. While CD73’ ADMSCs were dubbed big cells. In CD73 negative cells the expression of CD 105 and CD90 were partially positive and they lost CD73 expression.2. After myocardium nduction, the express rate of c-TnT is 45.6% in CD73+ ADMSCs, but in the CD73- subgroup is only 5.3%. CD73+ subsets was higher than negative subgroup in expression of TnT, Gata-4 gene (P< 0.01).3. After induction into fat cells, oil red O staining showed positive in CD73+ and CD73 "ADMSCs, there was no statistically significant difference (P> 0.05).4. After the osteoblast inducing, alizarin red staining showed positive in positive and negative subgroups, and there was no statistically significant difference about bone cells’ specificity gene OST and RUNX2 (P> 0.05).Conclusions1. CD73+ subset were mainly small cells, possessed stellate cells, triangle cells, slender and spindle cells, and expressed CD73, CD105 and CD90. While CD73- ADMSCswere dubbed big cells. In CD73 negative cells the expression of CD 105 and CD90 were partially positive and they lost CD73 expression.2. The two subpopulations were differentiated into adipocytes, osteoblasts and cardiomyocytesin vitro, with expression of a variety of cardiac-specific genes (e.g., GATA4 and cTNT) and osteogenic genes (OST and RUNX2). The percentage of cardiomyocyte transdifferentiation in CD73 positive cells was obviously higher than that in CD73 negative subset in vitro.In contrast, quantitative analysis revealed there were no differences about adipogenic and osteogenic differentiation between CD73+subpopulation and CD73- cells. All results demonstrated that CD73+ cell possessed preferable therapy effect for heart injury.