Study Of Cardiomyocyte Differentiation From Adipose-derived Stromal Cells

Stem cell therapy has great development potential in clinical application study of cardiovascular disease. Although some experimental results in vivo and in vitro are encouraging, the best source of stem cells and the molecular mechanism of stem cells differentiation into cardiomyocyte remain to be further studied and confirmed. Adipose-derived stromal cells (ADSCs) have attracted a wide spread attention of stem cell research field because of its abundance, easiness to collect and culture, less damage to the donor, multi-lineage differentiation potential and advantages of autologous transplantation. Although some researches have confirmed that ADSCs can differentiate into cardiomyocyte directly, the potential verified is limited to rabbits, mice and human currently, and the mechanism of ADSCs differentiation into cardiomyocyte is still unclear.In this study, ADSCs were isolated from subcutaneous adipose tissue located in inguinal of 6-8 weeks old mice, and cultured in DMEM medium supplied only with 20% newborn bovine serum (NBS), penicillin and streptomycin. We detected the expression of surface markers such as CD29、CD31、CD34and CD105 in ADSCs by flow cytometry, and analyzed the process of spontaneously differentiation by microscope. After differentiation, we analyzed the expression ofα-actin、MF20、Connexin45、cMHC、cTnI、Nkx2.5 and GATA4 by immunofluorescence staining ,immunohistostaining and flow cytometry; the results of immunofluorescence staining and Ca2+ imaging showed that E-C coupling became unlocked in ADSCs-derived cardiomyocyte. Then we cloned the CDS of cardiacα-actin and Nkx2.5 by Touchdown-PCR, and constructed pAd-Nkx2.5 and pAd-α-actin, which were used to infect primary culture mice ADSCs. We detected the efficiency of over-expression and cardiomyocyte markers by Real time-PCRf and Western Blot, and investigated the influence of over-expression of cardiacα-actin and Nkx2.5 on the differentiation of mice ADSCs into cardiomyocyte, the main results are as follows:1. Primary cultured mice ADSCs express CD29 and CD105, but not CD31 or CD34. They can differentiate into cardiomyocyte spontaneously under worse condition in vitro (DMEM+20% NBS, without any cytokines, 5-azacytidine or antioxidants), and these cells can contract spontaneously, express cardiomyocyte marker such asα-actin, MF20, Connexin45, cMHC, cTnI, Nkx2.5 and GATA4, what’s more, E-C coupling is unblocked in the contracted cells and obtain the characteristics of Ca2 + transients.2. In the process of ADSCs differentiation into cardiomyocyte, over-expression of Nkx2.5 andα-cardiac actin improved the expression of cardiomyocyte proteins (including GATA4、cTnI、Connexin45 and DHPR/RYR2), however, the contracted cardiomyocyte were reduced, which may due to the up-regulation ofβ-MHC.In addition, we isolated SVF from subcutaneous adipose tissue located in inguinal of 18-day old SD rat, and induced with 5-azacytidine and semi-solid methyl cellulose medium to differentiate into cardiomyocyte. After induction, we analyzed expression of cardiomyocyte specific genes such as GATA4, MEF-2C,α- MHC,β-MHC,α-cardiac actin, cTnT and ANP by PCR, and detected expression ofα-actin、MF20、Connexin45、cMHC、cTnI and Nkx2.5 by cell immuno chemical staining, cyto-immunofluorescence staining, immunohistochemistry and flow cytometry. The results of cyto-immunofluorescence staining and Ca2+ imaging proved that the E-C coupling in SVF-derived cardiomyocyte is unblocked. Then, ROCK specific inhibitor Y-27632 was added into the culture to block Rho/ROCK signal pathway, we detected the change of actin cytoskeleton before and after SVF differentiate into cardiomyocyte by Actin-Tracker Green staining. What’s more, the key kinase ROCK in the signal pathway of Rho/ROCK which plays an important role in regulating cytoskeleton was detected by Western Blot, and also three signal pathways related to cytoskeleton downstream of ROCK (JNK、p38 and Akt) . In a word, we studied the mechanism of cytoskeleton-related signal pathways in regulating rat ADSCs differentiate into cardiomyocyte. the main results are as follows:1. 5-azacytidine started the process of rat ADSCs differentiation into cardiomyocyte. ADSCs expressed some cardiomyocyte markers such as Desmin,α-actinin,α-actin, MF20 and Connexin45 after induction, and formed myotube-like structure but without spontaneous contraction.2. Rat SVF differentiated into contracting cardiomyocyte when cultured in semisolid methylcellulose medium in vitro, and these cells expressed cardiomyocyte-specific mRNA(GATA4, MEF-2C,α- MHC,β-MHC,α-cardiac actin, cTnT and ANP) and proteins (MF20, Connexin45, Nkx2.5, cTnI, cMHC andα-actin), and showed characteristics of Ca2+ transient.3. Cytoskeleton plays a very important role in the process of rat SVF differentiation into cardiomyocytes. Inhibition of cytoskeleton-related signal pathway ROCK reduced the expression of cardiomyocyte-marked proteins and the number of contracting cardiomyocytes, which may due to the inhabitation of JNKMAPK-phosphorylation, and improvement of p38MAPK- phosphorylation and Akt- phosphorylation.