Differentiation Of Porcine Amniotic Fluid Stem Cells Into The Cardiomyocytes And The Variation Of WNT Signaling Pathway Related Factors

It has been known that amniotic fluid stem cells (AFS) are able to differentiate into three germ layers in vitro. Myocardial cells derived from mesoderm, so AFS have the ablity to differentiate into cardiomyocytes. However, so far the induction effictiency of rhythmic contraction cell cluster derived from AFS are very low. Pig is a main domestic animal and has many biophysical and biochemical similarity to human beings. So the research on cardiomyocyte derived from poricine amniotic fluid stem cells (pAFS) is necessary and significant.1. Induction of cardiomyocyte differentiation from pAFSThe aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells (pAFS). For cardiac differentiation, the cells were formed embryoid bodies (EBs) firstly, then cultured in induced-medium including 5-azacytidine (5-aza) and vitamin C (Vc). We detected the specific markers of cardiomyocyte by immunocytochemistry, RT-PCR and transmission electron microscope. The results showed that some embryoid bodies beat rhythmically after 10 days of induction. Furthermore, analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest (33%) when induction using 0.1 mmol/L Vc and 5μmol/L 5-aza. Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressedα-actin, Tnni3. RT-PCR analysis also illustrated that TbX5, Gata4,α-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters. Especially, we observed basic structures of myocardium, such as myofilament, glycogen granule and so on by transmission electron microscope. In conclusion, 5- azacytidine and vitamin C could promote differentiation of pAFS into myocardium.2. Functional analysis of wnt/β-catenin signaling pathway on the cardiomyocytes differentiationIn order to learn the mechanism of cardiomyocyte derived pAFS, in this research, LiCl is an activator of Wnt/β-catenin signaling parth way, DKK-1 is an inhibitor, were used to treatment pAFS when pAFS were differentiating into cardiomyocyte.The results show that Wnt/β-catenin signaling parth way have the ablity to promote the differentiation of cardiomyocytes derived from pAFS to some extent.