Human Amniotic Fluid-derived C-kit~+Mesenchymal Stem Cells Growth Characteristics And Myocardial Differentiation Capacities In Vitro

Objective To investigate the biocharacteristics and myocardium differentiation capacityof human amniotic fluid-derived c-kit~+and c-kit-stem cells in vitro. MethodsThirteen samples of human amniotic fluid are routinely obtained from amniocenteses ofsecond trimester gestation (16-31weeks). C-kit~+amniotic fluid stem cells (AFSCs）were sorted by flow cytometer and were compared with c-kit-stem cells ofbiocharacteristics and adipogenic, osteogenic and myocardium differentiation capacities.Verification and differentiation was evaluated using RT-PCR，real-time PCR and Westonblot. Result Our fingdings revealed that the16to22weeks of gestation was the optimalduration to obtain c-kit~+stem cells. Following the c-kit sorting, c-kit~+cells had a similarmorphological and proliferative characteristics with the c-kit-AFSCs as demonstratedby growth curves. Both the c-kit~+and c-kit-AFSCs had mesenchymal stem cellscharacteristics through surface marker identification by flow cytometry,immunocytochemical analyses. They expressed mesenchymal stem cell markers（CD29,CD44, CD73, CD90, CD105）and not expressed hematopoietic stem cells makers(CD34, CD45). The cells were positive for Class I major histocompatibility (MHC)antigens (HLA-ABC), and negtive for MHC Class II (HLA-DR). Oct-4，Sox2andNanog were more positive expressed in c-kit~+AFSCs than in c-kit-AFSCs. Both twotypes of stem cells can differentiate along adipogenic and osteogenic lineages.However, the myocardium differentiation capacity is enhanced in c-kit~+AFSCs bydetecting the GATA-4, cTnT, α-actin, Cx43mRNA and protein expression throughreal-time PCR and Western blot analysis after myocardium induced. The c-kit-AFSCswere only detected with GATA-4, which investigated an early stage of immaturemyocardium like cells. Conclusion Both c-kit~+and c-kit-AFSCs have mesenchymalstem cells characteristics. They can differentiate along adipogenic and osteogeniclineages. The c-kit~+AFSCs could have potential clinical application for myogenesis in cardiac regenerative therapy.