[Bio-characteristics of c-kit+ human amniotic fluid-derived mesenchymal stem cells and their differentiation into cardiomyocytes in vitro].

OBJECTIVE

To investigate the bio-characteristics of c-kit+ human amniotic fluid-derived mesenchymal stem cells (HAFMSCs) and its capacity to differentiate into cardiomyocytes in vitro.

METHODS

Fifty samples of human amniotic fluid were obtained from amniocenteses or voluntary termination of pregnancy and were expanded in vitro. c-kit+ HAFMSCs were sorted by flow cytometry and were recultured in the same media. c-kit+ HAFMSCs were amplified and identified, then exposed to osteogenic, adipogenic, and myogenic media. The flow cytometry, and immunocytochemistry were used for identifying the cell phenotype, von Kossa staining for osteogenic differentiation, oil red O staining for adipogenic differentiation, and real-time fluorescent quantitative PCR for the expressions of NKx2.5, Tbx5, GATA-4, and alpha-MHC genes.

RESULTS

After the selection procedure, the percentage of c-kit+ HAFMSCs was 3.07% +/- 1.03% of the total adherent cells. The cells expressed MSCs markers (CD29, CD44, CD73, CD90, CD105), and did not express hematopoietic stem cells markers (CD34, CD45). The cells were positive for human leukocyte antigen (HLA)-ABC, and negative for HLA-DR. They also expressed Oct-4, which characterized the undifferentiated stem cell state. The growth curves of c-kit+ HAMFSCs at passages 5, 10, and 15 were similar. Lipid droplet was observed by oil red O staining and calcium deposition by von Kossa staining in the cells at 21 days after adipogenic and osteogenic induction. The myocardium special gene expressions of Tbx5, Nkx2.5, GATA-4, and alpha-MHC were significantly stronger after myogenic induction than those before myogenic induction (P < 0.05).

CONCLUSION

Selected c-kit+ HAFMSCs by flow cytometry is a group of pure MSCs, which has potential to differentiate into cardiomyocytes and can be used as seeding cells for myocardium regeneration treatment.