Bai Jing, Wang Yiru, Liu Lifeng, Chen Jie, Wang Yu
Department of Cardiology, the First Affiliated Hospital of General Hospital of Chinese PLA, Beijing, 100048, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):152-7.
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.
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.
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).
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.
研究c-kit+人羊水来源间充质干细胞（HAFMSCs）的生物学特性及其体外分化为心肌细胞的能力。
从羊膜穿刺术或自愿终止妊娠中获取50份人羊水样本，并在体外进行扩增。通过流式细胞术分选c-kit+ HAFMSCs，并在相同培养基中重新培养。对c-kit+ HAFMSCs进行扩增和鉴定，然后将其置于成骨、成脂和成肌培养基中。采用流式细胞术和免疫细胞化学鉴定细胞表型，用von Kossa染色鉴定成骨分化，油红O染色鉴定成脂分化，实时荧光定量PCR检测NKx2.5、Tbx5、GATA-4和α-MHC基因的表达。
经过筛选程序后，c-kit+ HAFMSCs占总贴壁细胞的百分比为3.07%±1.03%。这些细胞表达间充质干细胞标志物（CD29、CD44、CD73、CD90、CD105），不表达造血干细胞标志物（CD34、CD45）。细胞人白细胞抗原（HLA）-ABC呈阳性，HLA-DR呈阴性。它们还表达Oct-4，这表征了未分化的干细胞状态。第5、10和15代c-kit+ HAMFSCs的生长曲线相似。在成脂和成骨诱导21天后，油红O染色观察到细胞内有脂滴，von Kossa染色观察到钙沉积。成肌诱导后，Tbx5、Nkx2.5、GATA-4和α-MHC的心肌特异性基因表达明显强于成肌诱导前（P<0.05）。
通过流式细胞术筛选的c-kit+ HAFMSCs是一组纯间充质干细胞，具有分化为心肌细胞的潜力，可作为心肌再生治疗的种子细胞。
