通过形成类胚体诱导人羊水多能干细胞向心肌细胞分化

Differentiation of Human Amniotic Fluid Stem Cells into Cardiomyocytes through Embryonic Body Formation

摘要:

由人羊水中分离羊水多能性干细胞, 通过形成类胚体诱导其向心肌细胞分化。取人羊水标本进行体外培养, 分离得到人羊水干细胞, 已连续传代培养至42代, 采用免疫细胞化学、RT-PCR和流式细胞仪技术对羊水干细胞的生物学特性进行检测。取10~15代羊水干细胞, 悬浮培养使其形成类胚体, 进而向心肌细胞诱导分化。培养的羊水干细胞呈成纤维样, 表达部分胚胎干细胞特异标志基因, 悬浮培养可形成类胚体。类胚体碱性磷酸酶(AP)检测呈阳性, 表达三胚层特异标志基因fgf5、ζ-globin和a-fetoprotein。羊水干细胞形成类胚体后进行诱导, 得到a-actin阳性细胞, 表达心肌细胞特异标志基因Tbx5、Nkx2.5、GATA4和a-MHC。试验结果表明, 从人羊水标本中可分离得到具有胚胎干细胞特性的细胞, 经初步检测确定为羊水干细胞, 并能通过形成类胚体诱导其向心肌细胞分化。

关键词:人, 羊水干细胞, 类胚体, 心肌细胞, 诱导分化

Abstract:

To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer. hASCs at 10~15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, ζ-globin and α-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing α-actin positive and Tbx5, Nkx2.5, GATA4 and α-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.

Key words:human, amniotic fluid stem cells, embryonic body, cardiomyocytes, differentiation