Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 Japan.
Endocrinology. 2012 Sep;153(9):4336-45. doi: 10.1210/en.2012-1060. Epub 2012 Jul 9.
Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3'-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3',5'-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells.
虽然已经有报道称间充质干细胞和小鼠胚胎干细胞（ES 细胞）可分化为产生甾体类的细胞，但人 ES/诱导多能干细胞（iPS 细胞）分化为产生甾体类的细胞尚未见报道。本研究旨在建立诱导人 ES/iPS 细胞分化为产生甾体类的细胞的方法。我们首先尝试的方法是胚状体形成和进一步在贴壁板上培养。所得分化细胞表达编码甾体生成酶的 mRNA，包括类固醇急性调节蛋白、3β-羟类固醇脱氢酶、细胞色素 P450 酶（CYP）-11A1、CYP17A1 和 CYP19，并且在细胞培养基中检测到孕激素的分泌。然而，也检测到人绒毛膜促性腺激素的表达，提示分化细胞类似于滋养层细胞。我们接下来尝试了一种多步方法。第一步，人 ES/iPS 细胞被诱导分化为中胚层谱系。在 6-溴靛玉红-3'-肟（糖原合酶激酶-3β抑制剂）诱导分化 7 天后，人 ES/iPS 细胞已分化为表达胎肝激酶-1 和血小板衍生生长因子受体-α的中胚层谱系细胞。第二步，将编码甾体生成因子-1 的质粒 DNA 导入这些中胚层细胞，后者是甾体生成的主要调节因子。甾体生成因子-1 的强制表达和随后添加 8-溴腺苷 3'，5'-环单磷酸诱导中胚层细胞分化为甾体生成细胞谱系，并检测到 CYP21A2 和 CYP11B1 的表达，以及类固醇急性调节蛋白、3β-羟类固醇脱氢酶、CYP11A1 和 CYP17A1。此外，在培养基中检测到皮质醇的分泌，但未检测到人绒毛膜促性腺激素。这些发现表明，通过所述多步方法获得的产生甾体类的细胞不同于滋养层细胞，而是表现出肾上腺皮质细胞的特征。
