Differentiation of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells into Steroid-Producing Cells

Abstract

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.

Regulation of Steroidogenesis, Development, and Cell Differentiation by Steroidogenic Factor-1 and Liver Receptor Homolog-1

“…Our results also suggest that pluripotent stem cells can differentiate into steroidogenic cells by these factors after pre-differentiation into the mesodermal lineage. Consistent with this hypothesis, it was demonstrated by Sonoyama et al that human ES and iPS cells can differentiate into cortisol-producing cells by the expression of SF-1 only after pre-differentiation into mesodermal cells (Sonoyama et al, 2012). In addition, Jameson and colleague reported that pre-selection or EB formation is necessary to differentiate SF-1-introduced mouse ES cells into sex steroid-producing cells (Jadhav and Jameson, 2011).…”

Directed Mouse Embryonic Stem Cells into Leydig-Like Cells Rescue Testosterone-Deficient Male Rats In Vivo

“…Although several studies have attempted to differentiate stem cells, such as mesenchymal stem cells (MSCs) [13,14], ESCs [15][16][17] and induced pluripotent stem cells (iPSCs) [18], into steroid-producing cells, the efficiency and purity of the differentiating Leydig cells remain a barrier. These stem cells were induced to differentiate into steroid-producing cells through forced expression of steroidogenic factor-1 (SF-1), which is a transcriptional factor belonging to the nuclear receptor superfamily and is a tissue-specific regulator of the transcription of an array of genes that are involved in reproduction, steroidogenesis, and male sexual differentiation [19,20].…”

Generation of kidney tubular organoids from human pluripotent stem cells

“…Hence, KhES-1 cells were first differentiated into the cells of the primitive streak with a GSK-3β inhibitor (BIO, a WNT activator) (BIO(+); BIO-treated cultures) for 3 days, or without BIO (mock; BIO-untreated cultures) as a negative control (Fig. 1a)1429. Accordingly, the expression of the primitive streak marker BRACHURY 30 measured by real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was transiently increased, whereas the expression of the pluripotency marker OCT3/4 was down-regulated with BIO treatment (Fig.…”