Differentiation of human induced pluripotent stem cells into Leydig-like cells with molecular compounds

Xianwu Chen,Zhao Han,Shenzhi Zhao,Leikai Ma,Haitao Xi,Xiaoling Guo,Junzhao Zhao,Yong Chen,Zhangye Xu,Chao Li,Renshan Ge

doi:10.1038/s41419-019-1461-0

Abstract

Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.

Highlights

Leydig cells (LCs), which reside in the testis interstitium, were first identified in 1850 by Franz Leydig, and the name Leydig cells was coined after him
Differentiation of induced pluripotent stem cells (iPSCs) into Leydig-like cells Because a prerequisite for iPSC differentiation is the shutdown of the self-renewal machinery, iPSCs were pretreated in E7 medium for 2 days to encourage the spontaneous differentiation[25]
The necessity and optimum dose of all defined molecular compounds, which were required to induce the differentiation of iPSCs into Leydig-like cells, were screened and analyzed by comparing the secreted testosterone levels of iPSC-LCs, which were induced using different molecular compound combinations (Table S3 and S4). iPSCs often showed adherent clonal growth, and the clones gradually became larger with time

Summary

Introduction

Leydig cells (LCs), which reside in the testis interstitium, were first identified in 1850 by Franz Leydig, and the name Leydig cells was coined after him. Eutherian mammals develop at least two types of LCs: fetal Leydig cells and adult Leydig cells (ALCs) in the fetal and adult testis, respectively[1]. Male hypogonadism is a symptomatic clinical syndrome caused by testosterone deficiency, which is characterized by mood disturbance and fatigue, sexual dysfunction, decreased muscle mass and strength, decreased lean body mass and bone mineral density, and increased visceral fat[8,9,10]. These changes can be partially overcome by exogenous testosterone replacement therapy[11,12].

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