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

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.

An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

“…Such an approach has the potential to provide an in vitro model of the human gonad. To date, several studies have used pluripotent cells to induce gonad-like cells: bipotential gonads ( Sepponen et al., 2017 ), Sertoli or Leydig cells ( Bucay et al., 2009 ; Buganim et al., 2012 ; Chen et al., 2019 ; Rodríguez Gutiérrez et al., 2018 ; Jadhav and Jameson 2011 ; Kjartansdóttir et al., 2015 ; Yang et al., 2015 ; Yang et al., 2017 ; Yazawa et al., 2006 ), and germ cell lineages ( Hayashi et al, 2011 ; Irie et al, 2015 ; Hikabe et al, 2016 ; Shlush et al., 2017 ; Yamashiro et al., 2018 ; Gell et al., 2020 ). Some have overexpressed transcription factors to directly reprogram mouse or human fibroblasts into Sertoli and Leydig-like cells ( Buganim et al., 2012 ; Jadhav and Jameson 2011 ; Yang et al., 2015 , 2017 ; Yazawa et al., 2006 ).…”

A Novel Organoid Model of In Vitro Spermatogenesis Using Human Induced Pluripotent Stem Cells

“…Leydig cells (hLCs) were differentiated using a previously described method, [28] with a modification. The original protocol yielded 41.5% Leydig cells, likely because hiPSCs were spontaneously differentiated for 2 days prior to directed differentiation, allowing for endodermal and ectodermal lineage acquisition in addition to mesenchymal.…”

“…To date the use of hiPSCs to generate testicular organoids has yet to be investigated, likely because methods for the derivation of the major cellular components of the testicular niche from hiPSCs have only recently been established. [27][28][29][30] Using these derivation strategies, we engineered and evaluated a novel model of hiPSC-derived testicular organoids. We assessed their ability to mature and re-organize into testicular-like tissues and support spermatogenesis using immunohistochemical staining techniques and real time quantitative polymerase chain reaction (RT-qPCR) assays.…”

Differentiation of Peritubular Myoid-Like Cells from Human Induced Pluripotent Stem Cells

“…Therefore, in this study we hypothesized that substituting PDGFBB in place of PDGFAA into an existing Leydig cell differentiation protocol from hiPSCs(Chen, et al, 2019) would be enough to promote their differentiation into PTMs. We use immunocytochemistry and reverse transcription quantitative polymerase chain reaction (RT-qPCR) to show that cells with PTM-like or Leydig-like cell phenotypes and gene expression profiles can be alternatively derived from hiPSCs through the actions of PDGFBB or PDGFAA signaling.…”