Endosialin defines human stem Leydig cells with regenerative potential

Abstract

STUDY QUESTION Is endosialin a specific marker of human stem Leydig cells (SLCs) with the ability to differentiate into testosterone-producing Leydig cells (LCs) in vitro and in vivo? SUMMARY ANSWER Endosialin is a specific marker of human SLCs which differentiate into testosterone-producing LCs in vitro and in vivo. WHAT IS KNOWN ALREADY Human SLCs have been identified and isolated using the marker platelet-derived growth factor receptor α (PDGFRα) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mistakenly sorted as SLCs if PDGFRα or NGFR was used as a marker for human SLCs isolation. STUDY DESIGN, SIZE, DURATION Firstly, we re-evaluated the specificity of PDGFRα and NGFR for SLCs in adult human testes. Then we analysed the previously published single-cell sequencing data and found that endosialin may identify human SLCs. Subsequently, we sorted endosialin+ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialin+ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing medium. We next assessed the in vivo regenerative potential of human endosialin+ cells after xenotransplantation into the testes of immunodeficient mice. PARTICIPANTS/MATERIALS, SETTING, METHODS Single-cell sequencing analysis, immunofluorescence and flow cytometry were used to characterize human testis tissues. In vitro colony formation, multipotent differentiation (adipogenic, osteogenic and chondrogenic) and Leydig cell-lineage induction were used to assess stem cell activity. Xenotransplantation into 3-week-old immunodeficient mice was used to determine in vivo regenerative potential. Endpoint measures included testosterone measurements, cell proliferation, immunofluorescence, flow cytometry and quantitative RT–PCR. MAIN RESULTS AND THE ROLE OF CHANCE The results indicate that endosialin is a specific marker of SLCs compared with PDGFRα and NGFR. Additionally, endosialin+ cells isolated from human testes show extensive proliferation and differentiation potential in vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could differentiate into functional LCs that secreted testosterone in response to LH in a concentration-dependent manner in vitro. These self-renewal and differentiation properties reinforce the proposal that human testicular endosialin+ cells are SLCs. Furthermore, transplanted human endosialin+ cells appear to colonize the murine host testes, localize to peritubular and perivascular regions, proliferate measurably and differentiate partially into testosterone-producing LCs in vivo. LARGE SCALE DATA NA. LIMITATIONS, REASONS FOR CAUTION Owing to the difficulty in collecting human testis tissue, the sample size was limited. The functions of endosialin on SLCs need to be elucidated in future studies. WIDER IMPLICATIONS OF THE FINDINGS A discriminatory marker, endosialin, for human SLCs purification is a prerequisite to advance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism. STUDY FUNDING/COMPETING INTEREST(S) A.P.X. was supported by the National Key Research and Development Program of China (2017YFA0103802 and 2018YFA0107200). C.D. was supported by the National Natural Science Foundation of China (81971314) and the Natural Science Foundation of Guangdong Province, China (2018B030311039). The authors declare no conflict of interest.

Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

“…These characteristics are comparable with those of SLCs; thus, we investigated whether HTSCs have SLC properties by assessing the gene expression of SLC markers in isolated HTSCs. Human SLCs are positive for SF1, GATA-binding protein 4 (GATA4), STAR, and leukemia inhibitory factor receptor (LIFR) but negative for ALC markers, such as LHCGR and 3βHSD [ 18 , 19 , 32 ]. Consistent with this, HTSCs were positive for the SLC lineage marker (LIFR), a key transcription factor for LC development (GATA4), and steroidogenic markers (SF1 and STAR) but were negative for ALC markers (LHCGR and 3βHSD) (Fig.…”

“…D medium (consisting of PBGF-BB, LH, thyroid hormone, and IGF-1) was previously reported by Ge et al [31]. D medium is widely used to induce the differentiation of SLCs into LCs [5,18,19,[32][33][34][35]. F medium contains forskolin (FSK), which is a stimulator of adenylate cyclase that results in increased adenosine-3 0 ,5cyclic monophosphate (cAMP) production [36].…”

“…Recently, several studies have demonstrated that SLC transplantation is a promising therapy for male hypogonadism [ 5 , 18 , 19 ]. However, it has limitations in clinical use because of the lack of specific markers for isolation, the rare proportion of adult testes, and the necessity for supporting factors from other testicular somatic cells, such as Sertoli and peritubular myoid cells, that induce differentiation of SLCs into functional ALCs.…”

The mouse resource at National Resource Center for Mutant Mice

“…Besides broadly used to constitute cell-derived xenograft (CDX)and patient-derived xenograft (PDX)models (Yang et al 2019;Zheng et al 2020), NCG is also an ideal model for generation of human immune system (HIS), as sustainable long-term hematopoiesis of the engrafted human hematopoiesis stem cell (HSC) could be maintained in NCG. This model has been widely distributed over the world and applied in cancer research, regeneration medicine and infectious disease research (Cao et al 2017;He et al 2019;Lee et al 2017;Ludwik et al 2018;Poggio et al 2019;Wen et al 2018;Xia et al 2020), as well as drug efficacy evaluation in preclinical studies. Although the long-term engraftment of human immune cells, mainly the T cells, was achieved in NCG, other populations of immune cells were challenging to be reconstituted, largely due to the lack of essential cytokines or environment for proliferation, differentiation, and maturation of the human cells.…”

AAV-mediated gene therapy produces fertile offspring in theLhcgr-deficient mouse model of Leydig cell failure

“…Immunofluorescence staining was conducted as previously reported by our group 43 . The harvested testes were fixed with 4% PFA for 2 hours at 4 .…”