【human reproduction】内皮素-人睾丸间质干细胞标记物

翻译练习：

研究问题：内皮素能否成为具有体内外分化能力的睾丸间质干细胞（SLCs）特异性标记物？

研究结论：内皮素是具有体内外分化能力的睾丸间质干细胞特异性标记物

已知信息：血小板生长因子受体α（PDGFRα）或神经生长因子（NGFR）可用来鉴定人睾丸间质干细胞。然而，特异性并不高，因此，睾丸间质细胞和生殖细胞可被错误地认为是睾丸间质干细胞。

研究设计：首先，我们重新评估了PDGFRα及NGFR分离人睾丸间质干细胞的特异性。随后分析了前期发表的单细胞测序数据，发现内皮素可能成为鉴定人SLCs的标记物。因此，我们分离出内皮素阳性的细胞，并鉴定了其自我更新和多向分化能力。评估内皮素阳性的细胞体外分化为功能细胞的潜能，这些细胞通过分化诱导培养液刺激。之后，我们将内皮素阳性的细胞移植入免疫缺陷小鼠的睾丸中，以评估其体内再生潜能。

研究对象、材料，方法：采用单细胞测序分析，免疫荧光染色，流式细胞术鉴定人睾丸组织。体外克隆形成，多向分化能力，睾丸间质细胞诱导分化用来评估干细胞活力。3周龄免疫缺陷小鼠异种移植鉴定体内再生潜能。最终评估方法包括睾酮水平，细胞增殖，免疫荧光，流式及定量PCR。

主要结果：结果显示，与PDGFRα和NGFR相比，内皮素可作为SLCs特异性标记物。此外，内皮素阳性的细胞在体外显示出强大的增殖及分化潜能：这种自我更新能力是单个细胞形成球形克隆的能力。而且，这些细胞可在体外分化为功能睾丸间质细胞，这些细胞受LH作用以浓度依赖方式分泌睾酮。自我更新及分化能力进一步说明内皮素阳性的细胞为人睾丸间质干细胞。内皮素阳性的移植细胞定位在小鼠睾丸血管周区域，增殖并部分分化为分泌睾酮的睾丸间质细胞。

局限性：由于人睾丸组织采样困难，样本量受限。内皮素在睾丸间质干细胞中的作用还需深入研究。

结论的延伸意义：特异性标记物，内皮素作为SLCs分离纯化是进一步深入研究SLCs的先决条件，可进一步推动未来睾丸间质干细胞疗法在男性性腺功能减退中的临床应用。 

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 andin vivo.

WHAT IS KNOWN ALREADY: Human SLCs have been identified and isolated using the marker platelet-derived growth factor recep- tor a (PDGFRa) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mis- takenly sorted as SLCs if PDGFRa or NGFR was used as a marker for human SLCs isolation.

STUDY DESIGN, SIZE, DURATION: Firstly, we re-evaluated the specificity of PDGFRa 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 endosialinþ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialinþ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing me- dium. We next assessed the in vivo regenerative potential of human endosialinþ cells after xenotransplantation into the testes of immuno- deficient 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 chondro- genic) 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, immuno- fluorescence, 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 PDGFRa and NGFR. Additionally, endosialinþ cells isolated from human testes show extensive proliferation and differentiation potentialin vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could dif- ferentiate 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 endosialinþ cells are SLCs. Furthermore, transplanted human endosialinþ 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 ad- vance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism.