Supplementary MaterialsSupplementary Information. adiposity and others.1 In 2010 2010, the European Male Ageing Study reported that 17.0% of men aged 40C79 Rabbit Polyclonal to IRAK1 (phospho-Ser376) years had serum testosterone levels below the normal values, indicating the high prevalence of hypogonadism among middle-aged and elderly males.2 Epidemiological studies suggest that hypogonadism not only adversely affects patients’ quality of life, but also increases the risk of diabetes, 3 arteriosclerosis4 and dementia.5 Exogenous testosterone supplementation has some beneficial effects, including the improvement of sexual function, muscle mass, bone density and body composition.6, 7 However, it disrupts the hypothalamicCpituitaryCtesticular axis, and comes with the risk of serious side effects, such as erythrocytosis, lipid metabolism disturbance, infertility and others.8 In addition, as physiological requirements of testosterone vary in individuals,9 it is difficult for exogenous testosterone supplementation to meet the requirements of individualized treatment. Therefore, it becomes necessary to explore a new therapy for testosterone supplementation in a physiological pattern. Theoretically, because they are the primary source of testosterone,10 Leydig cell (LC) transplantation is a physiological therapy which could provide long-lasting delivery of testosterone. However, L-2-Hydroxyglutaric acid LCs account for only about 2C4% of the total testicular cell population in adult human testes.11 Moreover, LCs are terminally differentiated cells with no proliferation capacity.10 Therefore, it is difficult to apply LC transplantation therapy directly in clinic. LCs arise from undifferentiated stem Leydig cells (SLCs), which first develop in the neonatal testicular interstitium.12, 13, 14, 15 SLCs undergo phased transitions through progenitor and immature stages, and ultimately to terminally differentiated adult LCs stage.16 Fully grown males maintain a population of SLCs residing in the peritubular layer17 and/or testicular vasculature.12, 18 Adult SLCs, which are normally dormant, can regenerate new LCs to replace senescent or injured ones, thereby contributing to the maintenance of testicular homeostasis.18, 19 Recent studies on rodent SLCs have demonstrated that transplanted SLCs could replace the chemically disrupted or senescent LCs for testosterone production,13, 14, 20 indicating that SLC transplantation is a promising therapy for hypogonadism. In 2014, Landreh expand human SLCs. Here, we evaluate the use of p75 as a cell surface marker for identifying L-2-Hydroxyglutaric acid and isolating SLCs from human testes, and also demonstrate the stem cell characteristics of p75+ cells. We then demonstrate that transplanted p75+ SLCs can restore testosterone L-2-Hydroxyglutaric acid production and promote the recovery of spermatogenesis in EDS-treated rats (Figure 1a). These results suggest that p75 may serve as a putative surface marker for human SLC identification and isolation. Subsequently, we isolated p75+ cells from human adult testes by flow cytometry (Figure 1b). The sorted p75+ cells were seeded in specific serum-free expansion medium. After 1 day of culture, most cells adhered to the plastic wells. When adherent cells had propagated to 80% confluence, we dissociated these cells using collagenase type IV and transferred them to a new plate for further expansion. The p75+ cells formed small spheres, which subsequently became floating spheres and showed proliferation ability (Figure 1c). Open in a separate window Figure 1 The identification and isolation of p75+ cells from adult human testes. (a) p75+ cells abundantly expressed nestin but only negligibly expressed HSD3(Figure 2a). This indicated that cultured p75+ cells maintained their SLC identity. To further investigate their self-renewal capacity, we carried out single-cell sphere formation assays in which single-cell suspensions derived from P1 cytospheres were seeded into 96-well plates. Seeded single cells divided and formed spheres after 11 days of culture (Figure 2b). The cytosphere with a diameter equal to or greater than 50?expansion capacity of the p75+ cells, the cytospheres were enzymatically disassociated into single cells by collagenase IV for cell counting before each passage. Importantly, the p75+ cells could continuously proliferate for at least eight passages and expand by about L-2-Hydroxyglutaric acid 5000 times (Figure 2c). Taken together, these results demonstrate that the p75+ cells can expand and have clonogenic self-renewal capacity. Open in a separate window Figure 2 The proliferation and self-renewal capacity of p75+ cells. (a) Immunostaining showed that cultured spheres of p75+ cells maintained the expression of p75, nestin, PDGFRand LIFR, but showed only negligible expression of LHR and HSD3differentiation L-2-Hydroxyglutaric acid capacity of the p75+ cells To verify their plasticity, we cultured the p75+ cells under conditions known to favor osteogenic, adipogenic or chondrogenic differentiation (Figure 3a)..