小分子定向诱导iPSC分化为Leydig细胞机理研究

Exogenous androgen replacement therapy is the traditional treatment of male hypogonadism. However, long-term hormone therapy can cause a lot of complications and cannot maintain the circadian rhythm secretion of testosterone. Stem cell transplantation in the treatment of androgen deficiency gradually attracts attention, but the donor cell source is limited and there is immune rejection. Using small molecules to drive the induced pluripotent stem cells (iPSC) into Leydig cells (LC) is a viable approach. Our preliminary data show that the human iPSC derived from urine cells via reprogramming can differentiate into LC using small molecules. In this project, we will induce GFP-fluorescent-tagged single-celled iPSC into adult LC (iPSC-ALC) using small molecules and manual enrichment culture, and identify them by characterizing the gene, protein expression, and function and purity analysis. Meanwhile, the uniformed iPSC-ALC will be purified using the flow cytometry separation and transplanted into the ethane-dimethanesulfonate (EDS)-induced ALC-depleted testosterone-deficient male rat or nude mouse testis and the efficacy will be evaluated. The results of the study will provide the scientific basis for the future clinical application of autologous iPSC-ALC for transplantation in the treatment of male hypogonadism. This will have a significant theoretical and clinical value.

外源性雄激素替代疗法是治疗男性性功能低下的传统治疗方法，但是长期激素治疗会引起很多并发症且不能维持雄激素昼夜节律分泌。干细胞移植治疗雄激素缺乏症逐渐引起关注，但是供体细胞来源有限且存在免疫排斥反应。自体可诱导多功能干细胞（iPSC）通过小分子诱导成睾丸间质细胞是可行的方法。预实验结果显示，人源尿液细胞重编程来源的可诱导多功能干细胞（iPSC）可以在小分子诱导下分化成Leydig细胞（LC）。本项目拟采用小分子诱导带GFP荧光标签的单细胞状态iPSC分化为功能的成熟LC（iPSC-ALC），手工富集培养后对其进行基因、蛋白表达和功能的鉴定以及纯度分析，同时利用流式细胞分选纯化iPSC-ALC，最后进行乙烷二甲烷硫砜（EDS）消除ALC的性腺功能低下的雄性大鼠模型（或裸鼠）细胞移植并评价效果。研究结果将为临床采用自体iPSC-ALC移植治疗男性性功能低下提供科学依据，具有重要理论和临床价值。

Leydig细胞（LCs）在睾酮产生中发挥重要作用, 睾酮对调控男性生殖和发育至关重要。睾酮水平低会导致男性性腺机能减退。LC移植是治疗男性性腺功能减退的一种很有前途的替代疗法。然而, LCs的来源限制了该策略的临床应用。到目前为止, 已有报道通过基因转染诱导干细胞分化为LCs, 但安全和有效的诱导方法尚未见报道。在此, 我们报道了一种基于分子化合物的新型分化方法可以将人源可诱导多能干细胞 (iPSCs)诱导分化为Leydig样细胞。可诱导多能干细胞iPSC分化来源的Leydig样细胞 (iPSC-LCs)具备了睾酮合成功能, 有LCs类似的基因表达谱,能阳性表达睾丸间质细胞特异性标志蛋白质: LHCGR,STAR,SCARB1, SF-1, CYP11A1, HSD3B1和HSD17B3, 以及阴性表达iPSC特异性标记物: NANOG, OCT4和SOX2。将亲脂性红染料 (PKH26)标记的iPSC-LCs移植到大鼠睾丸中, 用二甲基磺酸乙酯EDS (75 mg/kg)选择性去除内源性的LCs, 移植后的iPSC-LCs在睾丸间质中存活并发挥作用, 加速血清睾酮水平和睾丸重量的恢复。总之, 这些结果表明, iPSCs能通过特定的分子化合物诱导分化为Leydig样细胞, 这将为进一步临床应用iPSC-LCs治疗性腺功能减退症奠定了安全的基础。