不同表面标记物人羊水干细胞对其分化为雌性生殖细胞的影响

Female germ cells could be derived from stem cells in vitro, which was helpful for research on human oogenesis. Previous study showed that the CD117-positive human amniotic fluid stem cells (hAFSCs) were induced to differentiate into primordial germ cells by adding porcine follicle fluid. Therefore, we speculated that the cell surface marker could be involved in the germ cells differentiation in vitro. In this study, the hAFSCs cell lines were isolated by different cell surface markers (CD117, CD29, and CD38), and infected with lentiviruses expressing STRA8 gene promoter which drive GFP expression. Female germ cell-like cells (GCLCs) were induced by adding human follicular fluid, cytokines and small molecules. while the characteristic and functions of female GCLCs would be screened out by the following criteria: expression of surface markers, expression of GFP protein, single cell transcriptome profile, methylation of imprinting genes, the level of germ cell protein, transplanting into kidney capsule and intracytoplasmic sperm injection. In conclusion, female germ cells in vitro differentiation system were established by specific stem cells and cytokines optimization, which will provide a new way for female infertility treatment.

干细胞体外定向诱导分化为雌性生殖细胞，在研究人卵母细胞的发生机制方面具有重要意义。前期研究证明，CD117阳性人羊水干细胞（human Amniotic fluid stem cells，hAFSCs）在猪卵泡液的诱导下能分化为原始生殖细胞，推测表面标记物可能参与了该分化过程。本研究通过分离具有不同表面标记物（CD117、CD29和CD38）的hAFSCs，用减数分裂关键基因STRA8启动子驱动的GFP慢病毒感染后，添加人卵泡液和细胞因子、小分子化合物对其进行诱导，以期获得雌性生殖细胞样细胞。通过对表面标记、GFP的启动、单细胞转录表达谱、印记基因甲基化、蛋白表达水平、体内移植分化、胞浆内单精子注射后是否发生卵裂等指标的鉴定，筛选出适于向雌性生殖细胞诱导分化的hAFSCs系，并建立稳定的雌性生殖细胞体外分化系统，为不孕患者的治疗提供了新的研究途径。

人多能干细胞在体外特定条件下能定向诱导分化为雌性生殖细胞，对研究人卵母细胞的发生机制具有重要意义。本研究采用了人的羊水来源的成体干干细胞，诱导多能细胞和胚胎干细胞作为种子细胞，来源于猪和牛的卵泡液、小分子化合物及细胞因子的组合作为诱导剂，对干细胞进行定向分化。结果显示，上述干细胞在给定条件下都能分化为卵母细胞，但后两者的分化效率更高，能清晰地观察到雌性生殖细胞的发育进程。在牛卵泡液的作用下，hPSCs首先在原始生殖细胞培养基中培养形成诱导性原始生殖细胞(iPGCs)，关键转录因子SOX17、PRDM1、TFAP2C在iPGCs中表达，同时RNA-Seq数据显示Hippo, TGF-beta和经典Wnt信号通路参与了早期原始生殖细胞的形成。将iPGCs在卵泡样细胞培养基中继续培养，形成诱导卵泡样结构(iFLs)，将iFLs收集后用卵母细胞成熟培养基培养成熟后。可发现GV期和MⅡ期卵母细胞，而且部分OLCs可自发形成孤雌胚胎。虽然体外受精的潜力尚未被证实，但是来源于hPSCs的OLCs为研究人类生殖细胞的形成和卵发生提供了新的手段。