组织细胞特异性转录因子直接转分化人脐带间充质干细胞为精原干细胞的研究

Male infertility caused by dyszoospermia is a serious reproductive health issue worldwide. Transplantation of spermatogonial stem cells (SSCs) is an optimal method for treating this disease, but a shortage of SSCs makes it impossible to use this method clinically. Recent studies have demonstrated that lineage-specific transcription factors could directly reprogram somatic cells into other lineages. Of them Bcl6b、Etv5、Lhx1、ID4、Plzf、Taf4b and Foxo1 are the key factors for maintaining development and function of SSCs. Our previous studies showed that human umbilical cord mesenchymal stem cells (HuMSCs) could differentiate into male germ-like cells but not enter meiosis. Our preliminary experiments found that the expression levels of Bcl6b, Etv5, Lhx1, ID4, Plzf, Taf4b and Foxo1 were significantly increased during the differentiation process of HuMSCs-derived iPS (induced pluripotent stem) cells into male germ-like cells, which suggested that such factors might associate with development of male germ cells. Furthermore, we successfully transfer the aforementioned factors into HuMSCs using electrotransfer method. In the current project, 1. RT-PCR, qRT-PCR, cellular immunofluorescence, bisulfite sequencing PCR, TRAP-PCR silver staining method to investigate the differentiation potential of HuMSCs into SSCs; 2. Gene expression chip, micoRNA array, comparative proteomics and the methods of gene overexpression and silencing are employed to screen the key regulators and the related signaling pathway, and to clarify their mechanism; the single intraperitoneal injection of busulfan, qRT-PCR and immunohistochemistry are carried out to explore the possibility that HuMSCs-derived SSCs develop into mature spermatozoa by assessing their survival, location and differentiation in testis. If the predicted results achieved, it would not only enrich basic theory of Andrology but also provide a new approach to treating male infertility caused by dyszoospermia, which has momentous theoretical significance and application prospect.

精原干细胞（SSCs）移植是治疗生精障碍的理想途径，但来源匮乏。研究发现：组织细胞特异性转录因子能直接转分化成体细胞为另一种细胞；Bcl6b等7个转录因子是SSCs发育的关键因子。我们已经证实HuMSCs能向生殖细胞分化；预实验发现Bcl6b等7因子在iPS细胞向男性生殖细胞分化中表达增高，可能介导生殖细胞的发育；预实验亦成功将Bcl6b等7因子导入HuMSCs中。在此基础上，本项目：1. 应用RT-PCR、qRT-PCR、细胞免疫荧光、BSP、TTRAP-PCR，研究HuMSCs转分化为SSCs的潜能；2. 应用表达谱芯片、micoRNA芯片、比较蛋白质组学、基因过表达和沉默技术，研究并阐明调控转分化的关键因子、通路和机制；3.移植HuMSCs源SSCs到睾丸中，检测细胞存活、定植、分化情况，探讨其分化为成熟精子的可能。研究将为精子发生提供新途径，为SSCs移植治疗男性不育症提供新方法。

研究发现组织细胞特异性转录因子能直接转分化成体细胞为另一种细胞。为解决生精障碍患者的生育需求，本课题将在SSCs发育和功能维持过程中发挥关键作用的转录因子（BCL6B、ETV5、LHX1、ID4、PLZF、TAF4B和FOXO1）分别或分组导入HuMSCs中，进行了一系列的研究，获得如下结果：.1.筛选出关键转录因子ID4、FOXO1，关键调控通路PI3K/Akt；.2.将ID4及FOXO1分别转入HuMSCs中，单细胞RNA测序及生信分析、特异性基因检测、流式细胞术及荧光原位杂交技术证实HuMSCs可成功向男性生殖细胞方向分化并突破减数分裂发育为男性单倍体生殖细胞；.3.基因过表达及siRNA干扰技术进一步确认ID4及FOXO1将HuMSCs直接转分化为男性单倍体生殖细胞可能是通过调控ID4-PI3K/Akt-FOXO1-PLZF信号通路实现；.4.BMP4诱导HuMSCs源iPSCs向男性生殖细胞方向分化的模型和HuMSCs细胞实验显示：ID4及FOXO1将HuMSCs直接转分化为男性单倍体生殖细胞与促进原始生殖细胞发育的关键基因SOX17高度相关；.5.男性自体单细胞生殖细胞的获得：本项目发现男性包皮可培养出大量间充质干细胞-包皮间充质干细胞（FKMSCs），其具备多向分化的潜能；ID4亦可成功将FKMSCs转分化为男性单倍体生殖细胞，为后续研究男性自体单倍体生殖细胞治疗生精障碍不育症奠定基础；.6.同时实验发现：HuMSCs及HuMSCs源胰岛细胞体内移植可改善T1D鼠的预后；HuMSCs通过抑制TNF-α、TGF-β/ERK1/2信号通路减轻DCM大鼠的心肌纤维化；CYGB-HuMSCs可通过抑制细胞凋亡及调节p38/MAPK通路改善HIBD大鼠的预后；.7.本项目在实施过程中申报相关发明专利3项。.本项目成功构建了转录因子ID4及FOXO1将HuMSCs和FKMSCs直接转分化为男性单倍体生殖细胞的技术体系，为男性生精障碍不育的治疗研究提供了有效的方法和手段；并初步阐明ID4及FOXO1将HuMSCs和FKMSCs直接转分化为男性单倍体生殖细胞可能是通过调控ID4-PI3K/Akt-FOXO1-PLZF信号通路实现的，还可能与SOX17高度相关。