利用miRNAs诱导小鼠iPS细胞形成精原干细胞样细胞

The induction of germ cells from iPS cells (iPSCs) is highly valuable both theoretically and practically. Researchers have been able to induce mouse iPSCs or ESCs into functional oocytes and spermatids. However, the induced spermatids are still quite different from mature sperms in terms of both morphology and function. My team has long been engaged in the in vitro propagation and differentiation of mouse spermatogonial stem cells (SSCs), and is able to amplify SSCs in large quantities for long terms. We have also induced mouse iPSCs into SSC-like cells (SSCLCs). We have profiled the expression of small RNAs in mouse spermatogenic cells and found that miR-202 is specifically expressed in SSCs and plays an essential role in maintaining SSC self-renewal. In the present application, we will first explore whether we are able to induce mouse iPSCs into SSCLCs that are highly self-renewable by overexpressing miR-202 and/or other miRNAs, and study the underlining mechanism. We will then try to finish the same induction using synthetic miRNA mimics. The accomplishment of this work will contribute to establishing a solid foundation whereby fully functional sperms can be derived.

将iPS细胞（iPSCs）诱导成生殖细胞具有重要的理论和应用价值。近来人们已经能够将小鼠的iPSCs或ESCs诱导形成具有功能的卵母细胞和精子细胞；但是所形成的精子细胞在形态和功能上与成熟的精子尚有较大差异。申请人团队能够将小鼠的精原干细胞（SSCs）在体外长期大量扩增并诱导其启动减数分裂；我们还成功地将小鼠iPSCs诱导成具有有限增殖能力的SSC-like细胞（SSCLCs）；系统地检测了小分子RNAs在生精细胞中的表达，发现miR-202在SSCs中高度表达并维持SSCs的自我更新。在本申请中，我们将首先探索是否可以通过表达外源miR-202和/或其他miRNAs来诱导小鼠iPSCs形成具有长期自我更新能力的SSCLCs，并对SSCLCs的功能进行严格鉴定；然后我们将尝试利用人工合成的miRNA类似物来实现该诱导过程。这些工作的完成将为我们最终在体外获得功能完全正常的精子奠定基础。

近年来人们已经可以将小鼠的多能干细胞或者胚胎干细胞诱导形成有功能的卵母细胞和精子细胞，但所形成的精子细胞在形态和功能上与成熟的精子尚有较大差异。本项目目标为建立稳定的培养平台和可靠的诱导体系，并通过建立报告细胞系统追踪分化路径，最终获得正常的生殖细胞。miRNAs作为一类在进化上保守的小RNAs通过序列匹配调控多个目标RNAs的稳定性和翻译过程，近年来有很多miRNA被报道在iPS形成、分化与转分化等过程发挥重要功能，具有很高的潜在应用价值。miR-202是一个雄性生殖系统特异表达的miRNA，其可能在多能干细胞向生殖细胞分化中行使功能。我们首先首先使用miR-202敲除小鼠探索了其在精子发生中的功能。我们发现miR-202能够防止过早的精原细胞分化与减数分裂起始，这一功能是通过调控DMRT6的表达实现的，miR-202/STRA8/DMRT6是减数分裂起始调控网络的一个节点。为了建立可靠培养体系，我们先后建立了Naïve与Formative胚胎干细胞的培养体系，并证明Naïve胚胎干细胞具有分化至原始生殖干细胞的能力；进一步我们建立了生殖细胞报告系统，为进一步获得生殖细胞奠定了良好的基础。