人类单倍体原始生殖细胞的获得及其分化机制研究

Primordial germ cells (PGCs) are the precursor cells of gametes, while human PGCs derived from stem cells can’t form authentic gametes because they lack the ability to initiate meiosis in vitro. Haploid stem cells are cells with only one set of genomes and the diploidy phenomenon can occur in the course of culturing them. Our study has found that the addition of six micromolecule inhibitors to the culture medium of human haploid embryonic stem cells (haESCs) is beneficial to the maintenance of haESCs haploidy. So we hypothesize that increased pluripotency might improve mitotic delays in haploid cells and thus facilitate haploidy’s maintenance, and stable haESCs can develop into haploid primordial germ cells (haPGCs) and eventually develop into oocytes, thus crossing the meiotic "barrier" and forming fertile gametes. The purpose of this study is to research the phenomenon of haESCs diploidy and the molecular mechanism of PGCs differentiation through the process of haESCs differentiation to haPGCs at the molecular, cellular and embryo levels. In this study, the molecular mechanism of PGCs differentiation is explored from the new perspective of acquiring haPGCs by inhibiting diploidy and taking advantage of the haPGCs to form fertile gametes by crossing meiosis, which can provide valuable resources for the research on the molecular mechanism of germ cell development.

原始生殖细胞（PGCs）是配子的前体细胞，而干细胞来源的人类PGCs由于在体外不能起始减数分裂而无法形成真正意义上的配子。单倍体干细胞是只有一套基因组的细胞，培养过程中有二倍化现象发生。我们研究发现：在人类单倍体胚胎干细胞（haESCs）的培养液中加入六种小分子抑制剂能够有利于haESCs单倍性的维持。因此我们提出假说：多能性的增加可能会改善单倍体细胞有丝分裂的延迟现象进而有利于单倍性的维持，单倍性稳定的haESCs可以分化到单倍体原始生殖细胞(haPGCs)再到卵母细胞，从而跨越减数分裂的“屏障”形成可受精的配子。本研究拟从分子、细胞和胚胎水平，利用haESCs分化haPGCs的过程探讨二倍化现象及PGCs分化的分子机制。本研究将从抑制二倍化获得haPGCs并利用haPGCs跨越减数分裂形成可受精配子这个新视点探讨PGCs分化的分子机制，为生殖细胞发育分子机制研究提宝贵资源。

单倍体胚胎干细胞（haESCs）已在许多物种中建立，并被广泛应用于正向和反向遗传筛选。哺乳动物中缺乏分化的单倍体细胞系类型，这是由于分化过程中自发的二倍体化阻碍了谱系特异性筛选。人类胚胎干细胞广泛应用于基础和临床前研究。在立项初期我们原计划利用优化的培养体系建立人类单倍体原始生殖细胞，为了研究课题的丰富性我们首先摸索培养分化体系建立人单倍体神经干细胞。在这项工作中，我们报告了在扩展多能培养基中的人类haNSC显示出更紧密的集落、更高的神经分化效率和更高的单倍体维持稳定性，这有效地促进了haNSC的衍生。人单倍体神经干细胞（haNSCs）可在体外诱导分化，并长期保持单倍体性和向神经元、神经胶质细胞分化的多能性。PiggyBac转染后，在haNSC基因组中有多个插入位点，且插入位点均匀分布在所有染色体上。这是第一个具有完整基因组、增殖能力和神经分化潜能的人类单倍体体细胞系，为隐性遗传和药物靶向筛选提供了细胞资源。