Generation of human oogonia from induced pluripotent stem cells in culture.,Nature Protocols

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Generation of human oogonia from induced pluripotent stem cells in culture.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-03-30 , DOI: 10.1038/s41596-020-0297-5
Chika Yamashiro _{1,

2} , Kotaro Sasaki _{2,

3} , Shihori Yokobayashi _{2,

4} , Yoji Kojima _{2,

4} , Mitinori Saitou _{1,

2,

4}

Affiliation

The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air-liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming.

中文翻译：

从培养物中诱导的多能干细胞生成人卵菌。

人类生殖细胞谱系起源于人类原始生殖细胞（hPGC）。hPGC经历了全基因组的表观遗传重编程，并分别分化为卵母细胞或生殖细胞，卵母细胞或精原细胞的前体。在这里，我们描述了一种在体外将人类诱导的多能干细胞（hiPSC）分化成卵黄质的方案。使用激活素A和WNT途径激动剂将hiPSC诱导为中胚层样细胞（iMeLC）。iMeLC或与发散性信号抑制剂一起培养的hPSC被包括骨形态发生蛋白4在内的细胞因子诱导成漂浮聚集体中的hPGC样细胞（hPGCLC）。hPGCLC与小鼠胚胎卵巢体细胞聚集形成异源重组卵巢将其在气液界面条件下培养约4个月，以使hPGCLC分化为卵母细胞和卵母细胞的即时前体状态。迄今为止，这是唯一从hPGCLC产生卵烟的方法。该协议适用于研究hPGC规范和表观遗传重编程的机制。

更新日期：2020-03-30

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