Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale

作者：Xiaoman Wang, Tingting Liao, Cong Wan, Xiaoyu Yang, Jiexiang Zhao, Rui Fu, Zhaokai Yao, Yaping Huang, Yujia Shi, Gang Chang, Yi Zheng, Fang Luo, Zhaoting Liu, Yu Wang, Xinliang Mao, Xiaoyang Zhao · 发表于：PeerJ · 年份：2019 · DOI：10.7717/peerj.6143 · 被引用次数：16 · 研究领域：Pluripotent Stem Cells Research、Mesenchymal stem cell research、Reproductive Biology and Fertility

BACKGROUND: The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. METHODS: The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. RESULTS: In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found ...