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

PeerJ

article

Xiaoman Wang¹  Tingting Liao⁶  Cong Wan²  Xiaoyu Yang⁷  Jiexiang Zhao²  Rui Fu¹  Zhaokai Yao²  Yaping Huang²  Yujia Shi²  Gang Chang⁸  Yi Zheng²  Fang Luo²  Zhaoting Liu²  Yu Wang¹  Xinliang Mao⁹  Xiao-Yang Zhao² 

[1] State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences;Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University;College of Life Sciences, University of the Chinese Academy of Sciences;Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, Southern Medical University;Guangzhou Regenerative Medicine and Health Guangdong Laboratory;Reproductive Medicine Center, Xiangya hospital, Central South University;State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University;Department of Biochemistry and Molecular Biology, Shenzhen University Health Science Center;School of Food Science and Engineering, South China University of Technology

关键词: Human primordial germ-like cells;    Induction system;    Methylcellulose;    hESCs;    hiPSCs;    Embryoids;   

DOI  :  10.7717/peerj.6143

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

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 that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. Discussion The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.

【 授权许可】

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