Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers
Mutsumi Kobayashi, Mutsumi Kobayashi, Misato Kobayashi, Misato Kobayashi, Junko Odajima, Keiko Shioda, Young Sun Hwang, Kotaro Sasaki, Pranam Chatterjee, Christian Kramme, Richie E. Kohman, George M. Church, Amanda R. Loehr, Robert S. Weiss, Harald Jüppner, Joanna J. Gell, Ching C. Lau, Toshi Shioda
Abstract
In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.