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Young Sun Hwang1,5, Shinnosuke Suzuki2,5, Yasunari Seita1,3,5, Jumpei Ito4, Yuka Sakata1, Hirofumi Aso4, Kei Sato4, Brian P. Hermann2 & Kotaro Sasaki1,*
1Institute for Regenerative Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 2Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.
3Bell Research Center for Reproductive Health and Cancer, Nagoya, Aichi, Japan.
4Division of Systems Virology, Department of infectious Disease Control, International Research Center for infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo 1088639, Japan.
5These authors contributed equally: Young Sun Hwang, Shinnosuke Suzuki, Yasunari Seita.
*Corresponding author
Abstract
Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.
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