Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells

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.

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program

“…Accordingly, hPSCs are induced into cells bearing properties similar to human primordial germ cells (hPGCs) ( 8 , 9 ), the founding population of the human germ-cell lineage that eventually gives rise to either spermatozoa or oocytes. The induced hPGC-like cells (hPGCLCs) are further differentiated into oogonia/early oocyte-like cells with appropriate epigenetic reprogramming in a reconstituted ovary culture ( 10 , 11 ), or into pro-spermatogonia-like cells in a reconstituted testis culture ( 12 ). Although further reconstitution of human germ-cell development remains a key challenge, these advances recapitulate a period of more than 10 wk of human germ-cell development, leading to a number of key findings with regard to the mechanism of human germ-cell development in general, and germ-cell specification in particular ( 8 , 9 , 13 , 14 , 15 , 16 , 17 ).…”

Endogenous retrovirus rewired the gene regulatory network shared between primordial germ cells and naïve pluripotent cells in hominoids

“…To characterize the transcriptome similarity between PGCs and naïve pluripotent cells, we compared the transcriptome signatures of in vitro -derived human PGCs (PGC-like cells; PGCLCs) and naïve embryonic stem cells (ESCs). We analyzed single-cell RNA sequencing (scRNA-Seq) datasets for in vitro -derived human male germ cells [Hwang et al ( 7 )] and for naïve and primed ESCs [Messmer et al ( 27 )] ( Fig. 1A ).…”

“…The Hwang et al dataset contains information for germ cells that were sequentially differentiated from primed iPSCs: incipient mesoderm-like cells (iMeLCs), PGCLCs, multiplying prospermatogonia-like cells (MLCs), and mitotically quiescent T1 prospermatogonia-like cells (T1LCs), which are formed via transitional cells (TCs) ( Fig. 1A ) ( 7 ). Dimension reduction analysis suggested that the global transcriptome is highly similar between PGCLCs and naïve ESCs, consistent with previous reports ( Fig.…”

“…(A) Dimension reduction analysis of scRNA-Seq data using UMAP ( 51 ). Data for in vitro -derived human male germline development [Hwang et al ( 7 )] and for naïve and primed ESCs [Messmer et al ( 27 )] were integrated and subsequently used. The 3,000 protein-coding genes that were the most differentially expressed among cells were used.…”

“…However, it is ethically challenging to routinely access human germ cells, particularly those from humans at early stages of development. Recent studies have established methodologies to derive human germ cells such as PGCs or more differentiated cells from human induced pluripotent stem cells (iPSCs) ( 4–7 ). These methods have enabled us to characterize the mechanisms of human germ cell development in detail.…”