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

Young Sun Hwang,Kei Sato,Yuka Sakata,Brian P Hermann,Hirofumi Aso,Jumpei Ito,Kotaro Sasaki,Yasunari Seita,Shinnosuke Suzuki

doi:10.1038/s41467-020-19350-3

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.

Highlights

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility
The germline is established as primordial germ cells (PGCs), and undergoes a complex cascade of developmental processes, that result in the formation of either spermatozoa or oocytes, depending on the sex-specific signals provided by the gonad[1]
To validate our in vitro reconstitution of human male GC development, we first required a precise understanding of the lineage trajectory of male GCs in vivo

Summary

Introduction

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. This system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. After the fetus is born, T1 migrate from the center of the seminiferous cords towards the periphery, which is the site of the spermatogonial stem cell (SSC) niche[7,8,9] As they migrate, T1 proliferate and differentiate into secondary transitional (T2)prospermatogonia (T2). Such spatiotemporal heterogeneity is unique to primates and suggests species-specific divergence of prospermatogonial specification

Methods

Results

Conclusion