Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Jingmei Hou,Zheng Li,Zuping He,Xiaobo Wang,Yang Liu,Shi Yang,Linhong Liu,Minghui Niu,Min Sun,Qingqing Yuan,Wenxian Zeng,Zijue Zhu

doi:10.1038/srep16922

Abstract

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

Highlights

Spermatogonial stem cells (SSCs) are a subpopulation of type A spermatogonia
Human male germ cells were separated from testicular tissues of obstructive azoospermic (OA) patients using two enzymatic digestions and followed by differential plating (Fig. 1A), and they were transfected with lentivirus called LentiEF1α -SV40LargeT-IRES-eGFP (Fig. 1B) by polybrene
The expression of SV40 large T antigen was driven by the promoter of EF1α, and eGFP was utilized as a reporter gene

Summary

Introduction

Spermatogonial stem cells (SSCs) are a subpopulation of type A spermatogonia. Studies on SSCs are of unusual significance in view of their unique characteristics[1]. We have recently shown that human SSCs can be induced to differentiate into haploid spermatids with fertilization and developmental capacity[7], reflecting that they can provide mature and functional male gametes for azoospermic patients with SSCs. Lastly and more importantly, a number of studies have demonstrated that SSCs can acquire pluripotency to become embryonic stem (ES)-like cells that are able to differentiate into all cell lineages of three germ cell layers[8,9,10,11,12,13]. Molecular, and functional assays in vitro and in vivo revealed that this cell line was human SSCs without Y chromosome microdeletions of numerous genes or tumor formation and it could be expanded with significant increases of cell number in vitro for over one and half years and colonized in vivo in the recipient mice. Our ability of establishing human SSC line could offer an unlimited cell source of human SSCs for their basic studies and great applications in regenerative and reproductive medicine

Methods

Results

Conclusion