Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules

Abstract

Studies of human germ cell development are limited in large part by inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells (PSCs) in vitro, differentiation of human germ cells from PSCs in vivo has not been reported. Here, we tested whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem cells (iPSCs). For this purpose, we derived integration-free iPSCs via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell-specific mRNA, VASA (OSKMV). All iPSC lines met classic criteria of pluripotency. Moreover, global gene expression profiling did not distinguish large differences between undifferentiated OSKM and OSKMV iPSCs; however, some differences were observed in expression of pluripotency factors and germ cell-specific genes, and in epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPSCs directly into the seminiferous tubules of germ cell-depleted immunodeficient mice revealed divergent fates of iPSCs produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation may contribute to tools for genetic analysis of human germ cell development.

Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies

“…Because multiple germ cell-related genes, especially PRDM14 , are expressed in control female and X chromosome aneuploidy iPSCs prior to differentiation, we hypothesized that xenotransplantation of these cells into murine seminiferous tubules would enable germ cell development in vivo . Xenotransplantation was previously demonstrated to promote formation of GCLCs from male iPSCs including iPSCs derived from azoospermic men1920; similarly, there is evidence that mouse PGCs have the potential to develop and progress through spermatogenesis or oogenesis49. Nonetheless, while mouse XX germ cells can enter spermatogenesis, they do not progress through meiosis, indicating the necessity of Y-specific transcription for further spermatocyte maturation5051.…”

“…Induced pluripotent stem cells (iPSCs) offer a source of patient-specific stem cells that can be used to study germ cell development in vitro and in vivo 151617181920. Studies using mouse embryonic stem cells (mESCs) have revealed key transcription factors for germ cell specification21.…”

Fate of iPSCs Derived from Azoospermic and Fertile Men following Xenotransplantation to Murine Seminiferous Tubules

“…AZF2 iPSCs were derived from normal human fetal foreskin fibroblasts (Stemgent) as previously described ((Durruthy Durruthy et al, 2014)). Fibroblasts were allowed to expand to 80–90% confluency before passaging and cryopreservation.…”

Spermatogonial stem cells and spermatogenesis in mice, monkeys and men

“…Human and monkey SSCs do not regenerate complete spermatogenesis when transplanted into mouse testes. However, they do migrate to the seminiferous tubule basement membrane and produce chains or networks of spermatogonia that persist for many months after transplantation (Hermann et al, 2009; Valli et al, 2014; Izadyar et al, 2011; Zohni et al, 2012; Nagano et al, 2001; Nagano et al, 2002; Hermann et al, 2007; Wu et al, 2009; Sadri-Ardekani et al, 2009; Sadri-Ardekani et al, 2011; Dovey et al, 2013; Clark et al, 2017; Durruthy Durruthy et al, 2014; Ramathal et al, 2014). It is not currently possible to recapitulate complete spermatogenesis from monkey or human cells using the xenotransplantation assays.…”