CD9 Is Expressed on Human Male Germ Cells That Have a Long-Term Repopulation Potential after Transplantation into Mouse Testes1

Abstract

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.

Spermatogonial stem cells and spermatogenesis in mice, monkeys and men

“…SSEA4 has not been used to isolate mouse SSCs, but is expressed by undifferentiated spermatogonia in monkey and human testes and has been used effectively to isolate transplantable SSCs (Zheng et al, 2014; Muller et al, 2008; Izadyar et al, 2011; Eildermann et al, 2012b; Smith et al, 2014). CD9 is expressed by a subpopulation of MAGEA4+ spermatogonia in human testes and can be used to isolate transplantable stem cells (Zohni et al, 2012). ITGA6, SSEA4 and CD9 are effective single markers for isolating primate spermatogonia because they clearly segregate the heterogeneous testis cell suspension into positive and negative fractions and have been tested functionally by xeno-transplantation into infertile mouse recipients.…”

“…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.…”

Evaluation of candidate spermatogonial markers ID4 and GPR125 in testes of adult human cadaveric organ donors

“…Furthermore, only a small number of markers are available to delineate the cell populations encompassing human SPG or SSCs, respectively (Waheeb and Hofmann, 2011). Xenotransplantation has been developed to assess SSC activity in human testicular cells (Nagano, et al, 2002; Zohni, et al, 2012). However, it remains quite unclear what fraction of germ cells in the adult human testis are SSCs.…”

“…Toward this end, a plethora of markers, either with internal or cell surface expression, have been developed for SSCs in model systems, such as the mouse; surface markers include Thy1, Itga6, CD9, Gfra1, and others (Nagano and Yeh, 2013). In humans, proposed SSC markers include CD9, GPR125, SSEA-4, ITGA6 (He, et al, 2010; Izadyar, et al, 2011; Zohni, et al, 2012). Only CD9 and SSEA-4 have been evaluated by xenotransplantation (Izadyar, et al, 2011; Zohni, et al, 2012).…”

Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside

“…Current SSC isolation methods are based on two-step enzymatic digestion [47]. Investigators have enriched human spermatogonial cells using magnetic activated cell sorting (MACS) with markers such as GFRA1 + [48], GPR125 + [49], SSEA4 + [50], and HLA-ABC − /CD9 + [51] or using fluorescence-activated cell sorting (FACS) by isolating EpCAM + /HLA-ABC − /CD49e − cells [52]. Ideally, isolation of pure SSCs is expected, but no specific marker has been found to identify the stem cells in testis [53].…”