Eliminating malignant contamination from therapeutic human spermatogonial stem cells

Abstract

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM + /HLA-ABC -/CD49e -(putative spermatogonia) and EpCAM -/HLA-ABC + /CD49e + (putative MOLT-4) cell fractions. The EpCAM + /HLA-ABC -/CD49e -fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM -/HLA-ABC + / CD49e + fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

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.…”

Transcriptional control of spermatogonial maintenance and differentiation

“…Human and mouse spermatogonia also use the same extrinsic factor—GDNF—for their growth in vitro [130]. To screen for other factors important for SSC self-renewal and differentiation, investigators can take advantage of the recent development of in vitro SSC culturing methods and the xenotransplantation assay for human SSC activity [118,121,130,132]. By utilizing the new tools that are available to the field, the transcription networks functioning in spermatogonia that promote their self-renewal and differentiation can be rapidly mapped in the near future.…”

Testicular Tissue Cryopreservation

“…in boys with leukaemia. As it was demonstrated in a rat model that transmission of leukaemia by transplanting testicular cells is possible [35], elimination of malignant cells both by cell sorting [36] and by in vitro cultures [37] may lower or even circumvent the risk for reintroducing cancer via transplantation.…”