As success rates in treating childhood cancers have improved, greater emphasis is now being placed on quality of life issues following successful treatment. Many cancer treatments can lead to infertility, but there are few methods to preserve the fertility of children who have not entered puberty. Spermatogonial stem cells (SSCs), which produce sperm cells, are present prior to the start of puberty. In theory, SSCs could be removed via biopsy prior to the start of treatment and then retransplanted following remission; however, there is a potential risk of reintroducing malignant material during transplantation. To overcome this hurdle, Serena Dovey and colleagues characterized the cell surface markers of human spermatogonia in testicular tissue from organ donors. They then developed a multi-parameter sorting approach to separate SSCs from MOLT-4 leukemia cells. Sorted SSCs exhibited spermatogonial colonizing activity (accompanying image), but did not form tumors, in a human-to-nude mouse xenotransplantation assay, demonstrating that FACS can be used to isolate and enrich human SSCs while removing malignant contaminants. The accompanying image is a cross-section from nude mouse testis demonstrating normal architexture of the seminiferous tubules stained with hematoxylin and eosin (H&E).
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.
Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig