Eliminating malignant contamination from therapeutic human spermatogonial stem cells
Serena L. Dovey, … , Joseph S. Sanfilippo, Kyle E. Orwig
Serena L. Dovey, … , Joseph S. Sanfilippo, Kyle E. Orwig
Published March 15, 2013
Citation Information: J Clin Invest. 2013;123(4):1833-1843. https://doi.org/10.1172/JCI65822.
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Technical Advance

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.

Authors

Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig

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Figure 2

SALL-4–positive spermatogonia are recovered in the EpCAMlo fraction of human testis cells.

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SALL-4–positive spermatogonia are recovered in the EpCAMlo fraction of h...
(A) Human testicular cells were stained with EpCAM-PE, and 3 populations were identified based upon EpCAM-PE staining intensity and side scatter of incident light. Negative gates were defined by analysis of human testis cells stained using PE-conjugated isotype control antibodies. (B–F) Following sorting, each fraction of cells was fixed and immunocytochemistry assessing SALL-4 expression was performed. Following SALL-4 staining, cells were counterstained with DAPI. Cells from at least 10 independent images were then counted based on DAPI staining and SALL-4 staining, respectively, to determine the percentage of cells expressing SALL-4. An unsorted fraction of cells was also stained with an isotype antibody to control for nonspecific binding to demonstrate specificity. (B) Relative SALL-4 expression in unsorted and EpCAM-sorted fractions. Bars indicate the mean percentage of SALL-4–positive cells (SALL-4–positive cells/total cells) in each fraction. Error bars represent SEM from 3 replicate sorting experiments. *P < 0.001, compared with unsorted cells. (C–F) Representative images from SALL-4 immunocytochemistry of unsorted and sorted fractions. Scale bar: 50 μm.