Eliminating malignant contamination from therapeutic human spermatogonial stem cells
Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig
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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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 5

The EpCAMlo /CD49e–/HLA-ABC– fraction of MOLT-4–spiked human testis cell suspension is enriched for human spermatogonia.

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The EpCAMlo /CD49e–/HLA-ABC– fraction of MOLT-4–spiked human testis cell...
(A) Human testicular cell suspensions were spiked with 10% MOLT-4 cells and then FACS sorted using EpCAM-PE, HLA-ABC-APC and CD49e-APC antibodies. (B) Fraction III in A was further analyzed with side scatter, as described in Figure 2, to identify the SSC fraction, Ep-CAMlo/side scatterhi (green, Fraction IIIa). Thus, only cells that (A) primarily fell within fraction III and (B) secondarily fell within fraction IIIa were collected. (C–F) Immunocytochemistry was performed to assess relative SALL-4 expression in unsorted and sorted fractions. We focused specifically on fractions II and IIIa (green), because this is where we expected to find MOLT-4 leukemia cells and human spermatogonia, respectively, based on data in Figures 2–4. Scale bar: 50 μm (C–E). Bars in F indicate the mean percentage of SALL-4–positive cells (SALL-4–positive cells/total cells) in each fraction. Error bars in F represent SEM from 6 replicate sorting experiments. (G) The human-to–nude mouse xenotransplantation assay was used to assess spermatogonial colonizing activity in unsorted (unspiked) and sorted (spiked) testis cell fractions (I, IIIa, and IV), as described in Figure 3. Bars indicate the mean number of colonies per 106 viable cells in each fraction. Error bars represent SEM from 6 replicate sorting experiments. *P < 0.001, compared with unsorted cells. A typical colony of human spermatogonia in recipient mouse seminiferous tubules is shown in the inset. Scale bar: 50 μm.