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.

Figure 1. Human-to–nude mouse xenotransplantation assay.

A rabbit anti-primate testis cell polyclonal antibody was previously generated that specifically recognizes antigens on primate (human and nonhuman) testis cells (37). (A) The antibody does not exhibit immunoreactivity with untransplanted mouse seminiferous tubules. (B) An isotype control antibody (rabbit IgG) does not exhibit immunoreactivity with mouse seminiferous tubules transplanted with human testicular cells. (C and D) The primate testis cell antibody cross-reacts with human testis cells and can be used to identify colonies of human spermatogonia in mouse seminiferous tubules 2 months after transplantation. Cells in colonies have a typical spermatogonial appearance, with large nuclear-to-cytoplasmic ratios, and are arranged as singles, pairs, and chains located on the basement membrane of seminiferous tubules. (E and F) The colonizing cells recognized by the primate testis cell antibody also express the germ cell marker VASA. Mouse seminiferous tubules are demarcated by dashed white lines. Scale bar: 100 μm.

Figure 2. SALL-4–positive spermatogonia are recovered in the EpCAM^lo fraction of human testis cells.

(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.

Figure 3. Human-to–nude mouse xenotransplant colonizing activity is enriched in the EpCAM^lo fraction of human testis cells.

Unsorted and EpCAM-sorted human testis cell fractions (see Figure 2) were transplanted into the testes of immune-deficient nude mice. Two months after transplant, colonies of human spermatogonia were identified in mouse recipient testes using the rabbit anti-primate testis antibody and Alexa Fluor 488–conjugated secondary antibody (green) (scale bar: 50 μm) (inset). Mouse seminiferous tubules are demarcated by dashed white lines. Colonies in each recipient testis were counted and normalized to 10⁵ viable cells transplanted per testis. *P < 0.001, compared with unsorted cells. Bars indicate the mean number of colonies per 10⁶ viable cells in each fraction. Error bars represent SEM from 3 replicate sorting experiments.

Figure 4. SALL-4–positive human spermatogonia do not express HLA-ABC or CD49e.

(A) To determine whether human spermatogonia express HLA-ABC, human testicular cell suspensions were stained with APC-conjugated HLA-ABC antibodies and sorted into positive and negative fractions by FACS. Negative gates were defined by analysis of human testis cells using APC-conjugated isotype control antibodies. (B–E) Following FACS, each fraction of cells was fixed and immunocytochemistry was performed to assess SALL-4 expression; then, fractions were counterstained with DAPI to quantify total cells. (B) The percentage of cells in each unsorted and sorted fraction that displayed SALL-4 staining (SALL-4⁺ green cells/DAPI-stained total cells). (F–J) A similar experiment was conducted using APC-conjugated CD49e antibodies. Scale bar: 50 μm (C–E and H–J). Bars in B and G indicate the mean percentage of SALL-4–positive cells (SALL-4–positive cells/total cells) in each fraction. Error bars in B and G represent SEM from 3 replicate sorting experiments. *P < 0.001, compared with unsorted cells.

Figure 5. The EpCAM^lo /CD49e^–/HLA-ABC^– fraction of MOLT-4–spiked human testis cell suspension is enriched for human spermatogonia.

(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-CAM^lo/side scatter^hi (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.

Figure 6. EpCAM^–/CD49e⁺/HLA-ABC⁺ cells form testicular tumors following transplantation into nude mice, but EpCAM^lo /CD49e^–/HLA-ABC^– cells do not form tumors.

(A and B) Unsorted spiked testicular cells and cells from fraction II (see Figure 5A) produced tumors in recipient mouse testes. (C) Cells from fraction IIIa (see Figure 5, A and B) that contained human spermatogonia colonizing the seminiferous tubule of nude mice (see Figure 5G) did not produce tumors.