title: 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.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Serena L
authors.initials: SL
authors.lastname: Dovey
authors.forename: Hanna
authors.initials: H
authors.lastname: Valli
authors.forename: Brian P
authors.initials: BP
authors.lastname: Hermann
authors.forename: Meena
authors.initials: M
authors.lastname: Sukhwani
authors.forename: Julia
authors.initials: J
authors.lastname: Donohue
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castro
authors.forename: Tianjiao
authors.initials: T
authors.lastname: Chu
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Sanfilippo
authors.forename: Kyle E
authors.initials: KE
authors.lastname: Orwig
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D002471:Cell Transformation, Neoplastic / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D007248:Infertility, Male / Q000628:therapy; D008297:Male; D051379:Mice; D008819:Mice, Nude; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000009:adverse effects*; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013736:Testicular Neoplasms / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism; D014183:Transplantation, Heterologous
chemical_list: D015703:Antigens, CD; C472575:SALL4 protein, human; D014157:Transcription Factors
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