Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions.
BACKGROUND
Chemotherapy and radiation treatments for cancer and other diseases can cause male infertility. There are currently no options to preserve the fertility of prepubertal boys who are not yet making sperm. Cryopreservation of spermatogonial stem cells (SSCs, obtained via testicular biopsy) followed by autologous transplantation back into the testes at a later date may restore fertility in these patients. However, this approach carries an inherent risk of reintroducing cancer.


METHODS
To address this aspect of SSC transplantation safety, prepubertal non-human primate testis cell suspensions were inoculated with MOLT4 T-lymphoblastic leukemia cells and subsequently sorted for cell surface markers CD90 (THY-1) and CD45.


RESULTS
Cancer cells segregated to the CD90-/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD45- fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD45-) revealed 0.1% contamination with cancer cells, which was sufficient to produce tumors in nude mice. We hypothesized that the contamination resulted from mis-sorting due to cell clumping and employed singlet discrimination (SD) in four subsequent experiments. Purity checks revealed no cancer cell contamination in the CD90+/CD45- fraction from three of the four SD replicates and these fractions produced no tumors when transplanted into nude mouse testes.


CONCLUSIONS
We conclude that spermatogonia can be separated from contaminating malignant cells by fluorescence-activated cell sorting prior to SSC transplantation and that post-sorting purity checks are required to confirm elimination of malignant cells.
Department of Obstetrics, Gynecology & Reproductive Sciences,  University of Pittsburgh School of Medicine, Pittsburgh, PA 15260,  USA.
Brian P
BP
Hermann
Meena
M
Sukhwani
Jennifer
J
Salati
Yi
Y
Sheng
Tianjiao
T
Chu
Kyle E
KE
Orwig
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D059247:Fertility Preservation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D012741:Sexual Maturation; D013093:Spermatogonia / Q000166:cytology* / Q000637:transplantation; D013737:Testis / Q000166:cytology*; D014021:Tissue Preservation
D014408:Biomarkers, Tumor