Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions

Abstract

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 CD902/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD452 fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD452) 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+/CD452 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.

Is transplantation of a few leukemic cells inside an artificial ovary able to induce leukemia in an experimental model?

“…Results differ between studies, but intratesticular [36] or intraperitoneal [37] injection of as few as 10 AML cells was found to induce disease in mice. The type of cell line used and its species-specific origin could also have an impact on the results.…”

“…BV-173 cells have a doubling time of 30-48 h, so 5 months appeared more than adequate for our purposes. The maximum evaluation period in previous studies was 4 months [36].…”

Functional sperm produced after spermatogonial stem cell transplantation into rhesus

“…So far, efficiency is rather low, but it might be enhanced by optimizing recipient preparation and by increasing the SSC population through enrichment strategies 7,8 or in vitro propagation. 9 These adaptations might increase the proportion of donor sperm and facilitate the production of donor offspring.…”

The Next Frontier: The Promise of in vitro Spermatogenesis Coupled with Intracytoplasmic Sperm Injection

“…For prepubescent boys with cancer and adult men who have no sperm to cryopreserve, one potential strategy is to collect, enrich, and cryopreserve their spermatogonial stem cells (SSCs) with the intent to transplant them back into their testes in the future to allow for spermatogenesis to occur in vivo. Successful SSC transplantation and restored spermatogenesis has been reported for a variety of mammalian species, including rodents, dogs, pigs, and goats [4]. The generation of sperm from transplanted SSCs, however, presupposes that the endogenous niche, or microenvironment, within the recipient testis is receptive to SSC colonization and differentiation, and is able to support the myriad of complex biological processes that occur during spermatogenesis, including meiotic maturation and spermatid elongation.…”

“…This scenario warrants a stringent cell sorting protocol, one that filters out cancerous cells from the SSC population. Hermann et al [4]. have described an innovative dual positive (CD90+) and negative (CD45-) selection strategy that uses fluorescence-activated cell sorting (FACS) to purify SSCs from a contaminated sample of nonhuman primate testis cells prior to transplantation [4].…”