br Discussion Xenotransplantation of human

Discussion Xenotransplantation of human and nonhuman primate adult testicular purchase glibenclamide into mouse testis is one of the major bioassays for germline potential. Using this assay together with FACS to isolate putative SSCs reveals that colony-forming potential is enriched in germ cells with an SSEA4, ITGA6, Thy1dim, EpCAMdim, or CD9 cell surface phenotype (Izadyar et al., 2011; Valli et al., 2014; Zohni et al., 2012). Specifically, xenotransplantation of unsorted adult testicular cells into busulfan-treated nude mice leads to ∼2.9–4.8 colonies per 100,000 cells transplanted, whereas sorting cells for ITGA6, Thy1, and EpCAM results in an average 9.6, 7.3, and 11.9 colonies per 100,000 cells transplanted (Valli et al., 2014). In the current study, the colony-forming potential from unsorted Carnegie stage 23 rhesus embryonic testes was 0.42 colonies per 100,000 cells transplanted (when averaging the results of all four replicates in Carnegie stage 23). However, at day 50 of embryo development, the average colony-forming potential was 1.25 colonies per 100,000 cells transplanted. In allogeneic mouse transplants, Chuma et al. (2005) reported spermatogenesis and teratoma-forming potential following transplantation of epiblast cells as well as PGCs at E8.5 into the seminiferous tubules of 5–8-day-old W (cKIT mutant) mouse pups. Just 2 days later, at E10.5, teratoma potential was lost when donor tissues containing PGCs were used (Chuma et al., 2005). Using the adult testis as a recipient, our data reveal that primate embryonic testes containing OCT4- and NANOG-positive PGCs do not yield teratomas after xenotransplantation into the adult testis. Therefore, repression of the latent pluripotency program in PGCs is not a requirement for xenotransplantation and colony-forming potential. We did identify atypical microscopic events that were relatively rare. It is not known whether these events are of germ cell origin, or whether they may correspond to somatic cells of the embryonic testis that were able to survive in the seminiferous tubule epithelium. Notably, these events were observed when either human or rhesus macaque embryonic testis were used as donor cells, and in both species the number of atypical events decreased with increasing embryonic age. This was opposite to typical colony-forming potential which appeared to increase with age. One possibility for this is PGC number. Our FACS experiments indicate that PGC number ranges from around 1,700 to ∼10,000 PGCs in Carnegie stage 23. Therefore, it is conceivable that the best transplants originated from testes with the highest number of PGCs. Alternatively, it is also conceivable that the microenvironment of the laminin-positive cords changes abruptly at the end of Carnegie stage 23, leading to a change in PGC colonizing ability. One way to distinguish these possibilities would be to sort PGCs at a range of developmental ages, and normalize PGC number prior to xenotransplant. Recently, transplanting undifferentiated hiPSCs into the seminiferous tubules of busulfan-treated adult nude mice yielded germ cell colonies as well as nonseminoma-type germ cell tumors including embryonal carcinoma, yolk sac tumors, and teratomas (Durruthy-Durruthy et al., 2014; Ramathal et al., 2014). From this work it was unclear whether these different outcomes were due to the seminiferous tubule microenvironment instructing hiPSCs to differentiate into the tumors, or whether the seminiferous tubule microenvironment first induced the differentiation of PGCLCs that subsequently differentiated into the nonseminoma-type germ cell tumors (Durruthy-Durruthy et al., 2014; Ramathal et al., 2014). In our study, we discovered that embryonic testes containing PGCs do not generate tumors. This argues that the tumors generated from hiPSCs/hESCs originate from the transplanted pluripotent cells and not a PGCLC intermediate.
Experimental Procedures
Author Contributions
Acknowledgments