Human PGCs are specified in the embryo between

Human PGCs are specified in the embryo between the second and third week of life after fertilization. Similar to mice, human PGCs are first localized outside of the embryo, then they migrate into the embryo during week 3, ultimately entering the genital ridges and indifferent gonads between the fourth and fifth week of life (Chiquoine, 1954; Witschi, 1948). Gonadal sex determination occurs between week 5 and 6, and male PGCs advance in differentiation toward pro-spermatogonia after week 10 post-fertilization (Gkountela et al., 2013; Guo et al., 2015). During the PGC stage of embryo development, both mouse and human PGCs express the tyrosine kinase receptor cKIT on their surface, with repression of cKIT occurring as PGCs differentiate into pro-spermatogonia (Gkountela et al., 2013; Høyer et al., 2005). In adults, cKIT is again expressed on a subset of differentiating spermatogonia just prior to entering meiosis (Unni et al., 2009). Recent work in the cynomologous (cy) macaque (Macaca fascicularis) suggests that cyPGCs are specified prior to primitive streak formation, at 11 days post-fertilization. At this time, the cyPGCs express cKIT as well as proteins encoding the pluripotency and PGC transcription factors OCT4, SOX17, TFAP2C, and PRDM1 (Sasaki et al., 2016). These transcription factors remain expressed in PGCs until after embryonic day 50. From embryonic day 50 to 70, expression of the pro-spermatogonial marker PLZF (ZBTB16) is initiated in cyPGCs, while genes associated with pluripotency including OCT4 and NANOG are repressed (Sasaki et al., 2016). Transplanting mouse germ purchase Digoxigenin-11-dUTP into the seminiferous tubules of adult mice was used to determine that spermatogenic potential is initiated as PGCs become pro-spermatogonia between E12.5 and E14.5 (Ohta et al., 2003). In contrast, using neonates as recipients, epiblast cells at E5.5, and embryonic tissues containing PGCs at E8.5, were shown to have both spermatogenic and teratoma-forming potential (Chuma et al., 2005), most likely on account of the latent pluripotency program of nascent PGCs (Matsui et al., 1992; Resnick et al., 1992). Curiously, in the neonatal recipients transplanted with E10.5 tissues, spermatogenesis occurred without teratoma formation. This suggests that neither differentiation into pro-spermatogonia, or repression of the latent pluripotency program per se are required for spermatogenic potential following PGC transplantation. In primates, the degree of latent pluripotency in PGCs is unclear given that neither cyPGCs, nor human PGCs, express SOX2 (Perrett et al., 2008; Sasaki et al., 2016). Testing this hypothesis by transplanting human PGCs into human testicles is not conceivable. In the monkey model, transplantable testicular stem cells can be identified and quantified using primate-to-nude mouse xenotransplantation, a method that was first described by Nagano and colleagues using monkey and human donor cells over 15 years ago (Nagano et al., 2001, 2002). Primate-to-nude mouse xenotransplantation is a quantitative bioassay that demonstrates the functional capacity of primate cells to engraft the basement membrane of mouse seminiferous tubules, proliferate to produce characteristic chains and networks of spermatogonia, and persist long term. Primate cells do not produce complete spermatogenesis in mouse tubules, probably due to species differences, but recapitulate many of the unique biological functions of spermatogonial stem cells (SSCs) that are not recapitulated by any other cell type. Based on these criteria, xenotransplantation to mouse seminiferous tubules has emerged as a routine bioassay for nonhuman primate and human SSCs (Dovey et al., 2013; Hermann et al., 2007, 2009; Izadyar et al., 2011; Maki et al., 2009; Sadri-Ardekani et al., 2009, 2011; Wu et al., 2009; Zohni et al., 2012). Xenotransplantation was recently extended to human fetal testis at 22 weeks of gestation (Durruthy-Durruthy et al., 2014), an age where fetal testes are enriched in pro-spermatogonia. In that study, 22 week fetal testicular cells produced colonies of primate cells in the mouse seminiferous tubules that were similar in morphology to those produced by adult human spermatogonia. At the other extreme, transplanting undifferentiated human iPSCs (hiPSCs) or human ESCs (hESCs) directly into the seminiferous tubules of busulfan-treated nude mice resulted in putative germ cell colonies accompanied by proliferating cell masses that correspond to embryonal carcinoma and yolk sac-like tumors (Durruthy-Durruthy et al., 2014; Ramathal et al., 2014), and occasionally teratomas (Durruthy-Durruthy et al., 2014). It is unclear whether xenotransplanting embryonic testes containing PGCs will yield colonies, tumors, or both.