br Results br Discussion We highlight the generation of

Results
Discussion We highlight the generation of human functional haploid spermatids from human SSCs of cryptorchid patients. Cryptorchidism is the most common etiologic factor for azoospermia in adults. Among men with untreated bilateral cryptorchidism, 89% eventually develop azoospermia (Chung and Brock, 2011). However, the exact causes of most cases of cryptorchidism remain unknown (Philibert et al., 2013). Our data indicate that cryptorchid patients have a normal chromosome karyotype with no mutations of INSL3, RXFP2, and AR genes. It has been suggested that a reduced total number of male germ eph receptor in cryptorchid testes is the cause of male infertility (Hadziselimovic and Herzog, 2001). Therefore, in vitro differentiation of human SSCs into haploid spermatids from cryptorchid testes could be an ideal method for treating infertility in cryptorchid patients. Here, we have shown obvious evidence for rescuing germ cell development in cryptorchid patients using in vitro techniques, as evidenced by our finding that human SSCs from cryptorchid patients can progressively differentiate into meiotic and haploid spermatids by treatment with RA and SCF. We first evaluated the spermatogenesis status of cryptorchid patients using immunohistochemistry with MAGEA4 and SCP3. Notably, human spermatogonia exist whereas meiotic male germ cells are rather rare or completely lost in the testis of cryptorchid patients, since there were numerous cells positive for MAGEA4, a marker for human spermatogonia (He et al., 2010), whereas fewer cells stained positively for SCP3, a hallmark for spermatocytes (West et al., 2006). This conclusion can also be verified by our observations that almost all freshly isolated male germ cells from cryptorchid patients were positive for UCHL1, a marker for human spermatogonia (He et al., 2010), and GFRA1, a surface hallmark for human SSCs (He et al., 2010). Our results are consistent with previous findings showing defective maturation of germ cells in cryptorchid patients (Agoulnik et al., 2012; Huff et al., 2001). Using multiplex PCR, we found that a number of Y chromosome genes, including SRY, sY254, sY127, sY86, sY134, sY84, and sY255, were present in cryptorchid patients, thus excluding Y chromosome microdeletion in cryptorchid patients. Therefore, testis tissues of these cryptorchid patients were chosen for differentiating into spermatocytes and haploid spermatids. The starting cells we used for differentiation were colony cells. We found that isolated cells from cryptorchid testes were able to proliferate and form colonies composed of numerous cells. We and others have revealed that the cells in these colonies were SSCs with proliferation potential (He et al., 2010; Sadri-Ardekani et al., 2009). It has been demonstrated by xenotransplantation that the cells in the colonies were actually human SSCs with self-renewal capacity (Sadri-Ardekani et al., 2009). The differentiation potential of human SSCs from cryptorchid testes was assessed by various types of approaches, including quantitative PCR, RT-PCR, immunocytochemistry, and meiotic spread assays. After treatment with RA and SCF, the expression of numerous genes for meiotic and haploid cells, including SYCP1, SYCP2, SYCP3, BOULE, PRM1, PRM2, TNP1, TNP2, and ACR (Holloway et al., 2008; Tedesco et al., 2011; West et al., 2006), in human SSCs was obviously enhanced. SCP3 can be used to measure the synaptonemal complex, while CREST is a hallmark for detecting centromeric regions and MLH1 has been utilized for measuring meiotic recombination frequency (Holloway et al., 2008). Our results, using these markers for meiosis and postmeiosis, clearly indicate that RA and SCF could induce human spermatogonia to enter the postmeiotic stage and eventually differentiate into haploid spermatids. RA has been shown to play an important role in triggering germ cells to enter meiosis (Niederreither and Dollé, 2008). We have previously reported that RA can act as a meiosis-inducing factor in the differentiation of iPSCs into male germ cells (Yang et al., 2012). SCF has been shown to be essential in spermatogonial differentiation as well as meiotic initiation (Feng et al., 2000). The SCF/KIT interaction plays a critical role in meiotic entry of differentiating spermatogonia (Rossi et al., 2008). Furthermore, the crosstalk between RA and the SCF pathway could stimulate differentiation of male germ cells toward the meiotic stages (Pellegrini et al., 2008). Consistent with these findings, we found that RA and SCF could efficiently induce the differentiation of human SSCs from cryptorchid testes into postmeiotic male germ cells.