Another potential source of stem cells is derived from precursor germ cells. In early embryonic development, a subset of pluripotent cells differentiate into primordial germ cells (PGCs) [55, 56]. These cells migrate, proliferate and colonize the genital ridge and represent a population of cells that will eventually further differentiate to form gametes. Initially discovered in mice, failure of PGCs to mitotically arrest following colonization leads to the formation of teratomas, tumors that contain cells representing all three germ layers: ectoderm, mesoderm, and endoderm . The first isolations and cultures of these proliferating PGCs yielded a multipotent cell line termed embryonal carcinoma cells. These cells are capable of being differentiated in culture into various cell types, including neurons and cardiomyocytes [58, 59]. It was also shown that culture of isolated PGCs prior to genital ridge colonization resulted in germ cell colonies that express numerous pluripotency markers akin to those of ESCs, such as OCT-4 [60, 61]. These unique cells, termed embryonic germ cells (EGCs) were shown to be highly pluripotent. EGCs have previously been an interesting cell source for studying gametogenesis in vitro because mouse EGCs appear to follow similar differentiation patterns as observed in in vivo gametogenesis . However, ethical concerns about obtaining human EGCs have tamed interest in this field.
Several groups have shown the ability of mouse, nonhuman primate and human ESCs to differentiate into germ cell lineages, specifically in vitro-derived PGCs (invPGCs) [63–79]. However, three groups in particular have demonstrated three different methodologies for faithfully deriving invPGCs from ESCs at higher efficiencies [63–65]. Yamauchi and colleagues  successfully differentiated cynomolgus monkey ESCs into germ cells by forming embryoid bodies (EBs) with retinoic acid and culturing these EBs for 28 days. At day 28, germ cells could be identified by positive immunostaining for SSEA1, VASA or DAZL. Furthermore, these researchers showed up-regulation of germ cell gene expression for CXCR4, NANOS1, NANOS2, NANOS3, VASA, PIWIL1 and TEKT1 upon EB formation with retinoic acid for 28 days. Likewise, this group was able to demonstrate that day 28 EBs grown in retinoic acid or bone morphogenetic protein (BMP)-4 elevated expression of the meiotic marker SCP1 but not SCP3. Kee and colleagues  showed that adherent differentiation with a BMP cocktail (BMP4, BMP7 and BMP8b) induced differentiation of hESCs into invPGCs in 7 to 14 days. Using a green fluorescent protein (GFP) transgene driven by the VASA promoter, these researchers showed that differentiation medium supplemented with BMPs resulted in increased expression of two PGC markers in differentiating hESCs: VASA and DAZL. Kee and colleagues also demonstrated that VASA-GFP+ cells could be isolated and cultured on mouse embryonic fibroblasts for 7 days to form invPGC colonies. These cultured cells also exhibited hypomethylation of the H19 locus, suggesting that these cells, like in vivo PGCs, undergo de-methylation prior to gametogenic progression . More importantly, Kee and colleagues demonstrated that over expression of DAZ family members (DAZ, DAZL and BOULE) in cultured invPGCs induces meiotic progression as determined by immunofluorescence staining for SCP3 and γH2AX . Even more striking, they demonstrated haploid formation by over-expression of the DAZL family members by the appearance of a small 1N peak in their propidium iodide FACS analysis and the expression of acrosin in a small fraction of cells. This remarkable discovery highlights the potential of driving gametogenesis in vitro from PSCs .
More recently, Amander Clark's group demonstrated a novel approach for rapidly and more efficiently differentiating hESCs into PGCs. Park and colleagues  showed that differentiation of hESCs on human fetal gonadal stromal cells significantly improved germ cell differentiation. Strikingly, these researchers showed that c-kit+/SSEA1+/VASA+ invPGCs (5% of the total population of cells) could be isolated from differentiated hESCs as early as 3 days of culture on human fetal gonadal stromal cells. Similarly to Kee and colleagues, Park and colleagues demonstrated that invPGCs exhibit imprint erasures and show expression of a wide range of germ cell markers [63, 65]. The work of Park and colleagues demonstrates progress towards a highly efficient methodology for generating PGCs from ESCs in vitro . Furthermore, Park and colleagues are the first group to differentiate human iPS cells into early germ cell lineages. These exciting results combined with the work of Kee and colleagues  and Yamauchi and colleagues  high light the similarities between in vivo PGCs and invPGCs illustrate the possibility of treating infertility by differentiating patient-matched ESCs into gametes or male germline stem cells for transplantation.
The ability to generate transplantable male germline stem cells or haploid gametes in culture has significant therapeutic implications for couples with infertility [80, 81]. The appeal of these approaches is enhanced by iPS cell and NT technologies, which would theoretically enable men to derive germline stem cells or sperm from their own skin cells in vitro. Thus, it is hypothetically possible for a man who is rendered infertile by toxic treatment for cancer (chemotherapy or radiation), and who did not cryopreserve semen prior to treatment, to father his own genetic children from germ cells derived from NT-ESCs or iPS cells. This potential can only be realized after extensive feasibility and safety studies are conducted, ideally in nonhuman primate models that are relevant to human physiology. There is a lack of consensus among species regarding the potential of PGCs to undergo spermatogenesis when introduced into seminiferous tubules (mouse PGCs can  and rat PGCs cannot (K Orwig, unpublished)). However, there is consensus in rodents and several large animal species that gonocytes and spermatogonia from neonate, pup and adult testes undergo spermatogenesis when trans planted into the testes of infertile recipients [82–87]. -Human PSCs can be differentiated into PGCs in the context of EBs [70, 88] or adherent differentiation cultures [63, 65, 67]. Similarly, two groups have reported macaque PSC to PGC differentiation in EBs [64, 89]. There are no reports of PSC to spermatogonial stem cell (SSC) differentiation, but several studies have reported PSC differentiation to haploid germ cells [63, 77, 78], suggesting a transient transition through an SSC-like intermediate. Thus, direct differentiation of PSCs to SSCs would provide a source of transplantable cells that could be used to ask important questions about the safety and efficacy of PSC-derived cells.
Interestingly, the postnatal mammalian testis itself may provide an alternative source of PSCs that bypasses the need for an embryonic intermediate or genetic manipulation. Several groups have shown the ability of germ cells in the mouse postnatal testis to produce PSCs in vitro [90–96]. Several recent studies have also provided evidence for PSCs derived from the adult human testis [97–100]. These cells arise in vitro from spermatogonia and can give rise to tissues of all three embryonic germ layers. Given that germ cells are responsible for initiating embryogenesis, it seems possible that germ cell factors could influence their ability to become pluripotent (for example, including expression of genes associated with pluripotency). Among the genes that are thought to form a core regulatory network in ESCs (OCT4, SOX2, and NANOG) , only OCT-4 is expressed by a few postnatal germ cells or cultured SSCs. Several reports have described a relatively small group of normal mouse spermatogonia that express OCT-4, including those in the adult testis, which could potentially be those that have the capacity to produce PSCs in vitro [102–107]. In human spermatogonia, though, only a few postnatal spermatogonia retain embryonic-expressed OCT-4, and this expression is lost after the first few months of infant life except in pathological conditions [108, 109]. In cultured SSCs, Oct-4 mRNA and protein can be detected, albeit at substantially lower levels than in ESCs [90, 95, 110, 111], and this feature may be required for long-term survival of SSCs in culture . Thus, the mechanisms that predispose spermatogonia (presumably SSCs) to acquire a pluripotent phenotype in a culture dish are unclear, but may involve similar gene expression features with other pluripotent cells (ESCs).