Emerging cell populations in the early embryo are challenging to investigate. Therefore, we used mouse ESCs from multiple genetic backgrounds to differentiate transgene-free, pre-gonadal stage PGCs where 100% of the single iPGCs express Blimp1 in vitro. Here we show that sorting for the cKitbright fraction of SSEA1+ cells at day 6 of differentiation when the population is first discernable yields an iPGC population with an identity suggestive of PGCs younger than e11.5.
One of the major challenges in the ESC and PGC fields has been to distinguish early progenitor PGCs from undifferentiated ESCs due to their similar expression patterns. Indeed, e11.5 PGCs isolated from the genital ridge prior to sex determination cluster very closely to undifferentiated ESCs in 2-dimensional principle component analysis after microarray 
. Therefore, it has been proposed that ESCs originate from a progenitor germ cell consistent with detectable expression of PGC-signature genes, including Dazl
and Tissue non specific alkaline phosphatase
in the undifferentiated state 
. Although our studies do not address the origin of ESCs, our data does indicate that a small nascent PGC-like population corresponding to about 10% of cells can be identified in an ESC culture in the self-renewing state, agreeing strongly with previous work which demonstrated that Dazl
null ESCs exhibit reduced expression of PGC-signature genes 
. However, our data also show that despite co-expression of germ cell genes in these 10% of cells, the transcript levels are significantly lower than the levels found in bona fide
PGCs isolated form the embryo between e9.5–e10.5 as well as the iPGCs. Taken together, our data argues that the majority of undifferentiated ESCs are not PGCs, and that a single cell analysis is critical to uncouple differences between ESCs and progenitor PGCs.
In the current study, we identified Id4 as a new marker enriched in iPGCs relative to undifferentiated ESCs. Id4 was recently found to be a germ line marker expressed in gonocytes and spermatogonia of postnatal and adult murine gonads 
. We extend these findings to show that Id4 is expressed during the earliest stage of germ line development, prior to gonadal colonization (). Interestingly, Id4
similar to Stella
constitutes a marker for defining PGC identity yet has no functional role in specifying PGC fate 
. However, by combining Id4, SSEA1, and Oct4 expression in day 6 EBs, we propose a model for germ line formation in vitro
that involves the generation of multiple SSEA1+/Oct4+ niches during EB formation, with Id4+ iPGCs emerging from within these niches (). We propose that similar to PGC development in the allantois of the embryo, the tight clustering of SSEA1+/Oct4+ cells creates a microenvironment in the EB to protect the iPGCs against somatic cell differentiation signals 
. Given that Id4+ cells constitute only a subpopulation of cells within SSEA1+ clusters, we hypothesize that the clusters are composed of a heterogeneous mixture of immature cells, including epiblast-like cells (Stella
negative), PGC precursors (Stella
positive) and definitive Id4-positive cKitbright
iPGCs (). Whether the SSEA1+/cKitbright
PGCs emerge from a subpopulation of the SSEA1+/cKitmid
fraction of cells remains to be determined. However, our data strongly argue that iPGCs do not differentiate from SSEA1+/cKitdim
cells, which have no colony forming potential, and express high levels of Hoxa1
, indicating commitment to a somatic fate.
Model for iPGC emergence from SSEA1/Oct4+ clusters in EBs.
Although our data suggest that the iPGCs are younger than e11.5 of development due to lack of Mvh protein expression, it is conceivable that iPGCs at day 6 are more similar to e11.5 in some aspects, but have not received the appropriate cues to express Mvh protein. The signals that promote Mvh protein expression in PGCs at e11.5 are not well understood, but one study has indicated that gonadal somatic cells are involved in this process 
. Lack of Mvh protein expression in our model suggests that the hanging drop EB system by day 6 of differentiation does not provide the necessary signals to promote developmental progression to Mvh protein-positive iPGCs. This result implies that progression of iPGCs in vitro
may require a gonadal niche to promote differentiation to the Mvh protein-positive stage. Indeed, while this manuscript was under review, Hayashi and colleagues demonstrated that a neonatal seminiferous tubule niche was necessary to promote differentiation of ESC-derived PGCs, which this group called PGC like cells (PGCLCs), into functional post-meiotic male germ cells 
. In these studies, PGCLCs were isolated using SSEA1 and Integrin Beta 3 and were hypothesized to be equivalent to e9.5 of development. Similar to this group, iPGCs isolated at day 6 also express significantly high levels of Integrin Beta 3
RNA (Figure S2
In the current study, we successfully acquired PGCs in the Blimp1
-positive stage of development. Blimp1
is not expressed in meiotic or post-meiotic cells and therefore our model is not useful for evaluating meiotic progression; however, we propose that this model can be used to successfully evaluate molecular events in PGC formation prior to gonadal colonization, gonadal reprogramming and sex determination. As an example of the utility of our model, one hundred male e10.5 embryos would be required to obtain approximately 100,000 PGCs via FACS, if we estimate that there are ~1,000 PGCs per embryo at this developmental age 
. In contrast, generating iPGCs equivalent to e9.5–10.5 of development using ESC differentiation required 50 plates of hanging drop EBs, which takes 1 hour to set up from only two wells of undifferentiated ESCs. This yields on average 150,000–175,000 Blimp1
positive iPGCs at day 6 of differentiation, resulting in more than 100-fold enrichment in cell numbers over embryonic dissections 
In conclusion, we propose that the ESC-to-PGC differentiation model is an essential tool for examining molecular events in PGC development. In this study we developed a model that specifically captures the Blimp1-positive stage of male PGC formation prior to the expression of Mvh protein. This period of germ cell development (prior to e11.5) is uniquely regulated in mammals and is not conserved with lower model organisms such as Drosophila, C. elegans, frog, and chick (for example, the role of Blimp1). Therefore, creating models that study the initial formation of mammalian PGCs such as the one described here, as well as extending this model to female ESC lines, will be critical to our understanding of the mechanisms that govern fundamental principles of inheritance via the germ line.