Skip to main content
Open Access Publications from the University of California


UCLA Electronic Theses and Dissertations bannerUCLA

Studying Niche Colonization and the Dynamics of Imprint Erasure During In Vitro Acquisition of Pluripotency in Germ Cells


Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. Scalable in vitro differentiation of PGCs from pluripotent cell types has emerged as a model to study PGC biology. The in vitro PGCs (iPGCs) obtained from ESCs through the embryoid body (EB) differentiation method correspond to a pregonadal stage of PGC development. Thus, extending the applications of this model to the study of more developmentally advanced germ cells requires maturation of iPGCs to different developmental stages. However, the maturation potential of iPGCs is unknown. In order to promote maturation of male iPGCs in vitro and in vivo we use a transplantable niche model where iPGCs are aggregated with male fetal gonadal cells and cultured prior to transplantation to induce de novo formation of the germ cell niche. Initial characterization of this model without iPGCs revealed that despite the different localization of germ and Sertoli cells within gonadal aggregates before transplantation, both of these cell types are found in physical contact inside the reconstructed germ cell niche. Furthermore, we determined that both germ and Sertoli cells mature in the gonadal transplants. Analyzing iPGCs in gonadal aggregates in vitro, we find that unlike the pregonadal PGCs of the E9.5 embryo, iPGCs do not express the gonadal stage marker MVH indicating they do not mature in vitro. Although transplantation of gonadal aggregates did not support niche colonization by either in vitro or embryo-derived exogenous germ cells, unlike iPGCs, PGCs from the embryo do not survive in the transplants. The surviving iPGCs remain in the extratubular space, disrupting the transplant morphology, and express proteins that uncommon in the germline indicating they have acquired a different cellular identity. To further examine similarities and differences between embryonic and ESC-derived PGCs, we analyzed their epigenetic stability at imprinting control centers (ICCs) in a PGC reprogramming assay in vitro using sequencing of bisulfite-treated DNA amplified by PCR. We find that both embryo and ESC-derived PGCs erase cytosine methylation at the Snrpn ICC. However, unlike in the PGCs of the embryo, this demethylation is faster and not stably maintained. Furthermore, we find that ESC-derived PGCs form colonies faster than E9.5 PGCs and this correlates with significantly less expression of Lats2, which indicates a lower barrier to reprogramming. Our results also indicate that, compared to E9.5 PGCs, iPGCs have significantly higher expression of the pluripotency-associated transcription factor Klf4. Interestingly, using this in vitro PGC reprogramming assay we evaluated the dynamics of imprint erasure during reprogramming embryonic PGCs in vitro, which to this date was unknown. We determined that cytosine methylation at ICCs is erased in E9.5 PGCs during the first four days of reprogramming. This is followed by de novo DNA methylation during the last days of PGC reprogramming in vitro. The extent of remethylation is locus-specific resulting in pluripotent EGCs with hypo and hypermethylated ICCs.

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View