In vitro derived female hPGCLCs are unable to complete meiosis in embryoid bodies.

Abstract The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs.