Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis

Abstract

STUDY QUESTIONWhich set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)?STUDY FINDINGWe validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12–13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM).WHAT IS KNOWN ALREADYThe dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well.STUDY DESIGN, SIZE, DURATIONThis immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development.PARTICIPANTS/MATERIALS, SETTING, METHODSWe have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12–13). The human material used was anonymously donated with informed consent from elective abortions without medical indication.MAIN RESULTS AND THE ROLE OF CHANCEWe observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs.LARGE SCALE DATANon-applicable.LIMITATIONS REASONS FOR CAUTIONMaterial to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available.WIDER IMPLICATIONS OF THE FINDINGSMost of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the ...

Specification and epigenetic resetting of the pig germline exhibit conservation with the human lineage

“…Changes in other histone and chromatin remodellers were also detected, such as the upregulation of components of MII complex (DPY30, RBBP5) and SWI/SNF proteins SMARCA5 and HLTF 1 0 S3C). Similar observations have been reported in Wk4 hPGCs and D4 hPGCLCs (Gell et al, 2020;Gkountela et al, 2013;Gomes Fernandes et al, 2018;Tang et al, 2015). By contrast, mouse PGCs show persistent H3K27me3 in gonadal PGCs (de Sousa Lopes et al, 2008;Seki et al, 2005), which might have a role in maintaining genomic integrity during the period of active DNA demethylation (Cotton et al, 2013 Posavec Marjanović et al, 2017 ).…”

“…Therefore, we sought to identify pPGC-specific membrane proteins by selecting pPGC-specific genes with relevant GO terms and/or curated in the Cell Surface Protein Atlas (Bausch-Fluck et al, 2015). As reported before in hPGCs and early cyPGCs (Gomes Fernandes et al, 2018;Sasaki et al, 2016;Tang et al, 2015), KIT and PDPN were upregulated in pre-migratory pPGCs (Fig. S2D).…”

Specification and epigenomic resetting of the pig germline exhibit conservation with the human lineage

“…The gene expression profile in soma g1 cells suggests that these cells may be undergoing epithelial-mesenchymal transition ( Pan et al., 2016 : Stemmler et al., 2019 ). In contrast, soma g2 cells in E14 embryos exhibit epithelial features with hallmark expression of the amnion-specific genes GATA3 , GATA2 , TFAP2A , TFAP2C , OVOL1 , and KRT7/8/1 8 ( Gomes Fernandes et al., 2018 ; Xiang et al., 2020 ) as well as the cell adhesion-related genes ITGA3 , PKP2 , PODXL , and AHNAK ( Saykali et al., 2019 ). Trajectory analysis confirmed the pseudo-temporal relationship among these cells, with soma g1 nascent mesoderm being closer to Epi cells, whereas soma g2 diverge from g1 and PGCs ( Figure S1 C).…”

Human blastocyst outgrowths recapitulate primordial germ cell specification events

“…Nevertheless, as this is a descriptive study, current findings should be considered only as preliminary. In the future, additional markers of human PGC specification (Gomes Fernandes et al ., 2018) as well as culture adaptations to improve embryo development may serve to further characterise the observed cell population. While additional high-throughput analyses may also be applied to validate the PGCLCs, the set of antibodies used highlights the high degree of lineage heterogeneity in 12 dpf blastocyst outgrowths, which will inherently limit efficiency and cell recovery.…”