STUDY QUESTIONWhat is the dynamics of expression of P-element induced wimpy testis-like (PIWIL) proteins in the germline during human fetal development and spermatogenesis?SUMMARY ANSWERPIWIL1, PIWIL2, PIWIL3 and PIWIL4 were expressed in a sex-specific fashion in human germ cells (GC) during development and adulthood. PIWILs showed a mutually exclusive pattern of subcellular localization. PIWILs were present in the intermitochondrial cement and a single large granule in meiotic GC and their expression was different from that observed in mice, highlighting species-differences.WHAT IS KNOWN ALREADYIn mice, PIWIL proteins play prominent roles in male infertility. PIWIL mouse mutants show either post-meiotic arrest at the round spermatid stage (PIWIL1) or arrest at the zygotene-pachytene stage of meiosis I (PIWIL2 and PIWIL4) in males, while females remain fertile. Recent studies have reported a robust piRNA pool in human fetal ovary.STUDY DESIGN, SIZE, DURATIONThis is a qualitative analysis of PIWILs expression in paraffin-embedded fetal human male (N = 8), female gonads (N = 6) and adult testes (N = 5), and bioinformatics analysis of online available single-cell transcriptomics data of human fetal germ cells (n = 242).PARTICIPANTS/MATERIALS, SETTING, METHODSHuman fetal gonads from elective abortion without medical indication and adult testes biopsies were donated for research with informed consent. Samples were fixed, paraffin-embedded and analyzed by immunofluorescence to study the temporal and cellular localization of PIWIL1, PIWIL2, PIWIL3 and PIWIL4.MAIN RESULTS AND THE ROLE OF CHANCEPIWIL1, PIWIL2 and PIWIL4 showed a mutually exclusive pattern of subcellular localization, particularly in female oocytes. To our surprise, PIWIL1 immunostaining revealed the presence of a single dense paranuclear body, resembling the chromatoid body of haploid spermatocytes, in meiotic oocytes. Moreover, in contrast to mice, PIWIL4, but not PIWIL2, localized to the intermitochondrial cement. PIWIL3 was not expressed in GC during development. The upregulation of PIWIL transcripts correlated with the transcription of markers associated with piRNAs biogenesis like the TDRDs and HENMT1 in fetal GC.LARGE SCALE DATANon-applicable.LIMITATIONS, REASONS FOR CAUTIONThis study is limited by the restricted number of samples and consequently stages analyzed.WIDER IMPLICATIONS OF THE FINDINGSIn the germline, PIWILs ensure the integrity of the human genome protecting it from ‘parasitic sequences’. This study offers novel insights on the expression dynamics of PIWILs during the window of epigenetic remodeling and meiosis, and highlights important differences between humans and mice, which may prove particularly important to understand causes of infertility and improve both diagnosis and treatment in humans.STUDY FUNDING/COMPETING INTEREST(S)M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011]; N.H. by China Scholarship Council (CSC) [No. 201307040026] and F.W. by Medical Personnel Training Abroad Project of...

SummaryPredicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondisjoined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5mCpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5mCpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5mCpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners.

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 ...

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2– and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.