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Greater transcultural and transdisciplinary engagement within Muslim contexts and deliberate inclusion of diverse Muslim voices in the development of international guidelines is required to improve understanding of the state of stem cell science, strengthen thinking about attendant ethical complexities, enhance compliance, deepen public deliberation, increase trust, and strengthen practice standards.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, are specified during weeks 2-3 after fertilization. Few studies on ex vivo and in vitro cultured human embryos reported plausible hPGCs on embryonic day (E) 12-13 and in an E16-17 gastrulating embryo. In vitro, hPGC-like cells (hPGCLCs) can be specified from the intermediary pluripotent stage or peri-gastrulation precursors. Here, we explore the broad spectrum of hPGCLC precursors and how different precursors impact hPGCLC development. We show that resetting precursors can give rise to hPGCLCs (rhPGCLCs) in response to BMP. Strikingly, rhPGCLCs co-cultured with human hindgut organoids progress at a pace reminiscent of in vivo hPGC devel-opment, unlike those derived from peri-gastrulation precursors. Moreover, rhPGCLC specification depends on both EOMES and TBXT, not just on EOMES as for peri-gastrulation hPGCLCs. Importantly, our study pro-vides the foundation for developing efficient in vitro models of human gametogenesis.

Summary Inhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium., Graphical Abstract, Highlights • Activin A and BMP4 expand potency of mouse ESCs • ASCs are hypermethylated and with stable genomic imprints • ASCs developmentally closed to E4.5–E6.5 in vivo epiblast • Hypermethylated ASCs directly derived from blastocyst by ABC/L medium, In this article, Bao and colleagues show that ASCs cultured in Activin A and BMP4, and in the absence of MEK1/2 inhibitor, possess reversible epigenetic changes that extend their developmental potency, distinct transcriptome pattern, and a stable hypermethylated epigenome. Importantly, they also derived novel hypermethylated ASCs from blastocysts in ABC/L medium.

Summary The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process., In this perspective, Rossant, Clark, and colleagues outline the process and deliberations that led to the updated ISSCR Guidelines for stem cell research involving the 14-Day rule, stem cell-based embryo models, and in vitro gametogenesis.

Summary The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here., The updated Guidelines for Stem Cell Research and Clinical Translation describe the ethical principles and best practices for basic, translational, and clinical research involving stem cells and human embryos. The updated Guidelines include new recommendations to address the recent scientific advances involving embryos, stem cell-based embryo models, chimeras, organoids, and genome editing.

Summary Post-implantation embryo development commences with a bilaminar disc in most mammals, including humans. Whereas access to early human embryos is limited and subject to greater ethical scrutiny, studies on non-primate embryos developing as bilaminar discs offer exceptional opportunities for advances in gastrulation, the germline, and the basis for evolutionary divergence applicable to human development. Here, we discuss the advantages of investigations in the pig embryo as an exemplar of development of a bilaminar disc embryo with relevance to early human development. Besides, the pig has the potential for the creation of humanized organs for xenotransplantation. Precise genetic engineering approaches, imaging, and single-cell analysis are cost effective and efficient, enabling research into some outstanding questions on human development and for developing authentic models of early human development with stem cells., Graphical abstract, Alberio et al. discuss conserved features of early development and the genesis of the germline in non-primates applicable to humans, with advantages of greater accessibility and lesser ethical concerns. The analysis at the resolution of single cells and genetic manipulations will provide insight into the evolution of gastrulating mammalian embryos for developing authentic in vitro models of early human development.

Summary Naive pluripotency can be maintained in medium with two inhibitors plus leukemia inhibitory factor (2i/LIF) supplementation, which primarily affects canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these three supplements alone is sufficient to maintain naive self-renewal remains unclear. Here we show that LIF alone in medium is sufficient for adaptation of 2i/L-ESCs to embryonic stem cells (ESCs) in a hypermethylated state (L-ESCs). Global transcriptomic analysis shows that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methyltransferases (DNMTs) play an important role in LIF-dependent mouse ESC adaptation and self-renewal. LIF-dependent ESC adaptation efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast stem cells, L-ESCs contribute to somatic tissues and germ cells in chimeras. L-ESCs cultured under such simple conditions as in this study would provide a more conducive platform to clarify the molecular mechanism of ESCs in in vitro culture., Graphical Abstract, Highlights • LIF alone supports ESC self-renewal and pluripotency in chemically defined media • L-ESCs re-establish the epigenetic state in LIF adaptation • DNMTs are important for LIF adaptation and L-ESC self-renewal • L-ESCs contribute to somatic tissues and germ cells in chimeras, In this article, Bao and colleagues show that LIF alone supports ESC self-renewal and pluripotency in chemically defined media. L-ESCs exhibit distinct transcriptional features and re-establish DNA hypermethylation. Moreover, DNMTs are important for L-ESC adaptation and self-renewal. Notably, L-ESCs also contribute to somatic tissues and germ cells in chimeras.

BACKGROUND: MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type. CONCLUSION/SIGNIFICANCE: These results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.

Background Specification of primordial germ cells in mice depends on instructive signalling events, which act first to confer germ cell competence on epiblast cells, and second, to impose a germ cell fate upon competent precursors. fragilis, an interferon-inducible gene coding for a transmembrane protein, is the first gene to be implicated in the acquisition of germ cell competence. Results Here, we describe four additional fragilis-related genes, fragilis2–5, which are clustered within a 68 kb region in the vicinity of the fragilis locus on Chr 7. These genes exist in a number of mammalian species, which in the human are also clustered on the syntenic region on Chr 11. In the mouse, fragilis2 and fragilis3, which are proximate to fragilis, exhibit expression that overlaps with the latter in the region of specification of primordial germ cells. Using single cell analysis, we confirm that all these three fragilis-related genes are predominant in nascent primordial germ cells, as well as in gonadal germ cells. Conclusion The Fragilis family of interferon-inducible genes is tightly associated with germ cell specification in mice. Furthermore, its evolutionary conservation suggests that it probably plays a critical role in all mammals. Detailed analysis of these genes may also elucidate the role of interferons as signalling molecules during development.

Early mammalian development entails a series of cell fate transitions that includes transit through naïve pluripotency to post-implantation epiblast. This subsequently gives rise to primordial germ cells (PGC), the founding population of the germline lineage. To investigate the gene regulatory networks that control these critical cell fate decisions, we developed a compound-reporter system to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with unbiased genome-wide CRISPR screening. This enabled identification of key genes both for exit from pluripotency and for acquisition of PGC fate, with further characterisation revealing a central role for the transcription factors Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in significantly impaired PGCLC development due to widespread activation (Nr5a2−/−) or inhibition (Zfp296−/−) of WNT pathway components. This leads to aberrant upregulation of the somatic programme or failure to appropriately activate germline genes in PGCLC, respectively, and consequently loss of germ cell identity. Overall our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate transitions.

Primordial germ cells (PGCs), the precursors of sperm and eggs, are established in perigastrulation-stage embryos in mammals. Signals from extra-embryonic tissues induce a unique gene regulatory network in germline-competent cells for PGC specification. This network also initiates comprehensive epigenome resetting, including global DNA demethylation and chromatin reorganization. Mouse germline development has been studied extensively, but the extent to which such knowledge applies to humans was unclear. Here, we review the latest advances in human PGC specification and epigenetic reprogramming. The overall developmental dynamics of human and mouse germline cells appear to be similar, but there are crucial mechanistic differences in PGC specification, reflecting divergence in the regulation of pluripotency and early development.

Pluripotent cells have the potential to differentiate into any cell type in the body. Their isolation and propagation from mouse embryos was pivotal for advances in understanding human development and disease.

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

Summary The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells., Graphical Abstract, Highlights • Germline deletion of Nanog reduces PGC numbers but does not abolish PGC development • Without Nanog, PGCLCs form ineffectively with less proliferation and more apoptosis • The Nanog target gene Esrrb can rescue PGCLC differentiation of Nanog−/− ESCs • Knockin of Esrrb at the Nanog locus restores PGC development efficiency, Although transcription factors functional in naive pluripotent cells are also expressed in primordial germ cells (PGCs), their PGC role remains unclear. Here, Zhang et al. show that, without Nanog, PGCs form ineffectively but that normal PGC development can be restored by induced expression of the NANOG target gene Esrrb.

The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.

Improved culture of non-human primate embryos reveals the establishment of the crucial framework for subsequent development of bodily tissues and the germline in fetuses, bringing us closer to comprehending the elusive development of early human embryos.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

MotivationA common class of behaviour encountered in the biological sciences involves branching and recombination. During branching, a statistical process bifurcates resulting in two or more potentially correlated processes that may under-go further branching; the contrary is true during recombination, where two or more statistical processes converge into one. A key objective is to identify the time of this bifurcation (branch time) from time series measurements e.g., comparing a control time series with a perturbed time series. Whilst statistical treatments for the two branch (control versus treatment) case exists, the ability to infer more complex branching structure from time series data remains open. Gaussian processes (GPs) represents an ideal framework for such analysis, allowing for nonlinear regression that includes a rigorous treatment of uncertainty. Currently, however, GP models only exist for two-branch systems. Here we highlight how arbitrarily complex branching processes can be built using the correct composition of covariance functions within a GP framework, thus outlining a general framework for the treatment of branching and recombination in the form of branch-recombinant Gaussian processes (B-RGPs). We first demonstrate the performance of B-RGPs compared to a variety of existing regression approaches, and demonstrate robustness to model misspecification. B-RGPs are then used to investigate the branching patterns ofArabidopsis thalianagene expression following inoculation with the hemibotrophic bacteria,Pseudomonas syringae DC3000, and a disarmed mutant strain, hrpA. By grouping genes according to the number of branches, we could naturally separate out genes involved in basal immune response from those subverted by the virulent strain, and show enrichment for targets of pathogen protein effectors. Finally, we identify two early branching genes WRKY11 and WRKY17, and showed that groups of genes that branched at similar times to WRKY11/17 were enriched for W-box binding motifs, and overrepresented for genes differentially expressed in WRKY11/17 knockouts, suggesting that branch time could be used for identifying direct and indirect binding targets of key transcription factors.Software is available from:https://github.com/cap76/BranchingGPs.

Naive hypomethylated embryonic pluripotent stem cells (ESCs) are developmentally closest to the preimplantation epiblast of blastocysts, with the potential to contribute to all embryonic tissues and the germline, excepting the extra-embryonic tissues in chimeric embryos. By contrast, epiblast stem cells (EpiSCs) resembling postimplantation epiblast are relatively more methylated and show a limited potential for chimerism. Here, for the first time, we reveal advanced pluripotent stem cells (ASCs), which are developmentally beyond the pluripotent cells in the inner cell mass but with higher potency than EpiSCs. Accordingly, a single ASC contributes very efficiently to the fetus, germline, yolk sac and the placental labyrinth in chimeras. Since they are developmentally more advanced, ASCs do not contribute to the trophoblast. ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor, followed by culturing in ABCL medium containing ActA, BMP4, CHIR99021 and leukemia inhibitory factor. Notably, ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes, as well as mesodermal somatic genes; Eomes, Eras, Tdgf1, Evx1, hand1, Wnt5a and distinct repetitive elements. Conversion of established ESCs to ASCs is also achievable. Importantly, ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints as compared to the hypomethylated inner cell mass of blastocysts and naive ESCs. Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2–3 of early post-implantation development$^{1}$. Using $\textit{in vitro}$ models of hPGC induction$^{2, 3, 4}$, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs$^{5}$. This may be due in part to the divergence in their pluripotency networks and early post-implantation development$^{6, 7, 8}$. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey $\textit{in vitro}$ models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program$^{9, 10, 11}$ and regulated by a balanced $\textit{SOX17–BLIMP1}$ gene dosage. Our combinatorial approach using human, porcine and monkey $\textit{in vivo}$ and $\textit{in vitro}$ models provides synthetic insights into early human development.

Embryonic stem cells (ESC) are characterised by the pluripotent capacity to generate all embryonic lineages. Here we show ESC can occupy a spectrum of distinct transcriptional and epigenetic states in response to varied extrinsic conditions. This spectrum broadly corresponds to a developmental continuum of pluripotency and is coupled with a gradient of increasing global DNA methylation. Each pluripotent state is linked with activation of distinct classes of transposable elements (TE), which in turn influence ESC through generating chimeric transcripts. Moreover, varied ESC culture parameters differentially license heterogeneous activation of master lineageregulators, including Sox1, Gata4 or Blimp1, and influence differentiation. Activation of Blimp1 is prevalent in 2i (without-LIF) conditions, and marks a dynamic primordial germ cell (PGC)-like sub-state, that is directly repressed by Klf4 downstream of LIF/STAT3 signalling. Thus, extrinsic cues establish a spectrum of pluripotent states, in part by modulating sub-populations, as well as directing the transcriptome, epigenome, and TE., Funding for this study came from a Wellcome Trust program grant (to M.A.S.), and Cancer Research UK (C6946/A14492)/Wellcome Trust (092096) core grants.

International audience; Meiosis is a critical phase in the life cycle of sexually reproducing organisms. Chromosome numbers are halved during meiosis, which requires meiosis-specific modification of chromosome behaviour. Furthermore, suppression of transposons is particularly important during meiosis to allow the transmission of undamaged genomic information between generations. Correspondingly, specialized genome defence mechanisms and nuclear structures characterize the germ line during meiosis. Survival of mammalian spermatocytes requires that the sex chromosomes form a distinct silenced chromatin domain, called the sex body. An enigmatic spherical DNA-negative structure, called the meiotic dense body, forms in association with the sex body. The dense body contains small non-coding RNAs including microRNAs and PIWI-associated RNAs. These observations gave rise to speculations that the dense body may be involved in sex body formation and or small non-coding RNA functions, e.g. the silencing of transposons. Nevertheless, the function of the dense body has remained mysterious because no protein essential for dense body formation has been reported yet. We discovered that the polycomb-related sex comb on midleg-like 1 (SCML1) is a meiosis-specific protein and is an essential component of the meiotic dense body. Despite abolished dense body formation, Scml1-deficient mice are fertile and proficient in sex body formation, transposon silencing and in timely progression through meiosis and gametogenesis. Thus, we conclude that dense body formation is not an essential component of the gametogenetic program in the mammalian germ line.

Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.

© 2016 Japan Society for Ova Research.Mammalian primordial germ cells (PGCs) are specified in the early post-implantation embryo. Attempts have been made to establish in vitro PGC development since the derivation of embryonic stem cells (ESCs) from blastocysts. Despite the advances made with mouse models, similar studies in human germ cell development have not progressed because practical and ethical reasons prevent the use of early human embryos. Recently, we and others developed a robust in vitro system for producing human primordial germ cell-like cells (hPGCLCs) from ESCs and induced pluripotent stem cells (iPSCs) by inducing competency for germ cells. Strikingly, the molecular mechanism for germline differentiation is not fully conserved between mouse and human, probably because of the differences in their early embryogenesis and regulation of the pluripotent state. Here, we present a review of the current status in the field of in vitro germ cell production from pluripotent stem cells, and discuss how its usefulness could be extended to clinical applications.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

During mammalian embryonic development, the trophectoderm and primitive endoderm give rise to extraembryonic tissues, while the epiblast differentiates into all somatic lineages and the germline. Remarkably, only a few classes of signaling pathways induce the differentiation of these progenitor cells into diverse lineages. Accordingly, the functional outcome of a particular signal depends on the developmental competence of the target cells. Thus, developmental competence can be defined as the ability of a cell to integrate intrinsic and extrinsic cues to execute a specific developmental program toward a specific cell fate. Downstream of signaling, there is the combinatorial activity of transcription factors and their cofactors, which is modulated by the chromatin state of the target cells. Here, we discuss the concept of developmental competence, and the factors that regulate this state with reference to the specification of mammalian primordial germ cells.

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.

During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.

Background Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated. Results Here, we show that ERG-associated protein with SET domain (ESET), a histone methyltransferase enzyme, maintains pluripotency through repression of Cdx2, a key trophectoderm determinant, by histone H3 lysine 9 trimethylation (H3K9me3) of the promoter region. Notably, this repression is mediated through the synergistic function of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4. ESET localises to the promyelocytic leukaemia (PML) nuclear bodies and is SUMOylated in ES cells. Interaction of ESET with Oct4 depends on a SUMO-interacting motif (SIM) in Oct4, which is critical for the repression of Cdx2. Conclusion Loss of ESET or Oct4 results in strikingly similar phenotypes both in ES cells with their differentiation into trophectoderm cells, and in early embryos where there is a failure of development of the pluripotent inner cell mass (ICM) of blastocysts. We propose that SUMOylated ESET-Oct4 complex is critical for both the initiation and maintenance of pluripotency through repression of differentiation, particularly of the trophectoderm lineage by epigenetic silencing of Cdx2.

Background Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes. Results Ago2 was deleted specifically in the growing oocytes. Although the Ago2-deficient oocytes are able to develop to mature oocytes, they have abnormal spindles and chromosomes that are unable to cluster together properly. This phenotype is very similar to the phenotype of Dicer-deficient oocytes. We examined the microRNA expression profile in the Ago2-deficient oocyte and found that the expression of most microRNAs was reduced by more than 80%. To determine the downstream genes that are regulated by Ago2, we used microarray analysis on Ago2-deficient oocytes and found that 512 genes were upregulated and 1,073 genes were downregulated (FC > 2, P < 0.05). Conclusion Our study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes.

SummarySpecification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal–mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

SummaryMore than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, “on/off” manner. Trim28+/D9 mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-“on” state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.Video Abstract

SummaryResetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5–7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.

Summary Investigations of the human germline and programming are challenging because of limited access to embryonic material. However, the pig as a model may provide insights into transcriptional network and epigenetic reprogramming applicable to both species. Here we show that, during the pre- and early migratory stages, pig primordial germ cells (PGCs) initiate large-scale epigenomic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and, potentially, base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3 as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis., Graphical Abstract, Highlights • Gene expression profiles of pig and human primordial germ cells are closely aligned • Pre-migratory pig PGCs undergo DNA demethylation, XCR, and histone remodeling • Identification of DNA demethylation-resistant loci in the pig germline, Zhu et al. show that pig primordial germ cells (PGCs) undergo DNA demethylation, histone remodeling, and X chromosome reactivation after specification. Pig PGCs retain few methylated loci after genome-wide demethylation, with potential for transgenerational inheritance. Species comparisons shows close similarities in transcriptional profiles of pig and human PGCs.