Nanog is the gateway to the pluripotent ground state.

Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.

Accessing the human trophoblast stem cell state from pluripotent and somatic cells.

Trophoblasts are specialized epithelial cells that perform critical functions during blastocyst implantation and mediate maternal-fetal communication during pregnancy. However, our understanding of human trophoblast biology remains limited since access to first-trimester placental tissue is scarce, especially between the first and fourth weeks of development. Moreover, animal models inadequately recapitulate unique aspects of human placental physiology. In the mouse system, the isolation of self-renewing trophoblast stem cells has provided a valuable in vitro model system of placental development, but the derivation of analogous human trophoblast stem cells (hTSCs) has remained elusive until recently. Building on a landmark study reporting the isolation of bona fide hTSCs from blastocysts and first-trimester placental tissues in 2018, several groups have developed methods to derive hTSCs from pluripotent and somatic cell sources. Here we review the biological and molecular properties that define authentic hTSCs, the trophoblast potential of distinct pluripotent states, and methods for inducing hTSCs in somatic cells by direct reprogramming. The generation of hTSCs from pluripotent and somatic cells presents exciting opportunities to elucidate the molecular mechanisms of human placental development and the etiology of pregnancy-related diseases.

Mechanisms of gene regulation in human embryos and pluripotent stem cells.

Pluripotent stem cells have broad utility in biomedical research and their molecular regulation has thus garnered substantial interest. While the principles that establish and regulate pluripotency have been well defined in the mouse, it has been difficult to extrapolate these insights to the human system due to species-specific differences and the distinct developmental identities of mouse versus human embryonic stem cells. In this Review, we examine genome-wide approaches to elucidate the regulatory principles of pluripotency in human embryos and stem cells, and highlight where differences exist in the regulation of pluripotency in mice and humans. We review recent insights into the nature of human pluripotent cells in vivo, obtained by the deep sequencing of pre-implantation embryos. We also present an integrated overview of the principal layers of global gene regulation in human pluripotent stem cells. Finally, we discuss the transcriptional and epigenomic remodeling events associated with cell fate transitions into and out of human pluripotency.

Recent insights into the naïve state of human pluripotency and its applications.

The past decade has seen significant interest in the isolation of pluripotent stem cells corresponding to various stages of mammalian embryonic development. Two distinct and well-defined pluripotent states can be derived from mouse embryos: "naïve" pluripotent cells with properties of pre-implantation epiblast, and "primed" pluripotent cells, resembling post-implantation epiblast. Prompted by the successful interconversion between these two stem cell states in the mouse system, several groups have devised strategies for inducing a naïve state of pluripotency in human pluripotent stem cells. Here, we review recent insights into the naïve state of human pluripotency, focusing on two methods that confer defining transcriptomic and epigenomic signatures of the pre-implantation embryo. The isolation of naïve human pluripotent stem cells offers a window into early developmental mechanisms that cannot be adequately modeled in primed cells, such as X chromosome reactivation, metabolic reprogramming, and the regulation of hominid-specific transposable elements. We outline key unresolved questions regarding naïve human pluripotency, including its extrinsic and intrinsic control mechanisms, potential for embryonic and extraembryonic differentiation, and general utility as a model system for human development and disease.

NANOG-dependent function of TET1 and TET2 in establishment of pluripotency.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.

Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.

Reprogramming capacity of Nanog is functionally conserved in vertebrates and resides in a unique homeodomain.

Pluripotency is a developmental ground state that can be recreated by direct reprogramming. Establishment of pluripotency is crucially dependent on the homeodomain-containing transcription factor Nanog. Compared with other pluripotency-associated genes, however, Nanog shows relatively low sequence conservation. Here, we investigated whether Nanog orthologs have the capacity to orchestrate establishment of pluripotency in Nanog(-/-) somatic cells. Mammalian, avian and teleost orthologs of Nanog enabled efficient reprogramming to full pluripotency, despite sharing as little as 13% sequence identity with mouse Nanog. Nanog orthologs supported self-renewal of pluripotent cells in the absence of leukemia inhibitory factor, and directly regulated mouse Nanog target genes. Related homeodomain transcription factors showed no reprogramming activity. Nanog is distinguished by the presence of two unique residues in the DNA recognition helix of its homeodomain, and mutations in these positions impaired reprogramming. On the basis of genome analysis and homeodomain identity, we propose that Nanog is a vertebrate innovation, which shared an ancestor with the Bsx gene family prior to the vertebrate radiation. However, cephalochordate Bsx did not have the capacity to replace mouse Nanog in reprogramming. Surprisingly, the Nanog homeodomain, a short sequence that contains the only recognizable conservation between Nanog orthologs, was sufficient to induce naive pluripotency in Nanog(-/-) somatic cells. This shows that control of the pluripotent state resides within a unique DNA-binding domain, which appeared at least 450 million years ago in a common ancestor of vertebrates. Our results support the hypothesis that naive pluripotency is a generic feature of vertebrate development.

Matrix remodeling maintains embryonic stem cell self-renewal by activating Stat3.

While a variety of natural and synthetic matrices have been used to influence embryonic stem cell (ESC) self-renewal or differentiation, and ESCs also deposit a rich matrix of their own, the mechanisms behind how extracellular matrix affects cell fate are largely unexplored. The ESC matrix is continuously remodeled by matrix metalloproteinases (MMPs), a process that we find is enhanced by the presence of mouse embryonic fibroblast feeders in a paracrine manner. Matrix remodeling by MMPs aids in the self-renewal of ESCs, as inhibition of MMPs inhibits the ability of ESCs to self-renew. We also find that addition of the interstitial collagenase MMP1 is sufficient to maintain long-term leukemia inhibitory factor (LIF)-independent mouse ESC (mESC) self-renewal in a dose-dependent manner. This remarkable ability is due to the presence of endogenously produced self-renewal-inducing signals, including the LIF-family ligand ciliary neurotrophic factor, that are normally trapped within the ECM and become exposed upon MMP-induced matrix remodeling to signal through JAK and Stat3. These results uncover a new role for feeder cells in maintaining self-renewal and show that mESCs normally produce sufficient levels of autocrine-acting pro-self-renewal ligands.

Generation of 3D Trophoblast Organoids from Human Naïve Pluripotent Stem Cells.

The human placenta is a transient organ that functions to support the needs of the fetus throughout gestation. Trophoblasts are the major epithelial cells found within the placenta and comprise a variety of distinct cell types with specialized roles in fetal-maternal communication. Our understanding of human trophoblast development remains limited due to ethical and legal restrictions on accessing first-trimester placental tissues, as well as the inability of common animal models to replicate primate placental development. It is therefore important to advance in vitro models of human trophoblast development as a basis for studying pregnancy-associated complications and diseases. In this chapter, we describe a protocol for generating 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs). The resulting stem-cell-derived trophoblast organoids (SC-TOs) contain distinct cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) cell types, which closely correspond to trophoblast identities in the human post-implantation embryo. We discuss methods for characterizing SC-TOs by immunofluorescence, flow cytometry, mRNA and microRNA expression profiling, and placental hormone secretion. Furthermore, SC-TOs can undergo differentiation into specialized 3D EVT organoids, which display robust invasion when co-cultured with human endometrial cells. Thus, the protocol described herein offers an accessible 3D model system of human placental development and trophoblast invasion.

Self-renewing human naïve pluripotent stem cells dedifferentiate in 3D culture and form blastoids spontaneously.

Human naïve pluripotent stem cells (hnPSCs) can generate integrated models of blastocysts termed blastoids upon switch to inductive medium. However, the underlying mechanisms remain obscure. Here we report that self-renewing hnPSCs spontaneously and efficiently give rise to blastoids upon three dimensional (3D) suspension culture. The spontaneous blastoids mimic early stage human blastocysts in terms of structure, size, and transcriptome characteristics and are capable of progressing to post-implantation stages. This property is conferred by the glycogen synthase kinase-3 (GSK3) signalling inhibitor IM-12 present in 5iLAF self-renewing medium. IM-12 upregulates oxidative phosphorylation-associated genes that underly the capacity of hnPSCs to generate blastoids spontaneously. Starting from day one of self-organization, hnPSCs at the boundary of all 3D aggregates dedifferentiate into E5 embryo-like intermediates. Intermediates co-express SOX2/OCT4 and GATA6 and by day 3 specify trophoblast fate, which coincides with cavity and blastoid formation. In summary, spontaneous blastoid formation results from 3D culture triggering dedifferentiation of hnPSCs into earlier embryo-like intermediates which are then competent to segregate blastocyst fates.

iPSC-based modeling of preeclampsia identifies epigenetic defects in extravillous trophoblast differentiation.

Preeclampsia (PE) is a hypertensive pregnancy disorder with increased risk of maternal and fetal morbidity and mortality. Abnormal extravillous trophoblast (EVT) development and function is considered to be the underlying cause of PE, but has not been previously modeled in vitro. We previously derived induced pluripotent stem cells (iPSCs) from placentas of PE patients and characterized abnormalities in formation of syncytiotrophoblast and responses to changes in oxygen tension. In this study, we converted these primed iPSC to naïve iPSC, and then derived trophoblast stem cells (TSCs) and EVT to evaluate molecular mechanisms underlying PE. We found that primed (but not naïve) iPSC-derived PE-EVT have reduced surface HLA-G, blunted invasive capacity, and altered EVT-specific gene expression. These abnormalities correlated with promoter hypermethylation of genes associated with the epithelial-mesenchymal transition pathway, specifically in primed-iPSC derived PE-EVT. Our findings indicate that abnormal epigenetic regulation might play a role in PE pathogenesis.

Modeling X-chromosome inactivation and reactivation during human development.

Stem-cell-based embryo models generate much excitement as they offer a window into an early phase of human development that has remained largely inaccessible to scientific investigation. An important epigenetic phenomenon during early embryogenesis is the epigenetic silencing of one of the two X chromosomes in female embryos, which ensures an equal output of X-linked gene expression between the sexes. X-chromosome inactivation (XCI) is thought to be established within the first three weeks of human development, although the inactive X-chromosome is reactivated in primordial germ cells (PGCs) that migrate to the embryonic gonads. Here, we summarize our current understanding of X-chromosome dynamics during human development and comment on the potential of recently established stem-cell-based models to reveal the underlying mechanisms.

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

A protein interaction network for pluripotency of embryonic stem cells.

Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.

Generating Trophoblast Stem Cells from Human Naïve Pluripotent Stem Cells.

The placenta is a transient organ that mediates the exchange of nutrients, gases, and waste products between the mother and the developing fetus and is indispensable for a healthy pregnancy. Epithelial cells in the placenta, which are termed trophoblasts, originate from the trophectoderm (TE) compartment of the blastocyst. The human trophoblast lineage consists of several distinct cell types, including the self-renewing and bipotent cytotrophoblast and the terminally differentiated extravillous trophoblast and syncytiotrophoblast. Despite the importance of trophoblast research, it has long been hindered by the scarce accessibility of primary tissue and the lack of a robust in vitro model system. Recently, a culture condition was developed that supports the isolation of bona fide human trophoblast stem cells (hTSCs) from human blastocysts or first-trimester placental tissues. In this chapter, we describe a protocol to derive bona fide hTSCs from naïve human pluripotent stem cells (hPSCs), thus presenting a robust methodology to generate hTSCs from a renewable and widely accessible source. This approach may be used to generate patient-specific hTSCs to study trophoblast-associated pathologies and serves as a powerful experimental platform to study the specification of human TE.

Induction of Human Naïve Pluripotency Using 5i/L/A Medium.

Prior to implantation, the cells in the mammalian epiblast constitute a naïve pluripotent state, which is distinguished by absence of lineage priming, freedom from epigenetic restriction, and expression of a unique set of transcription factors. However, human embryonic stem cells (hESCs) derived under conventional conditions have exited this naïve state and acquired a more advanced "primed" pluripotent state that corresponds to the post-implantation epiblast. We have developed a cocktail comprising five kinase inhibitors and two growth factors (5i/L/A) that enables induction of defining features of naïve pluripotency in primed hESCs. These conditions can also be applied to induce naïve pluripotency in patient-specific induced pluripotent stem cells (iPSCs). Here, we provide a detailed protocol for inducing naïve pluripotency in primed hESCs and iPSCs and methods for the routine validation of naïve identity. We also outline the use of two fluorescent reporter systems to track acquisition of naïve identity in live cells: (a) a GFP reporter linked to an endogenous OCT4 allele in which the primed-specific proximal enhancer has been deleted (OCT4-ΔPE-GFP); and (b) a dual-color reporter system targeted to both alleles of an X-linked gene that reports on the status of the X chromosome in female cells (MECP2-GFP/tdTomato). The conditions described herein have given insight into various aspects of naïve human pluripotent stem cells (hPSCs), including their unique transposon transcription profile, X chromosome status, and extraembryonic potential.

A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.