X chromosome reactivation dynamics reveal stages of reprogramming to pluripotency.

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

The human embryo selection arena is associated with transposable element activity.

Our current understanding of early human development is limited. A study in PLOS Biology found a previously undefined group of cells that diverges from the main lineages and undergo apoptosis through the activity of young transposable elements.

Keep the fate: how chromatin regulators safeguard embryonic stem cell identity.

Segregation of cells that form the embryo from those that produce the surrounding extra-embryonic tissues is critical for early mammalian development, but the regulatory layers governing these first cell fate decisions remain poorly understood. Recent work in The EMBO Journal identifies two chromatin regulators, Hdac3 and Dax1, that synergistically restrict the developmental potential of mouse embryonic stem cells and act as a lineage barrier to primitive endoderm formation.

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.

Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.

Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming.

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.

Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming.

Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.

Tox4 modulates cell fate reprogramming.

Reprogramming to induced pluripotency induces the switch of somatic cell identity to induced pluripotent stem cells (iPSCs). However, the mediators and mechanisms of reprogramming remain largely unclear. To elucidate the mediators and mechanisms of reprogramming, we used a siRNA-mediated knockdown approach for selected candidate genes during the conversion of somatic cells into iPSCs. We identified Tox4 as a novel factor that modulates cell fate through an assay that determined the efficiency of iPSC reprogramming. We found that Tox4 is needed early in reprogramming to efficiently generate early reprogramming intermediates, irrespective of the reprogramming conditions used. Tox4 enables proper exogenous reprogramming factor expression, and the closing and opening of putative somatic and pluripotency enhancers early during reprogramming, respectively. We show that the TOX4 protein assembles into a high molecular form. Moreover, Tox4 is also required for the efficient conversion of fibroblasts towards the neuronal fate, suggesting a broader role of Tox4 in modulating cell fate. Our study reveals Tox4 as a novel transcriptional modulator of cell fate that mediates reprogramming from the somatic state to the pluripotent and neuronal fate.This article has an associated First Person interview with the first author of the paper.

Murine iPSC-derived microglia and macrophage cell culture models recapitulate distinct phenotypical and functional properties of classical and alternative neuro-immune polarisation.

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX3CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX3CR1- iPSC-macrophages can easily be differentiated from newly established murine CX3CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX3CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX3CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.

Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes.

Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.

Mitosis gives a brief window of opportunity for a change in gene transcription.

Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

Nuclear reprogramming.

There is currently particular interest in the field of nuclear reprogramming, a process by which the identity of specialised cells may be changed, typically to an embryonic-like state. Reprogramming procedures provide insight into many mechanisms of fundamental cell biology and have several promising applications, most notably in healthcare through the development of human disease models and patient-specific tissue-replacement therapies. Here, we introduce the field of nuclear reprogramming and briefly discuss six of the procedures by which reprogramming may be experimentally performed: nuclear transfer to eggs or oocytes, cell fusion, extract treatment, direct reprogramming to pluripotency and transdifferentiation.

Histone variant macroH2A confers resistance to nuclear reprogramming.

How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

Epigenetic factors influencing resistance to nuclear reprogramming.

Patient-specific somatic cell reprogramming is likely to have a large impact on medicine by providing a source of cells for disease modelling and regenerative medicine. Several strategies can be used to reprogram cells, yet they are generally characterised by a low reprogramming efficiency, reflecting the remarkable stability of the differentiated state. Transcription factors, chromatin modifications, and noncoding RNAs can increase the efficiency of reprogramming. However, the success of nuclear reprogramming is limited by epigenetic mechanisms that stabilise the state of gene expression in somatic cells and thereby resist efficient reprogramming. We review here the factors that influence reprogramming efficiency, especially those that restrict the natural reprogramming mechanisms of eggs and oocytes. We see this as a step towards understanding the mechanisms by which nuclear reprogramming takes place.

Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency.

How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

Evaluation of Stem-Cell Embryo Models by Integration with a Human Embryo Single-Cell Transcriptome Atlas.

Single-cell RNA sequencing (scRNA-seq) revolutionized our understanding of the molecular processes of early development and provided us with the means to capture biological heterogeneity and assess the cellular composition in early embryos. Comparative analysis of the transcriptional landscapes of embryos with single-cell resolution allows us to better understand and improve stem-cell-based embryo models. However, proper comparison between different single-cell datasets acquired by different laboratories and through different technologies is imperative for adequate analysis and findings. In this chapter, we focus on the analysis of human blastoids, which model the blastocyst, and their integrative analysis with human embryo datasets and a 2D in vitro early development model system dataset, which models epiblast, extraembryonic mesoderm, and trophoblast cells.

Induction of Human Extraembryonic Mesoderm Cells from Naive Pluripotent Stem Cells.

The human extraembryonic mesoderm (EXM) is an important tissue in the postimplantation embryo which is specified before gastrulation in primates but not in rodents. EXM is mesenchymal and plays an important role in embryogenesis, including early erythropoiesis, and provides mechanical support to the developing embryo. Recently, it has been shown that self-renewing extraembryonic mesoderm cells (EXMCs) can be modeled in vitro by using human naive pluripotent stem cells. Here, we present a detailed step-by-step protocol to induce EXMCs from naive pluripotent stem cells in vitro.

Spatially Self-Organized Three-Dimensional Neural Concentroid as a Novel Reductionist Humanized Model to Study Neurovascular Development.

Although human pluripotent stem cell (PSC)-derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC-derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so-called concentroids. Such concentroids consisted of a pro-angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia-dense outer layer. Incorporating PSC-derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal-derived pericyte-like mural cells co-localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC-containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC-only concentroids. This PSC-derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood-brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics.