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Cellular reprogramming can manipulate the identity of cells to generate the desired cell types 1 – 3 . The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can enforce somatic cell reprogramming to pluripotent stem cells 4 – 7 . By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner 8 – 10 . However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome 2 , 11 , 12 and reduced plasticity 13 , 14 ; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans. Human somatic cells were reprogrammed to human chemically induced pluripotent stem cells that demonstrate key features of embryonic stem cells.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development 1 – 4 . However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization. Human fibroblasts are reprogrammed to generate blastocyst-like structures called iBlastoids, which recapitulate aspects of embryo implantation.

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human–rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.Enthusiasm for patient-specific therapies based on induced pluripotent stem cells (iPSCs) has risen in parallel with rapid advances in genome editing. This Review summarizes the progress in iPSC-based disease modelling over the past decade, with a focus on 3D organoid systems and chimeric models being exploited for new therapeutic approaches.

Key Points Regulation of pluripotency and early post-implantation embryonic development have diverged between humans and mice, which might affect the mechanism of primordial germ cell (PGC) specification. Specification of human and mouse PGCs occurs in response to extrinsic signals, including bone morphogenetic protein 2 (BMP2) and BMP4. Models of human PGC specification from pluripotent stem cells suggest that human PGCs originate from mesodermal precursors at the posterior epiblast during the onset of gastrulation, whereas mouse PGCs originate from the pre-gastrulation epiblast. The gene regulatory network for PGC specification and maintenance in humans and mice has diverged. Notably, SRY-box 17 (SOX17), a key endoderm specifier, is critical for PGC specification in humans but not in mice. PGCs undergo genome-wide DNA demethylation, which erases parental epigenetic memories and facilitates germ cell differentiation in humans and mice. Repressive histone modifications might safeguard PGC genome stability during global DNA demethylation. In early germline development, extra-embryonic signals trigger a regulatory network that induces the specification and subsequent epigenetic reprogramming of primordial germ cells, the precursors of sperm and eggs. Here, the authors review germline specification and reprogramming in humans, and discuss the crucial mechanistic differences between these processes in humans and mice. 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.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)—that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.Shi et al. profiled small non-coding RNAs (sncRNAs) through PANDORA-seq, which identified tissue-specific transfer RNA- and ribosomal RNA-derived small RNAs, as well as sncRNAs, with dynamic changes during induced pluripotent stem cell reprogramming.

Using a single-nucleus Hi-C protocol, the authors find that spatial organization of chromatin during oocyte-to-zygote transition differs between paternal and maternal nuclei within a single-cell zygote. Oocyte-to-zygote chromatin reorganization It has been difficult to investigate chromosome organization in early embryos with genomic techniques owing to the paucity of cellular material. Here, Kikuë Tachibana-Konwalski and colleagues have developed a single-nucleus Hi-C protocol, which they apply to investigate chromatin organization during the developmental transition from oocytes to zygotes in mice. They find that chromatin architecture is distinct in the paternal and maternal pronuclei within a single-cell zygote. Zygotic maternal nuclei contain topological domains and loops but no A–B compartments, whereas compartments can be observed in paternal nuclei. Clusters of contacts are variable between individual cells and do not always match topological domains across populations. The authors propose that the organization of zygotic maternal chromatin represents a transition state towards that of totipotent cells. Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism 1 . The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods 2 , 3 , 4 , 5 are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method 2 . Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin ‘ground state’ could potentially provide insights into reprogramming cells to a state of totipotency.

Base-resolution maps of DNA methylation in human gametes and early embryos offer novel insights into human methylation dynamics and the functional relationship between DNA methylation and gene expression. DNA methylation in the early embryo Global patterns of DNA methylation are drastically reprogrammed in primordial germ cells and early embryonic development in mammals. This reprogramming has been well characterized in mouse embryos, but a detailed understanding of DNA methylation dynamics in human embryos is lacking. Two papers published this week [in this issue of Nature ] reveal there is a massive loss of DNA methylation from most of the human genome immediately after fertilization, confirming that this epigenetic reprogramming is an evolutionarily conserved feature of development. Hongshan Guo et al . produced base-resolution maps of DNA methylation for human gametes and at several developmental stages of embryogenesis. Zachary Smith et al . obtained similar maps of DNA methylation at several developmental stages of early human embryogenesis and during derivation of human embryonic stem cell lines. The studies provide insights into differences between mouse and human methylation dynamics and the functional relationship between DNA methylation and the expression of genes and transposable elements. DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development 1 , 2 , 3 , 4 , 5 . However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development. In vitro culture has detrimental effects on transcriptomes and epigenetic programming of zygotes. Here the authors use microfluidic technology to co-culture bovine oviduct epithelial cells with zygotes and show that the transcriptomes and global methylation patterns of these zygotes are more similar to in vivo zygotes than to conventionally cultured zygotes.

Three papers in this issue of Nature use highly sensitive ChIP–seq assays to describe the dynamic patterns of histone modifications during early mouse embryogenesis, showing that oocytes have a distinctive epigenome and providing insights into how the maternal gene expression program transitions to the zygotic program. Chromatin states in embryogenesis Genomic analysis of chromatin states in early embryos has been technically difficult, owing to the limited number of cells available for analysis. Three papers in this issue of Nature use highly sensitive ChIP–seq assays to describe the dynamic patterns of histone modifications during early mouse embryogenesis. Arne Klungland and colleagues find that the oocyte genome is associated with broad non-canonical domains of histone H3K4me3 which seem to function in preventing deposition of DNA methylation. Wei Xie and colleagues find that the oocyte genome is associated with broad non-canonical domains of histone H3K4me3 which overlap with domains of low DNA methylation and seem to contribute to gene silencing. Shaorong Gao and colleagues map histone H3K4me3 and H3K27me3 modifications in pre-implantation embryos and focus on the re-establishment of histone modifications during zygotic genome activation. They find that the breadth of H3K4me3 domains is highly dynamic and that H3K4me3 re-establishes rapidly on promoter regions whereas H3K27me3 is mostly absent from these regions. Taken together—and with previously published work—these studies show that the oocyte has a distinctive epigenome and provide insights into how the maternal gene expression program transitions to the zygotic program. Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Dynamic histone modifications may have important roles in MZT 10 , 11 , 12 , 13 , but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps 14 or require a highly specialized microfluidics device that is not readily available 15 . We developed a micro-scale chromatin immunoprecipitation and sequencing (μChIP–seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~ 22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.