10 research outputs found
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Extracellular Signalling and Stem Cell Self-Renewal
In preimplantation mouse embryos, signalling and gene regulatory networks cooperate to determine lineage segregation, and modulating signalling in vitro allows for stem cell populations to be established from these lineages. Fibroblast growth factor (FGF) signalling triggers the differentiation of primitive endoderm (PrE) cells fated to contribute to the yolk sac, while cells unreceptive to FGF form the epiblast (Epi) that subsequently contributes to the embryo proper. In vitro, FGF signalling is required for preimplantation Epi-derived mouse ES cells to exit self-renewal. Conversely, in human ES cells and postimplantation Epi-derived mouse epiblast stem cells, FGF signalling is instead required for pluripotency maintenance. It remains unclear how these divergent outcomes arise, especially as these cells rely on a similar core pluripotency gene network.
This study demonstrates that ectopic expression of the PrE transcription factor Gata6 destabilises mouse ES cell pluripotency in vitro and upregulates PrE-associated genes independently of FGF signalling. As previous studies show that PrE specification is compromised in Fgf4-/- embryos, despite initiation of Gata6, this suggests FGF signalling and Gata6 cooperatively drive PrE specification in vivo. Characterising Gata6 function determines that it directly binds to both up- and downregulated gene targets and potently initiates reprogramming in multiple cell types, including human ES cells, suggesting it may also antagonise pluripotency in vivo.
Surprisingly, FGF stimulation negatively affects establishment of the pluripotent human Epi. Characterising alternative signalling pathways in the human embryo finds that modulating IGF signalling promotes proliferation of the human ICM, and similar to human ES cells, intact TGFβ/Nodal signalling is required for pluripotent gene expression in the Epi. Consequently, as signalling requirements in the human Epi appear somewhat distinct from both the mouse Epi and existing human ES cells, modulating embryo-specific signalling pathways may permit derivation of human ES cells that more accurately reflect the pluripotent Epi compartment
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An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.
The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis
Genome editing reveals a role for OCT4 in human embryogenesis.
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)
Defining the three cell lineages of the human blastocyst by single-cell RNA-seq
<p><strong>Abstract:</strong> Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNAsequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression<br>dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells<br>(hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with<br>mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.</p>
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<p><strong>Update: </strong>Boxplots contained in "Human lineages at blastocyst stage" include our own samples in addition to those from Yan et al that were used in Figs 3-6.</p
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An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.
The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis
Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.
Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells. Development 2015 Sep 15; 142(18):3151-65
Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease
Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention