Human embryo models: the importance of national policy and governance review

Integrated and non-integrated stem cell-based models of human embryos (SCB-EMs) are becoming widely adopted tools in biomedical research with distinct advantages over animal models for studying human development. Although SCB-EMs have tremendous benefits for research, they raise a number of social, ethical and legal questions which affect future research and widespread adoption in industry and clinical settings. The 2021 ISSCR guidelines for Stem Cell Research and Clinical Translation provide helpful guidance on many of these issues but do not have force in domestic law. Careful appraisal and development of national legal and ethical frameworks is crucial. Paving the way to better regulation provides an ethical and social foundation to continue using human embryo models and to fully realise their potential benefits for reproductive medicine

Dynamic proteomic profiling of extra-embryonic endoderm differentiation in mouse embryonic stem cells

During mammalian pre-implantation development, the cells of the blastocyst’s inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.This work was supported by the European Union 7th Framework Program (PRIME-XS project grant number 262067 to K.S.L., L.G and C.M.M), the Biotechnology and Biological Sciences Research Council (BBSRC grant number BB/L002817/1 to K.S.L and L.G.), as well as a HFSP grant (RGP0029/2010) and a European Research Council (ERC) Advanced Investigator grant to A.M.A.. C.S was supported by an EMBO long term fellowship and a Marie Curie IEF. L.T.Y.C. and K.K.N. were supported by the Medical Research Council (MRC, UK, MC_UP_1202/9) and the March of Dimes Foundation (FY11-436). We also thank Professor Steve Oliver and Dr. A.K.Hadjantonakis for helpful discussions and advice.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/stem.206

Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.

During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.This work was supported by the European Union 7th Framework Program (PRIME-XS project grant number 262067 to K.S.L., L.G and C.M.M), the Biotechnology and Biological Sciences Research Council (BBSRC grant number BB/L002817/1 to K.S.L and L.G.), as well as a HFSP grant (RGP0029/2010) and a European Research Council (ERC) Advanced Investigator grant to A.M.A.. C.S was supported by an EMBO long term fellowship and a Marie Curie IEF. L.T.Y.C. and K.K.N. were supported by the Medical Research Council (MRC, UK, MC_UP_1202/9) and the March of Dimes Foundation (FY11-436). We also thank Professor Steve Oliver and Dr. A.K.Hadjantonakis for helpful discussions and advice.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/stem.206

Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells.

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs

Self-organization of the human embryo in the absence of maternal tissues.

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.This work was supported by the Wellcome Trust grant to M.Z- G. Work in Dr. K.K.N lab was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK, the Medical Research Council and the Wellcome Trust. Dr. M.N.S. was initially supported by a Ramon Areces Spanish Foundation Fellowship, and subsequently by an EMBO Postdoctoral Fellowship. Dr. S.V was supported by a Post Doc Pool Grant from the Finnish Cultural Foundation. Dr. GR was supported by a Newton Fellowship.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Nature Publishing Group

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)

Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status

This work was funded by the BBSRC, grant BB/H088071/1 (www.bbsrc.ac.uk), MRC, grant G0700760 (www.mrc.ac.uk), Wellcome Trust, grant 095645/Z/11/Z (www.wellcome.ac.uk) and the EU through EpiGeneSys (www.epigenesys.eu) and Blueprint (www.blueprint-epigenome.eu). C.K. was funded by the DFG, personal fellowship KR 3317/2-1 (www.dfg.de) and CTR, personal short term fellowship (www.trophoblast.cam.ac.uk). M.J.H. received funding through grant NCI/NIH 2RO1 CA089426 (www.nih.gov)

Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse

AbstractEarly mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and differentiation. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription factors in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription factor OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in 128–256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription factor CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast growth factor receptor-1 (FGFR1), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription factor SOX17 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4–5dpf. SOX17 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription factor GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in differentiation mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human embryonic stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the outgrowth of TE-like cells, which impairs human embryonic stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation

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> <p> </p> <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