{"id":["DOI:10.1038/s41467-025-61830-x"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"publication_date":["2025-07-28"],"authors":{"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0009-0005-5450-8496":{"id":["ORCID:0009-0005-5450-8496"],"family_name":["McCarthy"],"personal_name":["Afshan"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}},"HDBItheme":["Early"]},"ORCID:0000-0003-4752-025X":{"id":["ORCID:0000-0003-4752-025X"],"family_name":["Woods"],"personal_name":["Laura"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-1330-2158":{"id":["ORCID:0000-0003-1330-2158"],"family_name":["Staneva"],"personal_name":["Desislava"],"affiliations":{"ROR:02hmmse52":{"organization_name":["The Loke Centre for Trophoblast Research"]}}},"ORCID:0000-0002-8178-3128":{"id":["ORCID:0000-0002-8178-3128"],"family_name":["Proks"],"personal_name":["Martin"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0002-8155-4296":{"id":["ORCID:0000-0002-8155-4296"],"family_name":["Salehin"],"personal_name":["Nazmus"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0009-0003-1386-3438":{"id":["ORCID:0009-0003-1386-3438"],"family_name":["Lea"],"personal_name":["Georgia"],"affiliations":{"ROR:02hmmse52":{"organization_name":["The Loke Centre for Trophoblast Research"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-3232-8436":{"id":["ORCID:0000-0002-3232-8436"],"family_name":["Huang"],"personal_name":["Qiulin"],"affiliations":{"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-2517-7972":{"id":["ORCID:0000-0003-2517-7972"],"family_name":["Linneberg-Agerholm"],"personal_name":["Madeleine"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"AUTHTEMP:00057":{"id":["AUTHTEMP:00057"],"family_name":["Faulkner"],"personal_name":["Alex"],"affiliations":{"ROR:05p40t847":{"organization_name":["Newcastle upon Tyne Hospitals NHS Foundation Trust"]}}},"AUTHTEMP:0002":{"id":["AUTHTEMP:0002"],"family_name":["Papathanasiou"],"personal_name":["Athanasios"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"ORCID:0000-0003-3510-8268":{"id":["ORCID:0000-0003-3510-8268"],"family_name":["Elder"],"personal_name":["Kay"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"AUTHTEMP:0003":{"id":["AUTHTEMP:0003"],"family_name":["Snell"],"personal_name":["Phil"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall Clinic"]}}},"AUTHTEMP:0004":{"id":["AUTHTEMP:0004"],"family_name":["Christie"],"personal_name":["Leila"],"affiliations":{"ROR:04qg15997":{"organization_name":["Bourn Hall 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Trust"]}},"HDBItheme":["Early"]},"ORCID:0000-0001-8607-5748":{"id":["ORCID:0000-0001-8607-5748"],"family_name":["Roy"],"personal_name":["Anindita"],"affiliations":{"ROR:052gg0110":{"organization_name":["University of Oxford"]},"ROR:02khxwt12":{"organization_name":["MRC Molecular Haematology Unit"]},"ROR:0187kwz08":{"organization_name":["National Institute for Health Research"]}}},"ORCID:0000-0003-1580-7491":{"id":["ORCID:0000-0003-1580-7491"],"family_name":["Brickman"],"personal_name":["Joshua M."],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]}},"abstract":["Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner."],"datasets":{"GEO:GSE250614":{"id":["GEO:GSE250614"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"description":["Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naive human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner."],"authors":{"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]}},"tags":["sequencing"]},"GEO:GSE297052":{"id":["GEO:GSE297052"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"description":["Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.\nDay 5 human embryos were treated with 5µM Ulixertinib (Cambridge Bioscience); 0.1% DMSO for 36 hours and single-cell RNA sequencing was performed using the SMART-seq protocol from Takara."],"authors":{"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]},"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-8178-3128":{"id":["ORCID:0000-0002-8178-3128"],"family_name":["Proks"],"personal_name":["Martin"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0002-8155-4296":{"id":["ORCID:0000-0002-8155-4296"],"family_name":["Salehin"],"personal_name":["Nazmus"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0003-1580-7491":{"id":["ORCID:0000-0003-1580-7491"],"family_name":["Brickman"],"personal_name":["Joshua M."],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0002-0975-1121":{"id":["ORCID:0000-0002-0975-1121"],"family_name":["Biggins"],"personal_name":["Laura"],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]}}}},"tags":["sequencing"]},"GEO:GSE297478":{"id":["GEO:GSE297478"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst [mouse_ERKi_scRNAseq]"],"description":["Day 3.25 mouse embryos were treated for 24 hours with 5µM Ulixertinib or 0.1% DMSO and collected at day 4.25 for single-cell RNA sequencing using the SMART-seq protocol from Takara."],"authors":{"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]},"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-8178-3128":{"id":["ORCID:0000-0002-8178-3128"],"family_name":["Proks"],"personal_name":["Martin"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0002-8155-4296":{"id":["ORCID:0000-0002-8155-4296"],"family_name":["Salehin"],"personal_name":["Nazmus"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0003-1580-7491":{"id":["ORCID:0000-0003-1580-7491"],"family_name":["Brickman"],"personal_name":["Joshua M."],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}}},"tags":["sequencing"]},"GEO:GSE239843":{"id":["GEO:GSE239843"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst [RNA-seq I]"],"description":["Human d6.5 blastocysts incubated with Ulixertinib to suppress ERK signalling or with DMSO as a control. Embryos were dissected by immunosurgery and single-cells collected for analysis by RNA-seq."],"authors":{"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-4752-025X":{"id":["ORCID:0000-0003-4752-025X"],"family_name":["Woods"],"personal_name":["Laura"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]}},"tags":["sequencing"]},"GEO:GSE250613":{"id":["GEO:GSE250613"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst [RNA-seq II]"],"description":["Naïve hESCs derived in PXGL or UXGL media following human embryo treatment with either Ulixertinib or DMSO. The embryo-derived naïve hESCs were transcriptionally profiled by bulk RNA-seq analysis."],"authors":{"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]},"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-4752-025X":{"id":["ORCID:0000-0003-4752-025X"],"family_name":["Woods"],"personal_name":["Laura"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-1330-2158":{"id":["ORCID:0000-0003-1330-2158"],"family_name":["Staneva"],"personal_name":["Desislava"],"affiliations":{"ROR:02hmmse52":{"organization_name":["The Loke Centre for Trophoblast Research"]}}}},"tags":["sequencing"]},"EGA:EGAD50000001475":{"id":["EGA:EGAD50000001475"],"title":["PBAT sequencing of human embryonic stem cells"],"description":["To assess the DNA methylation landscape of naïve human embryonic stem cells derived from ERKi or control blastocysts using PXGL or UXGL media."],"authors":{}},"DOI:10.6084/m9.figshare.28597145":{"id":["DOI:10.6084/m9.figshare.28597145"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"description":["Confocal microscopy images (.lif) of human embryos from the manuscript: Simon et. al., (2025) Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst.\nFGF experiments (related to Fig. 1a):\n\n    Experiments 220318, 220512, 220520, 220705, 241203, and 241223\n    Control vs FGF (250ng/ml FGF4 + Heparin, 500ng/ml FGF4 + Heparin, and 750ng/ml FGF4 + Heparin) treated Day 5 human embryos for 36 hours.\n    Immunofluorescence staining for GATA4 (A488, hypoblast), NANOG (A594, epiblast), and GATA3 (A647, trophectoderm) and counterstained with DAPI.\n    Imaged on Leica SP8 Scanning Confocal Microscopy.\n\npERK experiments (related to Fig. 2a):\n\n    Experiments 211021, 211105 and 220120\n    Control Day 5, Day 6 and Day 6.5 human embryos\n    Immunofluorescence staining for SOX2 (A488, epiblast), pERK (A594) and OTX2 (A647, hypoblast) and counterstained with DAPI.\n    Imaged on Leica SP8 Scanning Confocal Microscopy.\n\nERKi experiments (related to Fig. 2d and 4a):\n\n    Experiments 220205, 220225, and 220311\n    Control (0.1% DMSO) vs ERKi (5μM Ulixertinib) treated Day 5 human embryos for 36 hours.\n    Immunofluorescence staining for GATA4 (A488, hypoblast), NANOG (A594, epiblast), and GATA3 (A647, trophectoderm) and counterstained with DAPI.\n    Imaged on Leica SP8 Scanning Confocal Microscopy.\n\n    Experiment 231212\n    Control (0.1% DMSO) vs ERKi (5μM Ulixertinib) treated Day 5 human embryos for 36 hours.\n    Immunofluorescence staining for KLF17 (A488 - naive pluripotency), KLF4 (A555, failed), and SOX2 (A647, epiblast) and counterstained with DAPI.\n    Imaged on Leica SP8 Scanning Confocal Microscopy.\n\nPlease see manuscript for detailed materials and methods\nDownstream quantification of fluorescence intensities performed with an image analysis pipeline available on GitLab: https://gitlab.developers.cam.ac.uk/ctr/ctr-bioinformatics/niakan-lab/simon-et-al-2025"],"authors":{"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}}}},"DOI:10.5281/zenodo.15128175":{"id":["DOI:10.5281/zenodo.15128175"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"description":["Processed embryo scRNAseq data"],"authors":{"ORCID:0000-0001-9614-1403":{"id":["ORCID:0000-0001-9614-1403"],"family_name":["Simon"],"personal_name":["Claire S."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-1646-4734":{"id":["ORCID:0000-0003-1646-4734"],"family_name":["Niakan"],"personal_name":["Kathy K."],"affiliations":{"ROR:01d5qpn59":{"organization_name":["Babraham Institute"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:05nz0zp31":{"organization_name":["Wellcome/MRC Cambridge Stem Cell Institute"]},"ROR:04tnbqb63":{"organization_name":["The Francis Crick Institute"]}},"HDBItheme":["Early"]},"ORCID:0000-0003-4752-025X":{"id":["ORCID:0000-0003-4752-025X"],"family_name":["Woods"],"personal_name":["Laura"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"AUTHTEMP:00057":{"id":["AUTHTEMP:00057"],"family_name":["Faulkner"],"personal_name":["Alex"],"affiliations":{"ROR:05p40t847":{"organization_name":["Newcastle upon Tyne Hospitals NHS Foundation Trust"]}}},"ORCID:0000-0002-8178-3128":{"id":["ORCID:0000-0002-8178-3128"],"family_name":["Proks"],"personal_name":["Martin"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0002-8155-4296":{"id":["ORCID:0000-0002-8155-4296"],"family_name":["Salehin"],"personal_name":["Nazmus"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0009-0003-1386-3438":{"id":["ORCID:0009-0003-1386-3438"],"family_name":["Lea"],"personal_name":["Georgia"],"affiliations":{"ROR:02hmmse52":{"organization_name":["The Loke Centre for Trophoblast Research"]},"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0003-2517-7972":{"id":["ORCID:0000-0003-2517-7972"],"family_name":["Linneberg-Agerholm"],"personal_name":["Madeleine"],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}},"ORCID:0000-0003-1580-7491":{"id":["ORCID:0000-0003-1580-7491"],"family_name":["Brickman"],"personal_name":["Joshua M."],"affiliations":{"ROR:04txyc737":{"organization_name":["Novo Nordisk Foundation"]},"ROR:035b05819":{"organization_name":["University of Copenhagen"]}}}},"tags":["sequencing"]}},"code":{"SWHID:swh:1:dir:f193b7d168bf727dee933ac636887eed2a6b6644":{"id":["SWHID:swh:1:dir:f193b7d168bf727dee933ac636887eed2a6b6644"],"title":["Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst"],"description":{},"authors":{}}},"HDBItheme":["Early"]}