{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:09:39Z","timestamp":1792786179578,"version":"4.5.0"},"posted":{"date-parts":[[2025,9,25]]},"group-title":"Developmental Biology","reference-count":95,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T00:00:00Z","timestamp":1758758400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["postdoctoral fellowship"],"award-info":[{"award-number":["postdoctoral fellowship"]}],"id":[{"id":"https:\/\/ror.org\/00hasdx88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Seattle Hub for Synthetic Biology","award":["CZIF2023-008738"],"award-info":[{"award-number":["CZIF2023-008738"]}]},{"name":"Alex's Lemonade Stand Foundation","award":["19-15730"],"award-info":[{"award-number":["19-15730"]}],"id":[{"id":"https:\/\/ror.org\/038ja4880","id-type":"ROR","asserted-by":"crossref"}]},{"award":["1UM1HG011586"],"award-info":[{"award-number":["1UM1HG011586"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01HG010632"],"award-info":[{"award-number":["R01HG010632"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"The Allen Family Philanthropies","award":["Allen Discovery Center for Cell Lineage Tracing"],"award-info":[{"award-number":["Allen Discovery Center for Cell Lineage Tracing"]}],"id":[{"id":"https:\/\/ror.org\/01degd278","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Brotman Baty Institute for Precision Medicine","id":[{"id":"https:\/\/ror.org\/03jxvbk42","id-type":"ROR","asserted-by":"crossref"}]},{"name":"The Royalty Research Fund"},{"name":"Howard Hughes Medical Institute","award":["Howard Hughes Medical Institute Investigator"],"award-info":[{"award-number":["Howard Hughes Medical Institute Investigator"]}],"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Human gastruloids are a powerful class of stem cell-derived models that recapitulate key features of early embryonic development, including symmetry breaking and the emergence of three germ layers\n                  <jats:sup>1\u20133<\/jats:sup>\n                  . However, they lack anterior embryonic structures and coordinated axial organization\n                  <jats:sup>4\u20136<\/jats:sup>\n                  . To address this limitation, we pre-patterned human pluripotent stem cells (hPSCs) by exposing them to either anterior (FGF2) or posterior (CHIR99021 [CHIR] &amp; retinoic acid [RA]) cues. Upon mixing, these dual-patterned hPSCs interacted and self-organized into elongated structures with both anterior and posterior features\u2014which we term anterior-posterior (AP) human gastruloids. Anteriorly pre-treated cells robustly intercalated into posteriorly pre-treated cells, collectively giving rise to a continuum of neural tissues\u2014including a brain-like domain, a neural tube-like structure, and neuro-mesodermal progenitors (NMPs)\u2014with segmented somites arrayed bilaterally. Single cell RNA sequencing (scRNA-seq) revealed that human AP gastruloids contain cell types resembling the midbrain-hindbrain boundary (MHB), regionalized hindbrain structures (\n                  <jats:italic>i.e.<\/jats:italic>\n                  rhombomeres 1\u20138), regionalized neural crest (\n                  <jats:italic>i.e.<\/jats:italic>\n                  cranial, vagal, trunk)\n                  <jats:sup>7,8<\/jats:sup>\n                  and head mesoderm. Transcriptomic comparisons to primate embryos revealed that human AP gastruloids most closely resemble Carnegie stage 11 (CS11) embryos. While they lack a notochord and full dorsal-ventral polarity, human AP gastruloids recapitulate key spatial and temporal features of early neurulation and somitogenesis. Perturbation of folic acid metabolism or rho-associated kinase (ROCK) signaling induced spinal cord defects, phenocopying aspects of spina bifida and other neural tube defects, highlighting this model\u2019s potential for studying congenital disorders\n                  <jats:sup>9<\/jats:sup>\n                  . AP gastruloids may serve as a simple, robust, scalable platform for modeling coordinated human AP body axis development. More broadly, our results suggest that controlled interactions between differentially prepatterned progenitors can initiate self-organization of complex body axis features. The \u201cpattern-and-mix\u201d strategy may serve as a generalizable framework for assembling spatially organized stem cell models of mammalian development.\n                <\/jats:p>","DOI":"10.1101\/2025.09.25.678678","type":"posted-content","created":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T07:00:18Z","timestamp":1758870018000},"source":"Crossref","is-referenced-by-count":1,"title":["Dual-patterned pluripotent stem cells self-organize into a human embryo model with extended anterior-posterior patterning"],"prefix":"10.64898","author":[{"given":"Zukai","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengxiang","family":"Qiu","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"},{"name":"Department of Molecular and Systems Biology, Dartmouth College, Hanover, NH, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Connor A.","family":"Kubo","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stella","family":"Xu","sequence":"additional","affiliation":[{"name":"Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riza M.","family":"Daza","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva","family":"Nichols","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anh","family":"Vo","sequence":"additional","affiliation":[{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"},{"name":"Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary B.","family":"O\u2019Neill","sequence":"additional","affiliation":[{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"},{"name":"Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Choli","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jay","family":"Shendure","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences, University of Washington, Seattle, WA, USA"},{"name":"Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA"},{"name":"Seattle Hub for Synthetic Biology, Seattle, WA, USA"},{"name":"Brotman Baty Institute for 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