TY - JOUR
T1 - Complete human day 14 post-implantation embryo models from naive ES cells
AU - Oldak, Bernardo
AU - Wildschutz, Emilie
AU - Bondarenko, Vladyslav
AU - Comar, Mehmet-Yunus
AU - Zhao, Cheng
AU - Aguilera-Castrejon, Alejandro
AU - Tarazi, Shadi
AU - Viukov, Sergey
AU - Pham, Thi Xuan Ai
AU - Ashouokhi, Shahd
AU - Lokshtanov, Dmitry
AU - Roncato, Francesco
AU - Ariel, Eitan
AU - Rose, Max
AU - Livnat, Nir
AU - Shani, Tom
AU - Joubran, Carine
AU - Cohen, Roni
AU - Addadi, Yoseph
AU - Chemla, Muriel
AU - Kedmi, Merav
AU - Keren-Shaul, Hadas
AU - Pasque, Vincent
AU - Petropoulos, Sophie
AU - Lanner, Fredrik
AU - Novershtern, Noa
AU - Hanna, Jacob H.
N1 - The J.H.H. Laboratory was funded by Pascal and Ilana Mantoux; the Flight Attendant Medical Research Institute (FAMRI); a MBZUAI-WIS Program grant; the Israel Science Foundation (ISF)–Regular Research grant 1220/20; Minerva Stiftung grant; the Israel Cancer Research Fund (ICRF)–Research Professorship grant; Kimmel Stem Cell Research Center grants (2019-2023) at the Weizmann Institute, United states; Israel Binational Science Foundation (BSF) research grant 2017094; a sponsored research programme by RenewalBio; the Dr Barry Sherman Institute for Medicinal Chemistry; and the Helen and Martin Kimmel Institute for Stem Cell Research. This work in the Hanna Laboratory was also funded/co-funded by the European Union: ERC-COG-2022 #101089297–ExUteroEmbryogenesis (which funded mouse SEM-related experiments only) and ERC-CoG-2016 #726497–CELLNAIVETY. The views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. The F.L. Laboratory is supported by the Ming Wai Lau Center for Reparative Medicine; Ragnar Söderbergs Stiftelse; Wallenberg Academy Fellow; the Center for Innovative Medicine; and the Karolinska Institutet SFO Stem Cells and Regenerative Medicine. The S.P. Laboratory is supported by the Swedish Research Council and the Swedish Society for Medical Research, the Emil och Wera Cornells Stiftelse, the Canadian Institutes of Health Research (CIHR, PJT-178082), Fonds de recherche du Québec, and The Natural Sciences and Engineering Research Council of Canada (NSERC). S.P. holds the Canada Research Chair in Functional Genomics of Reproduction and Development (950-233204). Research in the Pasque Laboratory was supported by the Research Foundation–Flanders (FWO; Odysseus Grant G0F7716N; G0C9320N and G0B4420N.); the KU Leuven Research Fund (BOFZAP grant StG/15/021BF. and C1 grant C14/21/119); a FWO Ph.D. Fellowship to T.X.A.P. (11N3122N); Pandarome project 40007487 (G0I7822N) (funded by the FWO and F.R.S.-FNRS) under the Excellence of Science (EOS) programme. V.B. is supported by the Weizmann Postdoctoral Excellence Fellowship of the Feinberg Graduate School of Science. We thank Y. Stelzer for assistance on using his previously published pipeline for scRNA-seq analysis of the placental compartment in mouse SEMs (Extended Data Fig. 3a,b); and the Weizmann Institute Board and Management for providing critical financial and infrastructural support to the Hanna Lab. J.H.H. thanks Y. Groner for his scientific guidance and discussions. We acknowledge the Virtual Human Embryo (VHE) for permission to use VHE images (https://www.ehd.org/virtual-human-embryo/) in Figs. 2a, 3j,k, 4c and 5e. Author contributions: B.O and E.W established the SEM aggregation conditions and protocols for ex utero culture, designed and conducted most of the wet lab work and contributed on manuscript elaboration. B.O established the human stem cell conditions, and inductions. E.W and V.B conducted most embryo immunostaining and confocal imaging. V.B made light sheet microscopy analysis and helped writing the manuscript. B.O. generated cell lines with assistance from S.V and optimized the final SEM protocol. A.A.C conducted some aggregation experiments and roller culture adaptation for the system, ACCELERATED immunostaining and microscopy and sample preparation for 10X scRNA-seq experiments. M.Y.C helped on human stem cell culture expansion and SEM protocol optimizations and reproducibility. C.Z conducted the sc-RNAseq comparative analysis to previous human datasets, under the supervision of F.L and S.P. T.X.A.P conducted integrative scRNA-seq analysis of TE and ExEM-like cells with existing 2D reference datasets under the supervision of V.P. S.T contributed on optimization of lineage inductions. R.C generated MEF and other critical reagents for stem cell maintenance. S.A and D.L conducted immunostainings and RT-PCR. F.R, C.J, M.R assisted in immunostainings. N.L assisted on lentivirus production and flow cytometry experiments. E.A supervised flow cytometry and sorting experiments. T.S helped in bioinformatic analysis. S.V generated plasmids. Y.A. assisted in light-sheet microscope operation and data analysis. A.A.C, M.K., M.C. and H.K.S performed RNA library preparation and sequencing. S.A provided input on optimizing light sheet microscopy experimentation and analysis. N.N. – conducted and supervised bioinformatics analyses. B.O., E.W and M.Y.C independently reproduced human SEM generation in the Hanna lab. J.H.H. conceived the idea for the project, supervised data analysis and manuscript writing.
PY - 2023/10/19
Y1 - 2023/10/19
N2 - The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation 1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking 1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs) 3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions) 4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.
AB - The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation 1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking 1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs) 3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions) 4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.
UR - http://www.scopus.com/inward/record.url?scp=85173067123&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s41586-023-06604-5
DO - https://doi.org/10.1038/s41586-023-06604-5
M3 - مقالة
SN - 1476-4687
VL - 622
SP - 562
EP - 573
JO - Nature
JF - Nature
IS - 7983
ER -