@article{2b97375afcfa4b29b60f3561d74beca4,
title = "Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development",
abstract = "Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.",
keywords = "NODAL signaling, amnion, human embryoid, mesoderm, microfluidics, primate development, primitive streak, primordial germ cell, single-cell transcriptome, trophoblast",
author = "Yi Zheng and Yan, {Robin Zhexuan} and Shiyu Sun and Mutsumi Kobayashi and Lifeng Xiang and Ran Yang and Alexander Goedel and Yu Kang and Xufeng Xue and Esfahani, {Sajedeh Nasr} and Yue Liu and {Resto Irizarry}, {Agnes M.} and Weisheng Wu and Yunxiu Li and Weizhi Ji and Yuyu Niu and Chien, {Kenneth R.} and Tianqing Li and Toshihiro Shioda and Jianping Fu",
note = "Funding Information: Studies of μPASE are supported by the Michigan-Cambridge Research Initiative (J.F.), the National Institutes of Health ( R21 NS113518 and R21 HD100931 , J.F.), the National Science Foundation ( CMMI 1917304 and CBET 1901718, J.F.), and the 21st Century Jobs Trust Fund received through the Michigan Strategic Fund from the State of Michigan (Grant CASE-315037, J.F.). Studies of human blastocysts are supported by the Open Project of Yunnan Provincial Reproductive and Obstetrics and Gynecology Clinical Medicine Center ( zx2019-01-01 and 2020LCZXKF-SZ04 , L.X.). Non-human primate monkey studies are supported by the National Key Research and Development Program of China ( 2016YFA0101401 ), the Major Basic Research of Yunnan from China ( 2019FY002 ), and a Grant for Swedish-Chinese Collaboration from the Swedish Research Council (Dnr 539-2013-7002 ). M.K. and T.S. are partially supported by the RICBAC Foundation . K.R.C. is partially supported by a Grant for Distinguished Professors from the Swedish Research Council (Dnr 541-2013-8351 ). A.M.R.I. is partially supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE 1256260. A.M.R.I. and S.N.E. are partially supported by the University of Michigan Rackham Predoctoral Fellowship. The Lurie Nanofabrication Facility at the University of Michigan is acknowledged for support with microfabrication. Funding Information: In the original publication of the CS7 human gastrula (Tyser et al., 2021), a UMAP plot was generated with all of the cells in the human gastrula, including those at relatively late developmental stages, such as “Hemogenic Endothelial Progenitors” and “Erythrocytes,” which could negatively affect the resolution of cell clustering analysis, especially for those closely related cell lineages. To address this issue, we re-generated a UMAP plot using only the “Epiblast,” “Primitive Streak,” “Nascent Mesoderm,” “Emergent Mesoderm,” “Amniotic/embryonic ectoderm,” and “PGC” clusters from the human gastrula dataset (Figure 4D). Interestingly, this UMAP plot reveals distinct cell clusters corresponding to Meso and AM, supported by feature plots showing expression patterns of key Meso and AM markers (Figures 4D and 4E). Notably, human PGCs identified in the original publication are clustered together with AM cells in the UMAP plot (Figure 4D), supporting their close lineage relation. We further isolated the Epi, AM, and PGCs from the CS7 human gastrula to generate a diffusion map (Figure 4F). Surprisingly, AM and PGC seemingly display lineage bifurcation trajectories from the Epi (Figure 4F), similar to μPASEs in Figure 3A. In addition, as shown in Figure 4G, pseudotime gene expression dynamics of AM cells from the CS7 human gastrula are highly consistent with those of AMLCs in μPASEs in Figure 2D. However, because of the low number of PGCs, AM and PGC lineage bifurcation is not as evident as that shown in the μPASE diffusion map (Figure 3A), and we could not obtain pseudotime gene expression dynamics for PGCs.Studies of μPASE are supported by the Michigan-Cambridge Research Initiative (J.F.), the National Institutes of Health (R21 NS113518 and R21 HD100931, J.F.), the National Science Foundation (CMMI 1917304 and CBET 1901718, J.F.), and the 21st Century Jobs Trust Fund received through the Michigan Strategic Fund from the State of Michigan (Grant CASE-315037, J.F.). Studies of human blastocysts are supported by the Open Project of Yunnan Provincial Reproductive and Obstetrics and Gynecology Clinical Medicine Center (zx2019-01-01 and 2020LCZXKF-SZ04, L.X.). Non-human primate monkey studies are supported by the National Key Research and Development Program of China (2016YFA0101401), the Major Basic Research of Yunnan from China (2019FY002), and a Grant for Swedish-Chinese Collaboration from the Swedish Research Council (Dnr 539-2013-7002). M.K. and T.S. are partially supported by the RICBAC Foundation. K.R.C. is partially supported by a Grant for Distinguished Professors from the Swedish Research Council (Dnr 541-2013-8351). A.M.R.I. is partially supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE 1256260. A.M.R.I. and S.N.E. are partially supported by the University of Michigan Rackham Predoctoral Fellowship. The Lurie Nanofabrication Facility at the University of Michigan is acknowledged for support with microfabrication. Y.Z. and J.F. conceived and initiated the project. Y.Z. designed and performed microfluidic embryoid experiments and conducted scRNA-seq data analyses and interpretations. R.Z.Y. conducted CellChat analysis and participated in data analysis. S.S. participated in microfluidic embryoid experiments and repeated experiments with additional cell lines. M.K. and T.S. established and characterized NODAL-KO hPSC lines. L.X. Y. Li, and T.L. performed human embryo experiments. R.Y. A.G. W.J. Y.N. and K.R.C. performed monkey embryo experiments. X.X. S.N.E. Y. Liu, and A.M.R.I. helped with microfluidic embryoid experiments. W.W. helped with scRNA-seq data analyses. Y.Z. and J.F. wrote the manuscript. J.F. supervised the study. All authors edited and approved the manuscript. Two patents related to this work have been filed (US20190321415/WO2018106997 by J. Fu, Y. Zheng, and S.N. Esfahani; US2020049721/PCT/US20/49721 by J. Fu and Y. Zheng). Publisher Copyright: {\textcopyright} 2022 Elsevier Inc.",
year = "2022",
month = sep,
day = "1",
doi = "10.1016/j.stem.2022.08.009",
language = "English (US)",
volume = "29",
pages = "1402--1419.e8",
journal = "Cell Stem Cell",
issn = "1934-5909",
publisher = "Cell Press",
number = "9",
}