Self-patterning of human stem cells into post-implantation lineages

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1–13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4–7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.


Statistics
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The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

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A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Single cell RNA sequencing is performed using 10x Genomic Chromium system The code generated in this study is provided at https://github.com/Smith-Lab-YSCC/hExtra_embryoid.For microscopy data, samples were imaged with the Leica STELLARIS 5 microscope in tilescan mode, using a HC PL APO CS2 40x/1.10water objective.

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Life sciences study design
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Sample size
No statistical methods were used to predetermine sample size.Sample sizes were based on similar studies performed on stem cell-based platforms modelling embryos (e.g.Data exclusions Culture experiments: on principle, data were only excluded for failed experiments, reasons for which included suboptimal culture conditions.
Spheroid structures displaying a clear inner and outer compartment were used for single cell RNA sequencing analysis.For data collection in embryonic symmetry breaking occurence, only structures that display an inner, embryonic compartment were counted.

Replication
Each result described in the paper is based on at least two independent biological replicates but very often an experiment is based on more.Randomization For experiments were our samples were exposed to chemical inhibitors, samples were randomly allocated to control and experimental groups.

Blinding
The majority of the experiments performed are descriptive, and therefore samples were not assigned to experimental groups.For inhibitor treatment experiments, control untreated and experimental treated groups were analysed.In the inhibitor treated, and knock-out groups, blinding was not possible, as inhibitor-treated/KO structures were easily distinguished based on morphological criteria.For all groups, healthy samples of representative stages and status were allocated to ensure comparability.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Figure legends indicate the number of independent experiments performed in each analysis.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Research guidelines for submitting code & software for further information.The scRNA-seq data for human extra-embryoids generated in this study have been deposited in the GEO database under accession code GSE208195.Published human gastrula in vivo, human post-implantation embryos in vitro, and Cynomolgus monkey in vitro embryo datasets used in this study were obtained fromTyser  et al. 2021, Xiang et al. 2020, and Ma et al. 2019under accession numbers E-MTAB-9388 (processed data available at http://www.human-gastrula.net),GSE136447, and GSE130114 respectively.For WGBS data, previously published hESC samples obtained from GSE126958 and the placenta samples obtained from GSE152104.Source data are provided with this paper.All other data is available upon request.