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.

The asymmetric morphology of the mammalian heart is essential to its function as the organ of pulmonary and systemic double circulation. Left- right asymmetry is established by a leftward flow in the node that results in the asymmetric expression of Nodal. This triggers a cascade of asymmetric expression of downstream genes, such as Pitx2c, in the lateral plate mesoderm that gives rise to the first morphologically recognizable primordial heart structure, the cardiac crescent. Relatively little is known about gene expression asymmetries in the cardiac crescent that might underpin asymmetric cardiac morphogenesis. To systematically identify asymmetrically expressed genes, we performed a single-cell transcriptional analysis of manually dissected left and right halves of the cardiac crescent at stages spanning symmetry breaking. This revealed both left and right-sided genes that have not previously been implicated in left-right symmetry breaking. Some of these were expressed in multiple cell types but showed asymmetric expression in only a sub-set of cell types. We validated these findings using multiplexed in situ Hybridization Chain Reaction (HCR) and high- resolution volume imaging to characterize the expression patterns of select genes. Using Dnahiv/iv mutant embryos that show randomized situs, we established that all the genes tested tracked the asymmetric expression of Pitx2c, indicating their asymmetric expression also arose from the early asymmetries at the node. This study provides a high-fidelity molecular characterization of left- right symmetry breaking during cardiac crescent formation, providing a basis for future mechanistic studies on asymmetric cardiac morphogenesis.