Summary The earliest aspects of human embryogenesis remain mysterious. To model patterning events in the human embryo we used colonies of human embryonic stem cells (hESCs) grown on micropatterned substrate and differentiated with BMP4. These gastruloids recapitulate the embryonic arrangement of the mammalian germ layers and provide an assay to assess the structural and signaling mechanisms patterning the human gastrula. Structurally, high-density hESCs lateralize their TGF-β receptors to their lateral side in the center of the colony, while maintaining apical localization of receptors at the edge. This relocalization insulates cells at the center from apically applied ligands while maintaining response to basally presented ones. Additionally, BMP4 directly induces the expression of its own inhibitor, Noggin, generating a reaction-diffusion mechanism that underlies patterning. We develop a quantitative model that integrates edge sensing and inhibitors, to predict human fate positioning in gastruloids, and potentially the human embryo.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions for the rapid differentiation of hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of 6 pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 days of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

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In vertebrates, the development of the nervous system is triggered by signals from a powerful 'organizing' region of the early embryo during gastrulation. This phenomenon--neural induction--was originally discovered and given conceptual definition by experimental embryologists working with amphibian embryos. Work on the molecular circuitry underlying neural induction, also in the same model system, demonstrated that elimination of ongoing transforming growth factor-β (TGFβ) signaling in the ectoderm is the hallmark of anterior neural-fate acquisition. This observation is the basis of the 'default' model of neural induction. Endogenous neural inducers are secreted proteins that act to inhibit TGFβ ligands in the dorsal ectoderm. In the ventral ectoderm, where the signaling ligands escape the inhibitors, a non-neural fate is induced. Inhibition of the TGFβ pathway has now been demonstrated to be sufficient to directly induce neural fate in mammalian embryos as well as pluripotent mouse and human embryonic stem cells. Hence the molecular process that delineates neural from non-neural ectoderm is conserved across a broad range of organisms in the evolutionary tree. The availability of embryonic stem cells from mouse, primates, and humans will facilitate further understanding of the role of signaling pathways and their downstream mediators in neural induction in vertebrate embryos.

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