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.

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.

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Protein variability in single cells has been studied extensively in populations, but little is known about temporal protein fluctuations in a single cell over extended times. We present here traces of protein copy number measured in individual bacteria over multiple generations and investigate their statistical properties, comparing them to previously measured population snapshots. We find that temporal fluctuations in individual cells exhibit the same properties as those previously observed in populations. Scaled fluctuations around the mean of each trace exhibit the universal distribution shape measured in populations under a wide range of conditions and in two distinct microorganisms; the mean and variance of the traces over time obey the same quadratic relation. Analyzing the individual protein traces reveals that within a cell cycle protein content increases exponentially, with a rate that varies from cycle to cycle. This leads to a compact description of the trace as a 3-variable stochastic process -exponential rate, cell cycle duration and value at the cycle start- sampled once a cycle. This description is sufficient to reproduce both universal statistical properties of the protein fluctuations. Our results show that the protein distribution shape is insensitive to sub-cycle intracellular microscopic details and reflects global cellular properties that fluctuate between generations.

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9-engineered live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism.