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In this article, we discuss the ethics of human embryoids, i.e., embryo-like structures made from pluripotent stem cells for modeling natural embryos. We argue that defining our social priorities is critical to design a consistent ethical guideline for research on those new entities. The absence of clear regulations on these emerging technologies stems from an unresolved debate surrounding natural human embryo research and one common opinion that one needs to solve the question of the moral status of the human embryo before regulating their surrogate. The recent NIH funding restrictions for research on human embryoids have made scientists even more unlikely to raise their voices. As a result, the scientific community has maintained a low profile while longing for a more favorable socio-political climate for their research. This article is a call for consistency among biomedical research on human materials, trying to position human embryoids within a spectrum of existing practice from stem cell research or IVF to research involving human subjects. We specifically note that the current practices in infertility clinics of freezing human embryos or disposing of them without any consideration for their potential benefits contradicts the assumption of special consideration for human material. Conversely, creating human embryoids for research purposes could ensure that no human material be used in vain, always serving humankind. We argue here that it is time to reconsider the full ban on embryo research (human embryos and embryoids) beyond the 14-day rule and that research on those entities should obey a sliding scale combining the completeness of the model (e.g., complete vs. partial) and the developmental stage: with more advanced completeness and developmental stage of the considered entity, being associated with more rigorous evaluation of societal benefits, statements of intention, and necessity of such research.

Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers. Organizing centers act to pattern surrounding tissues during embryogenesis through the secretion of morphogens. Here the authors model human organizers using light stimulus to geometrically confine SHH expression in differentiating hESCs, generating spatially resolved proximal distal patterns.

This paper demonstrates that micropatterned human embryonic stem cell colonies can acquire spatial patterns reminiscent of those in the embryo and proposes their use to study early developmental processes in the human system. Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model.

Embryonic stem cells derived from the inner cell mass can differentiate to all embryonic lineages of any adult cell type and to a limited extent to extra-embryonic tissues. A study now allows the generation of pig and human stem cells with enhanced differentiation potential towards all embryonic and extra-embryonic fates, one step closer to the totipotent state of the fertilized egg.

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. Embryonic stem cells can renew themselves to generate more stem cells, or specialize to become any type of cell found in an adult. They therefore hold great potential for studying how we develop from a single cell into a complex organism made of many different cell types. In a key stage of development, individual cells form into organized tissues. The earliest phase of tissue organization involves the formation of three ‘germ layers’. Human embryonic stem cells allow us to recreate this early stage of embryo development in the lab. When grown in confined spaces, the cells organize into clusters that can then develop germ layers. Previous work using these clusters showed that a network of signaling proteins – including one called WNT – trigger human embryonic stem cells to form the initial clusters. Then, another signaling protein called ACTIVIN tells the cells to specialize to form the two inner germ layers. But in experiments that apply only the ACTIVIN signal, the cells instead keep dividing to make more stem cells. ACTIVIN can trigger the activity of a protein called SMAD. To visualize how cells respond to ACTIVIN in real time, Yoney et al. used a gene editing technique called CRISPR to add fluorescent tags to SMAD in human embryonic stem cells. The results show that the ACTIVIN response triggers a peak in the amount of SMAD in the cell’s nucleus that then decreases over several hours. This briefly activated several genes that are known to help to form germ layers. However, this gene activity was not maintained for long enough to cause the stem cells to specialize and organize into layers. Yoney et al. then repeated the experiments on cells that had previously been exposed to WNT signaling proteins. The germ layer gene activity was maintained in this case, leading to the cells specializing and forming the inner two germ layers. This suggests that the cells somehow remembered the WNT signal, and this memory changed how they responded to ACTIVIN. The next step is to understand how cells store the memory of the WNT signal. As well as aiding our understanding of development, it could also help us to understand situations where signaling goes wrong, such as cancer. The technique used here to follow signals in real time could also be used to study other biological signaling processes.

This protocol describes how to differentiate and image human embryonic stem cells on micropatterned colonies to create radially organized domains of the germ layers mimicking embryonic gastrulation in vitro . Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into 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. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped, submillimeter colonies. After 42 h 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 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 (LN-521) as extracellular 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.

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP’s potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington’s Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP’s transcriptional activity can partially rescue the HD phenotype.

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington’s disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening. Micropatterning generates the four ectodermal cell types of human neurulation.

Breaking the anterior–posterior symmetry in mammals occurs at gastrulation. Much of the signalling network underlying this process has been elucidated in the mouse; however, there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro three-dimensional model of a human epiblast whose size, cell polarity and gene expression are similar to a day 10 human epiblast. A defined dose of BMP4 spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial-to-mesenchymal transition. We show that WNT signalling and its inhibitor DKK1 play key roles in this process downstream of BMP4. Our work demonstrates that a model human epiblast can break axial symmetry despite the absence of asymmetry in the initial signal and of extra-embryonic tissues or maternal cues. Our three-dimensional model is an assay for the molecular events underlying human axial symmetry breaking. Simunovic et al. use human embryonic stem cells to generate a three-dimensional model of a human pre-gastrulation epiblast and show that anterior–posterior symmetry breaking can be induced by BMP4 and WNT signalling.