Deep-learning analysis of micropattern-based organoids enables high-throughput drug screening of Huntington's disease models.

Organoids are carrying the promise of modeling complex disease phenotypes and serving as a powerful basis for unbiased drug screens, potentially offering a more efficient drug-discovery route. However, unsolved technical bottlenecks of reproducibility and scalability have prevented the use of current organoids for high-throughput screening. Here, we present a method that overcomes these limitations by using deep-learning-driven analysis for phenotypic drug screens based on highly standardized micropattern-based neural organoids. This allows us to distinguish between disease and wild-type phenotypes in complex tissues with extremely high accuracy as well as quantify two predictors of drug success: efficacy and adverse effects. We applied our approach to Huntington's disease (HD) and discovered that bromodomain inhibitors revert complex phenotypes induced by the HD mutation. This work demonstrates the power of combining machine learning with phenotypic drug screening and its successful application to reveal a potentially new druggable target for HD.

Huntingtin CAG expansion impairs germ layer patterning in synthetic human 2D gastruloids through polarity defects.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFß signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFß receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.

Depletion of aneuploid cells in human embryos and gastruloids.

Chromosomal instability leading to aneuploidy is pervasive in early human embryos1-3 and is considered as a major cause of infertility and pregnancy wastage4,5. Here we provide several lines of evidence that blastocysts containing aneuploid cells are worthy of in vitro fertilization transfer. First, we show clinically that aneuploid embryos can lead to healthy births, suggesting the presence of an in vivo mechanism to eliminate aneuploidy. Second, early development and cell specification modelled in micropatterned human 'gastruloids' grown in confined geometry show that aneuploid cells are depleted from embryonic germ layers, but not from extraembryonic tissue, by apoptosis in a bone morphogenetic protein 4 (BMP4)-dependent manner. Third, a small percentage of euploid cells rescues embryonic tissue in mosaic gastruloids when mixed with aneuploid cells. Finally, single-cell RNA-sequencing analysis of early human embryos revealed a decline of aneuploidy beginning on day 3. Our findings challenge two current dogmas: that a single trophectoderm biopsy at blastocyst stage to perform prenatal genetic testing can accurately determine the chromosomal make-up of a human embryo, and that aneuploid embryos should be withheld from embryo transfer in association with in vitro fertilization.

Self-organizing neuruloids model developmental aspects of Huntington's disease in the ectodermal compartment.

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.

A 3D model of a human epiblast reveals BMP4-driven symmetry breaking.

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.

WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids.

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.

Synthetic embryology: controlling geometry to model early mammalian development.

Differentiation of embryonic stem cells in vitro is an important tool in dissecting and understanding the mechanisms that govern early embryologic development. In recent years, there has been considerable progress in creating organoids that model gastrulation, neurulation or organogenesis. However, one of the key challenges is reproducibility. Geometrically confining stem cell colonies considerably improves reproducibility and provides quantitative control over differentiation and tissue shape. Here, we review recent advances in controlling the two-dimensional or three-dimensional organization of cells and the effect on differentiation phenotypes. Improved methods of geometrical control will allow for an even more detailed understanding of the mechanisms underlying embryologic development and will eventually pave the way for the highly reproducible generation of specific tissue types.

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning.

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.

Chromosomal instability during neurogenesis in Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication. We conclude that disrupted neurogenesis during development is an important, unrecognized aspect of HD pathogenesis. To address the function of normal HTT protein we generated HTT+/- and HTT-/- lines. Surprisingly, the same phenotype emerged in HTT-/- but not HTT+/- lines. We conclude that HD is a developmental disorder characterized by chromosomal instability that impairs neurogenesis, and that HD represents a genetic dominant-negative loss of function, contrary to the prevalent gain-of-toxic-function hypothesis. The consequences of developmental alterations should be considered as a new target for HD therapies.

Channeling of Branched Flow in Weakly Scattering Anisotropic Media.

When waves propagate through weakly scattering but correlated, disordered environments they are randomly focused into pronounced branchlike structures, a phenomenon referred to as branched flow, which has been studied in a wide range of isotropic random media. In many natural environments, however, the fluctuations of the random medium typically show pronounced anisotropies. A prominent example is the focusing of tsunami waves by the anisotropic structure of the ocean floor topography. We study the influence of anisotropy on such natural focusing events and find a strong and nonintuitive dependence on the propagation angle which we explain by semiclassical theory.

A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization.

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 localize their receptors to transforming growth factor ß at 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. In addition, 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.

Self-organization of human embryonic stem cells on micropatterns.

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.

Distribution of the fittest individuals and the rate of Muller's ratchet in a model with overlapping generations.

Muller's ratchet is a paradigmatic model for the accumulation of deleterious mutations in a population of finite size. A click of the ratchet occurs when all individuals with the least number of deleterious mutations are lost irreversibly due to a stochastic fluctuation. In spite of the simplicity of the model, a quantitative understanding of the process remains an open challenge. In contrast to previous works, we here study a Moran model of the ratchet with overlapping generations. Employing an approximation which describes the fittest individuals as one class and the rest as a second class, we obtain closed analytical expressions of the ratchet rate in the rare clicking regime. As a click in this regime is caused by a rare, large fluctuation from a metastable state, we do not resort to a diffusion approximation but apply an approximation scheme which is especially well suited to describe extinction events from metastable states. This method also allows for a derivation of expressions for the quasi-stationary distribution of the fittest class. Additionally, we confirm numerically that the formulation with overlapping generations leads to the same results as the diffusion approximation and the corresponding Wright-Fisher model with non-overlapping generations.

Intensity fluctuations of waves in random media: what is the semiclassical limit?

Waves traveling through weakly random media are known to be strongly affected by their corresponding ray dynamics, in particular in forming linear freak waves. The ray intensity distribution, which, e.g., quantifies the probability of freak waves is unknown, however, and a theory of how it is approached in an appropriate semiclassical limit of wave mechanics is lacking. We show that this limit is not the usual limit of small wavelengths, but that of decoherence. Our theory, which can describe the intensity distribution for an arbitrary degree of coherence is relevant to a wide range of physical systems, as decoherence is omnipresent in real systems.

Universal statistics of branched flows.

Even very weak correlated disorder potentials can cause extreme fluctuations in Hamiltonian flows. In two dimensions this leads to a pronounced branching of the flow. Although present in a great variety of physical systems, a quantitative theory of the branching statistics is lacking. Here, we derive an analytical expression for the number of branches valid for all distances from a source. We also derive the scaling relations that make this expression universal for a wide range of random potentials. Our theory has possible applications in many fields ranging from semiconductor to geophysics.