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 TGFβ 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 TGFβ 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

Evolutionarily Conserved Transcriptional Co-Expression Guiding Embryonic Stem Cell Differentiation

Understanding the molecular mechanisms controlling pluripotency in embryonic stem cells (ESCs) is of central importance towards realizing their potentials in medicine and science. Cross-species examination of transcriptional co-expression allows elucidation of fundamental and species-specific mechanisms regulating ESC self-renewal or differentiation.We examined transcriptional co-expression of ESCs from pathways to global networks under the framework of human-mouse comparisons. Using generalized singular value decomposition and comparative partition around medoids algorithms, evolutionarily conserved and divergent transcriptional co-expression regulating pluripotency were identified from ESC-critical pathways including ACTIVIN/NODAL, ATK/PTEN, BMP, CELL CYCLE, JAK/STAT, PI3K, TGFbeta and WNT. A set of transcription factors, including FOX, GATA, MYB, NANOG, OCT, PAX, SOX and STAT, and the FGF response element were identified that represent key regulators underlying the transcriptional co-expression. By transcriptional intervention conducted in silico, dynamic behavior of pathways was examined, which demonstrate how much and in which specific ways each gene or gene combination effects the behavior transition of a pathway in response to ESC differentiation or pluripotency induction. The global co-expression networks of ESCs were dominated by highly connected hub genes such as IGF2, JARID2, LCK, MYCN, NASP, OCT4, ORC1L, PHC1 and RUVBL1, which are possibly critical in determining the fate of ESCs.Through these studies, evolutionary conservation at genomic, transcriptomic, and network levels is shown to be an effective predictor of molecular factors and mechanisms controlling ESC development. Various hypotheses regarding mechanisms controlling ESC development were generated, which could be further validated by in vitro experiments. Our findings shed light on the systems-level understanding of how ESC differentiation or pluripotency arises from the connectivity or networks of genes, and provide a "road-map" for further experimental investigation

Tryptophan Scanning Analysis of the Membrane Domain of CTR-Copper Transporters

Membrane proteins of the CTR family mediate cellular copper uptake in all eukaryotic cells and have been shown to participate in uptake of platinum-based anticancer drugs. Despite their importance for life and the clinical treatment of malignancies, directed biochemical studies of CTR proteins have been difficult because high-resolution structural information is missing. Building on our recent 7Å structure of the human copper transporter hCTR1, we present the results of an extensive tryptophan-scanning analysis of hCTR1 and its distant relative, yeast CTR3. The comparative analysis supports our previous assignment of the transmembrane helices and shows that most functionally and structurally important residues are clustered around the threefold axis of CTR trimers or engage in helix packing interactions. The scan also identified residues that may play roles in interactions between CTR trimers and suggested that the first transmembrane helix serves as an adaptor that allows evolutionarily diverse CTRs to adopt the same overall structure. Together with previous biochemical and biophysical data, the results of the tryptophan scan are consistent with a mechanistic model in which copper transport occurs along the center of the trimer

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

Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter 1 (Ctr1) protein is a critical router of FGF signals during early embryogenesis. Ctr1 both promotes the differentiation and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr1 involves the activation of the Ras–MAP kinase cascade and appears to be independent of its role in copper transport. Mouse embryonic stem (ES) cells deficient for Ctr1 (Ctr1−/−) retain characteristics of pluripotency under conditions that favor differentiation in wild-type ES cells, indicating a conserved role for Ctr1 during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr1 functions as a key regulator of the differentiation capacity of both stem and progenitor cell populations