Modeling mammalian gastrulation with embryonic stem cells

Understanding cell fate patterning and morphogenesis in the mammalian embryo remains a formidable challenge. Recently, in vivo models based on embryonic stem cells (ESCs) have emerged as complementary methods to quantitatively dissect the physical and molecular processes that shape the embryo. Here we review recent developments in using embryonic stem cells to create both two and three-dimensional culture models that shed light on mammalian gastrulation.Comment: 18 pages, 1 figur

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

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

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

Beyond defoaming:the effects of antifoams on bioprocesses productivity

Antifoams are often added to bioprocesses with little knowledge of their impact on the cells or product. However, it is known that certain antifoams can affect the growth rates of both prokaryotic and eukaryotic organisms in addition to changing surface properties such as lipid content, resulting in changes to permeability. This in turn can be beneficial to a recombinant protein production system for soluble proteins, as has been demonstrated by increased secretion of a-amylase and GFP, or achievement of greater yields of protein due to increased biomass. However, in some cases, certain concentrations of antifoams appear to have a detrimental effect upon cells and protein production, and the effects vary depending upon the protein being expressed. These findings emphasise the importance of optimising and understanding antifoam addition to bioprocesses

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

Geometrical confinement controls the asymmetric patterning of brachyury in cultures of pluripotent cells

International audienceDiffusible signals are known to orchestrate patterning during embryogenesis, yet diffusion is sensitive to noise. The fact that embryogenesis is remarkably robust suggests that additional layers of regulation reinforce patterning. Here, we demonstrate that geometrical confinement orchestrates the spatial organisation of initially randomly positioned subpopulations of spontaneously differentiating mouse embryonic stem cells. We use micropatterning in combination with pharmacological manipulations and quantitative imaging to dissociate the multiple effects of geometry. We show that the positioning of a pre-streak-like population marked by brachyury (T) is decoupled from the size of its population, and that breaking radial symmetry of patterns imposes polarised patterning. We provide evidence for a model in which the overall level of diffusible signals together with the history of the cell culture define the number of T+ cells, whereas geometrical constraints guide patterning in a multi-step process involving a differential response of the cells to multicellular spatial organisation. Our work provides a framework for investigating robustness of patterning and provides insights into how to guide symmetry-breaking events in aggregates of pluripotent cells

Synthèse de complexes de métaux de transition optiquement purs et étude photophysique en présence d'ADN

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Vers un contrôle magnétique de la polarité cellulaire

L'imagerie In Vivo a montré comment l'établissement et la maintenance de la polarité cellulaire dépend de mécanismes complexes au travers lesquels des voies de signalisation sont régulées à l'échelle sub-cellulaire. L'intégration de ces réseaux dans l'espace intracellulaire d'une cellule polarisé n'est pas bien connue. Nous avons développé une approche permettant de sonder et perturber localement des voies de signalisation au sein de cellules vivantes : des nanoparticules magnétiques (NPMs), fonctionnalisées avec des protéines d'intérêt, sont insérées dans le cytoplasme de cellules adhérentes où elles se comportent comme des plateformes solides de signalisation. En exerçant des forces magnétiques, les NPMs sont transportées dans le cytosol pour localiser leur activité de signalisation. Nous montrons que des NPMs de différentes tailles, de 50 nm à 500 nm, peuvent être utilisées pour créer différentes distributions de protéines actives. Alors que les plus grosses particules nécessitent des forces importantes pour être transportées (> 10pN), elles permettent de créer des perturbations ponctuelles précises à la périphérie cellulaire. A l'opposé, de plus petites NPMs diffusent vite dans le cytosol (~1 m /s) et sont utilisées pour créer des gradients d'activité à l'extension comparable à la taille de la cellule. Nous avons appliqué notre approche aux voies de signalisation associées aux Rho-GTPases. En fonctionnalisant les NPMs avec des GEFs ou des GTPases actives nous avons démontré que la voie de signalisation qui lie Rac1 à la polymérisation d'actine est spatialement contrainte dans les parties protrusives des cellules. La création de gradients de GTPases est enfin discutéeIn vivo imaging has shown how the establishment and maintenance of cell polarity relies on complex mechanisms by which signaling cascades become regulated at sub-cellular levels. How signaling networks are spatio-temporally coordinated into a polarized cell is not elucidated. In this context, we developed a new tool to locally probe and perturb signaling pathways inside living cells. In our approach, magnetic nanoparticles (MNPs) functionalized with active proteins are inserted in the cytosol of mammalian cells where they behave as solid signaling platforms. By exerting magnetic forces, MNPs are manipulated in the cytosol to position their signaling activity at different subcellular locations. We show that MNPs of different sizes, from 50nm to 500nm in diameter, can be used to generate different spatial perturbation patterns. While the biggest MNPs are trapped in internal structures of the cell and require large forces (>10 pN) to be displaced, they allow us to create precise point-like perturbations at the cell periphery. At the opposite, smallest MNPs diffuse fast in the cytosol (~1 m /s) and are used to create gradient of signaling activity spanning over the whole cell. We apply our approach to the Rho-GTPase signaling network which orchestrate cell polarity and migration. MNPs were functionnalized with GEF or active GTPase. We demonstrated that the pathway linking Rac1 to actin polymerization is spatially restricted to the protrusive areas of the cell by transporting TIAM1 particles at different subcellular locations while monitoring GTPase activation and actin polymerization. The creation of intracellular GTPase gradient on cell polarity is finally discussedPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF