Periodic Reporting for period 1 - EpiFold (Engineering epithelial shape and mechanics: from synthetic morphogenesis to biohybrid devices)
Reporting period: 2021-01-01 to 2022-06-30
All surfaces of our body, both internal and external, are covered by thin cellular layers called epithelia. Epithelia are responsible for fundamental physiological functions such as morphogenesis, compartmentalization, filtration, transport, environmental sensing and protection against pathogens. These functions are determined by the three-dimensional (3D) shape and mechanics of epithelia. However, how mechanical processes such as deformation, growth, remodeling and flow combine to enable functional 3D structures is largely unknown. Here we propose technological and conceptual advances to unveil the engineering principles that govern epithelial shape and mechanics in 3D, and to apply these principles towards the design of a new generation of biohybrid devices. By combining micropatterning, microfluidics, optogenetics and mechanical engineering we will implement an experimental platform to (1) sculpt epithelia of controlled geometry, (2) map the stress and strain tensors and luminal pressure, and (3) control these variables from the subcellular to the tissue levels. We will use this technology to engineer the elementary building blocks of epithelial morphogenesis and to reverse-engineer the shape and mechanics of intestinal organoids. We will then apply these engineering principles to build biohybrid devices based on micropatterned 3D epithelia actuated through optogenetic and mechanical control. We expect this project to enable, for the first time, full experimental access to the 3D mechanics of epithelial tissues, and to unveil the mechanical principles by which these tissues adopt and sustain their shape. Finally, our project will set the stage for a new generation of biohybrid optomechanical devices. By harnessing the capability of 3D epithelia to sense and respond to chemical and mechanical stimuli, to self-power and self-repair, and to secrete, filter, digest and transport molecules, these devices will hold unique potential to power functions in soft robots.
As written in the work plan, the project begun with the development of new technologies to map and control epithelial shape, stress and luminal pressure in 3D. We designed pressurized epithelia with circular, rectangular and ellipsoidal footprints and developed a computational method to map the stress tensor in these epithelia. This method establishes a direct correspondence between epithelial shape and mechanical stress without assumptions of material properties. We also developed a microfluidic chip and a computational framework to engineer 3D epithelial tissues with controlled shape and pressure. In the setup, an epithelial monolayer is grown on a porous surface with circular low adhesion zones. On applying hydrostatic pressure, the monolayer delaminates into a spherical cap from the circular zone. This simple shape allows us to calculate epithelial tension using Laplace’s law. Through this approach, we subject the monolayer to a range of lumen pressures at different rates and hence probe the relation between strain and tension in different regimes, while computationally tracking actin dynamics and their mechanical effect at the tissue scale. Imaging and segmentation are performed either through scanning confocal microscopy or through light sheet imaging. To achieve spatiotemporal control over epithelial folding, we created an optogenetic version of Shroom3, which causes apical constriction by recruiting ROCK to apical junctions.
We have also applied these technologies to the study of the mechanics of intestinal organoids. Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Within EpiFold, we mapped the three-dimensional cell-matrix and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We showed that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements established that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model showed that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we showed that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Thus, EpiFold unveiled how patterned forces enable folding and collective migration in the intestinal crypt.
We have also applied these technologies to the study of the mechanics of intestinal organoids. Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Within EpiFold, we mapped the three-dimensional cell-matrix and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We showed that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements established that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model showed that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we showed that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Thus, EpiFold unveiled how patterned forces enable folding and collective migration in the intestinal crypt.
EpiFold has already gone beyond the state of the art by developing tools to map and manipulate epithelial shape and mechanics. We have applied these tools to study how epithelia of different shape deform and how intestinal organoids fold. In the next reporting period, we expect to further develop our tools and to apply them to engineer the shape and mechanics of epithelial monolayers and organoids and to develop the first epifluidic components.