Generation Of Reporter Human Pluripotent Stem Cell Lines To Study Cardiac Development And Disease
Maria Gallo, Pluripotency for organ regeneration Group
The development and the use of human pluripotent stem cells (hPSCs) represent an effective tool to recapitulate characteristics related to tissue differentiation, morphogenesis and conversely, human diseases. Through their inherent capacity to differentiate towards the three germ layers and their easy manipulation, it has been possible to generate three dimensional (3D) self-organized organ-like structures, the so-called organoids. In this context, the advent of CRISPR-Cas9 technology has now allow to incorporate permanent or transient changes in the genome of living organisms and cells. Our laboratory has developed a cellular platform, named iCRISPR2 (iC2), that allows to perform highly efficient genome editing in hPSCs through the insertion of an inducible Cas9 under the endogenous TET/ON promoter. This platform allowed us the generation of reporter, knock-out and knock-in hPSCs lines. By exploiting the generation of cardiac reporter cell lines it has been possible to define new approaches for the generation of self-assembled cardiac-like organoids. Cardioids have been characterized at conventional molecular biology techniques, confocal microscopy and functional analysis. Moreover, taking advantages of specific cell culture conditions emulating early stages of diabetic cardiomyopathy it has been possible to establish a platform for the interrogation of transcriptional and functional changes during diabetic disease.
Mechanics of epithelial layers subjected to controlled pressure
Nimesh Chahare, Integrative cell and tissue dynamics Group
Epithelial sheets form specialized 3D structures suited to their physiological roles, such as branched alveoli in the lungs, tubes in the kidney, and villi in the intestine. To generate and maintain these structures, epithelia must undergo complex 3D deformations across length and time scales. How epithelial shape arises from active stresses, viscoelasticity, and luminal pressure remains poorly understood. To address this question, we 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. Slow pressure changes relative to the actin dynamics allow the tissue to accommodate large strain variations. However, under sudden pressure reductions, the tissue develops buckling patterns and folds with different degrees of symmetry-breaking to store excess tissue area. These insights allow us to pattern epithelial folds through rationally directed buckling. Our study establishes a new approach for engineering epithelial morphogenetic events.