911勛圖

Suspension-derived Cardiac Organoids to model Inherited Arrhythmias

Self-assembly of hiPSCs enables the development of tissue-like structures called organoids. Initial cardiac organoid (CO) models demonstrated the ability to mimic aspects of human heart development, including myocardial walls, fibroblasts, and endothelial cells (ECs) surrounding one or more cavities, and an epicardium. These COs were predominantly grown in static culture in multiwell dishes and induced through complex sequences of chemical cues. Here we used Wnt modulation and suspension culture to generate suspension-derived COs (sCOs), which enabled upscaling of organoid production and led to the discovery that small changes in conditions create unique developmental niches and induce novel tissues, such as a structure resembling the proepicardium. We used directed differentiation by Wnt modulation and stirred spinner flasks to produce sCOs. Spinner flasks were inoculated with hiPSCs and spontaneously formed embryoid bodies (EBs) were differentiated and after 4-5 days in culture we observed a secondary sphere emerge on sCOs. This secondary sphere consisted of an outer epithelial layer enclosing mesenchymal (VIM+) and multipotent (SOX2+) cells, resembling a proepicardium. The proepicardial-sphere (PS) continued to grow out of the initial EB by forming a ���stalk���. Over time the stalk bent thereby enabling contact and fusion of the PS to the initial EB, which by this time point had developed into a spontaneously beating myocardium enclosing a cavity. Fusion of the PS to the myocardium resulted in the spread of mesenchymal cells (VIM+) over its surface, which subsequently differentiated into epicardial cells (WT1+). At later time points, non-CMs and ECs (CD31+) populated the CO, and the ECs formed networks resembling vasculature. This process mimics normal heart development, in which epicardial cells undergo epithelial-mesenchymal transition, migrate into the myocardium, and differentiate to form non-cardiomyocytes. Here we present a novel cardiac organoid model, sCOs, that is fully derived in suspension and can be readily scaled. We show that typical Wnt modulation, coupled with appropriate physical culture conditions, creates developmental niches that spontaneously generate proepicardial-like structures, which subsequently form the epicardium and epicardium-derived lineages. Furthermore, we apply sCO technology to model Arrhythmogenic Cardiomyopathy (ACM) and revealed a previously unknown disease pathology contributing to ACM development, namely epicardial function.