An in vitro model for cardiac organoid production: The combined role of geometrical confinement and substrate stiffness.

Induced pluripotent stem cells (iPSCs), carrying the patient's genetic background, open the path to advanced in vitro modeling. The feasibility of recapitulating complex pathophysiological scenarios depends on iPSC's ability to differentiate into the plurality of specific organ resident cells, on their maturation and networking. To this end, a strong interest has arisen in organoids, 3D structures, obtained by exploiting iPSC natural capability to self-assemble and rebuild organ parts. In this study, we describe the characterization of a novel iPSC-based cardiac organoid (CO) model, generated by a high-throughput and cost-effective method. Organoids were obtained by culture onto substrates of known stiffness, under geometrical confinement and inducing cardiac differentiation by small-molecule-based modulation of Wnt pathway. COs were characterized using a multi-omic approach (including bulk/single-cell RNA-sequencing, and proteomic analysis), immunofluorescence, electrophysiology (patch clamp), and optical recording-based contraction measurements. Results showed that COs recapitulate relevant cardiac features, including spontaneous contraction, multicellularity (e.g., cardiomyocytes, fibroblasts, epicardial layer) and chamber organization. Moreover, modulation of environmental mechanical cues showed a significant effect on organoid cardiac features. In particular, culturing organoids onto substrates of low stiffness, in the range of that characterizing the embryonal surrounding, enriched the gene sets related to cardiac maturity and cardiomyocyte ultrastructure. Functionally, different cardiac-specific ionic currents and consistent spontaneous action potentials were recorded upon patch-clamp of cardiomyocytes dissociated from COs. Finally, the beating rate of the whole COs was monitored non-destructively via video recording and quantified, demonstrating their response to clinically used chronotropic compounds, supporting the feasibility of future implementation of the proposed COs as in vitro platform for drug testing.