Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues -termed Cardiac MicroRings (CaMiRi) -in custom 3D-printmolded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.Screening using engineered heart tissue is limited by the high cost associated with the large cell numbers required for high-fidelity in vitro cardiac models, the lack of long-term function and viability of the tissues, and the challenge of imaging for data acquisition. Miniaturization of self-organizing cardiac organoids is a strategic approach to address these challenges using photolithography-and micromachining-based fabrication techniques 1-12 . Unfortunately, some of these systems are difficult to manufacture on a macroscale, and production often requires a high level of skill. Furthermore, these methods are restrictive to specific design geometries (for instance, vertical walls spanning either the micrometer or centimeter scale but not both) and are occasionally expensive to prototype (due to requiring access to a cleanroom, cost of materials, and time). Another challenge typically associated with these microfluidic-and microstructure-based devices is adaptation to existing tissue culture equipment such as microscope stage adapters and liquid dispensing tools. Potential complications may also arise upon the

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