Abstract Body: Background: Theoretically, 3D multi-cellular cardiac spheroids (3D-MCS) composed of CMs, ECs, and CFs could mimic important features of the human heart microenvironment. We developed injectable 3D-MCSs derived from hiPSCs and investigated their therapeutic potential for cardiac regeneration.

Methods: We designed scaffold-free, self-aggregating 3D-MCSs using ultra-low attachment microcavity plates to produce uniformly sized spheroids, and confirmed that the three cell types (CMs, ECs, CFs) were distributed throughout the 3D-MCS structure. We evaluated the structural and functional properties of CMs across different 3D-MCS composition groups, including an analysis of contractility and responsiveness using genetically encoded calcium and voltage indicators expressed in CMs. Cellular engraftment was monitored by seeding spheroids onto a collagen gel matrix, and electrophysiological properties were assessed by recording field potentials with multi-electrode arrays. Finally, we transplanted each type of 3D-MCS via intramyocardial injection into infarcted hearts and compared their therapeutic efficacy using echocardiography and pressure-volume analysis.

Results: The structural and functional properties of CMs differed among the 3D-MCS groups. Spheroids containing CFs formed smaller aggregates more rapidly, and their CMs exhibited a more mature phenotype compared to CF-free spheroids. The intramyocardial transplantation of MCS-MEF was followed up for eight weeks, during which it led to a significant improvement in cardiac function and a reduction in adverse remodeling compared to the control and other 3D-MCS groups. Notably, as early as one week post-transplantation, MCS-MEF grafts exhibited a significantly higher engraftment rate and greater vascularization relative to other groups. Furthermore, by the end of the follow-up period, the MCS-MEF group demonstrated a markedly higher CM retention rate, and the grafted CMs exhibited an adult-like morphology with well-organized gap junctions compared to other 3D-MCS groups. These findings suggest that reconstructing the heart microenvironment with appropriate cardiac cell components and their interactions can substantially enhance myocardial regeneration in infarcted hearts.

Conclusions: We developed 3D-MCSs derived from hiPSCs to recapitulate human heart microenvironment and provided compelling evidence that this injectable platform of MCS-MEF can be a promising tool to enhance cardiac repair on MI hearts.