3D Tri-culture Model Promotes Enhanced Mechanical Forces and Sustained Long-Term Contractility of Human Pluripotent Stem Cell-Derived Cardiomyocytes

BACKGROUND: Engineered myocardial tissue represents a promising methodology for cardiac repair and regeneration, but the survival and contractility of the cardiomyocytes (CMs) in the engineered tissue remains a limiting factor. Since native CMs interact with endothelial and stromal cells, we generated a three-dimensional (3D) engineered myocardial tissue to study the role of cell-cell interactions on CM survival and function. HYPOTHESIS: Using human pluripotent stem cells (hPSCs) as a therapeutic cell source, we hypothesize that 3D tri-culture of hPSC-derived CMs (hCMs), hPSC-derived endothelial cells (hECs), and human amniotic mesenchymal stem cells (hAMSCs) will enable sustained, long-term contractility of the hCM population. METHODS AND RESULTS: We engineered myocardial tissues by culturing combinations of the three cell populations, within a 3D hydrogel for up to 6 weeks as follows: 1) hCMs only (mono-culture), 2) hCMs + hECs (co-culture), 3) hCMs + hAMSCs (co-culture), and 4) hCMs + hECs + hAMSCs (tri-culture). Timelapse imaging was used to compare contractility frequency and beating area, troponin T protein expression using immunofluorescence staining, and cardiac gene expression by qPCR. Quantification of contractility demonstrated enhanced function of hCMs in tri-culture. Expression of cTNT was up-regulated in the tri-culture vs. other culture conditions, which was supported by qPCR measurement of cardiac genes related to maintenance of CM structure (TNNT2 or β-myosin heavy chain, P CONCLUSION: These findings demonstrate that the 3D tri-culture system enabled significantly greater long-term contractility, sustained survival, and enhanced cardiac lineage-related gene expression of hCMs when compared to mono- and co-culture conditions. This study highlights the role of 3D hydrogels and cell-cell interactions to maintain cardiac phenotype and survival.