Abstract 12234: A Three-Dimensional Culture System Composed of Hipsc Derived Cms, Ecs, Smcs, and Cardiac Fibroblasts Promotes Hipsc-cm Maturation

Abstract

Introduction: Cardiomyocytes (CMs), endothelial cells (ECs), smooth muscle cells (SMCs), and cardiac fibroblasts (CFs) differentiated from human induced-pluripotent stem cells (hiPSCs) are the fundamental components of cell-based regenerative myocardial therapy and can be used as in-vitro models for mechanistic studies and drug testing. However, newly differentiated hiPSC-CMs tend to more closely resemble fetal CMs than the mature CMs of adult hearts, and current techniques for improving CM maturation can be both complex and labor-intensive. Hypothesis: CMs tend to develop a more mature phenotype when cultured as spheroids in a three-dimensional (3D) environment, rather than as two-dimensional monolayers, and the activity of ECs, SMCs, and CFs promote both CM maturation and electrical activity. Methods: hiPSCs were differentiated into CMs (hiPSC-CMs) using a belly dancer shaker in 3D condition and seeded into 96-Well of Nunclon Sphera-Treated, U-Shaped-Bottom Microplate (Thermo Fisher Scientific). hiPSC differentiated endothelial cells (hiPSC-ECs), smooth muscle cells (hiPSC-SMCs), and cardiac fibroblasts (hiPSC-CFs) were added to each well to produce cardiac spheroids. Quantitative polymerase chain reaction (qPCR), western blot, immunostaining, bioenergetics, transmission electron microscopy, field potential, and contractility of cardiac spheroids were characterized. Results: The inclusion of hiPSC-ECs, -SMCs, and CFs in CM spheroids increased spheroid size and improved cell viability. hiPSC-ECs, -SMCs, and -CFs promoted hiPSC-CMs maturation in cardiac spheroids: a. gene expression levels of TNNI3, N-cadherin, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1a) significantly increased; b. Contractile fibers were better organized and mitochondria were better aligned; c. ATP level, the ratio of NAD:NADH, and cAMP level were greater in cardiac spheroids; d. Field-potential duration and conduction velocity were greater in CMs in cardiac spheroids. Conclusion: we developed a simple and reproducible 3D-culture-based process for generating cardiac spheroids containing more mature hiPSC-CMs in the presence of ECs, SMCs, and CFs.