Abstract
SummaryCardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.
Highlights
Dialogue between stromal, vascular, and tissue-specific cells is essential for maintaining tissue homeostasis
We demonstrated that cardiac fibroblasts (CFs), expressing connexin 43 (CX43) gap junction protein, were most effective in supporting human induced pluripotent stem cells (hiPSCs)-CM maturation, and this was mediated by cyclic AMP
To induce CF differentiation, hiPSC-Epicardial cells (EPIs) were dissociated on day and re-plated in medium with basic fibroblast growth factor (FGF2) (10 ng/mL) for 8 days, refreshing on day and every 2 days thereafter (Figure 1A)
Summary
Vascular, and tissue-specific cells is essential for maintaining tissue homeostasis. Human induced pluripotent stem cells (hiPSCs) can. 862 Cell Stem Cell 26, 862–879, June 4, 2020 a 2020 The Author(s). Article ll (legend continued on page) Cell Stem Cell 26, 862–879, June 4, 2020 863. Subtraction of values lower than the result of the sum of the average and standard deviation were performed in each slice. Classification of nuclei based on the average intensity value in fitted 2D ellipses was performed for all channels. A cell with an average intensity of KI67 higher than zero was assumed as proliferative. A cell with an average intensity of COL1A1 higher than zero was considered a fibroblast. Distance between nuclei in the sample and its 12 closest neighbors was computed. The median value was calculated for each nuclei as the most probable distance
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