Supplementary MaterialsVideo S1 Vector Flow Evaluation of MTs, Linked to Figure?4 mmc7. Motion Movement analysis software could be requested by getting in touch with L.G.J.Tertoolen@lumc.nl. Overview Cardiomyocytes (CMs) from human being induced pluripotent stem cells (hiPSCs) are functionally immature, but that is improved by incorporation into built tissues or pressured contraction. Right here, we demonstrated that tri-cellular mixtures of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in quickly built, 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 Z-Ile-Leu-aldehyde 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. (Carvajal-Vergara et?al., 2010, Caspi et?al., 2013, DellEra et?al., 2015, Dudek et?al., 2013, Giacomelli et?al., 2017c, Moretti et?al., 2010, Te Riele et?al., 2017, Siu et?al., 2012, Wang et?al., 2014) and to some extent predict cardiotoxicity of pharmacological compounds and key pathways in disease (Cross et?al., 2015, Sala et?al., 2017, van Meer et al., 2019). Relatively mature hiPSC-CMs have only been convincingly observed in 3D scaffold-based cultures or engineered heart tissues (EHTs) (Lemoine et?al., 2017, Mannhardt et?al., 2016, Ronaldson-Bouchard et?al., 2018, Tiburcy et?al., 2017) with escalating forced contraction enhancing maturation such that transverse (T-) tubule-like structures become evident (Ronaldson-Bouchard et?al., 2018, Tiburcy et?al., 2017). T-tubules normally develop postnatally to regulate Ca2+ homeostasis, excitation-contraction coupling, and electrical activity of the heart (Brette and Orchard, 2007). However, EHTs require specific expertise, specialized apparatus, gelation substrates, and analysis tools (Mathur et?al., 2015) and are thus complex solutions for most academic laboratories and pharma applications. Moreover, monotypic cell configurations do not recapitulate how stromal or vascular cells might affect the behavior of CMs and mediate disease or drug-induced phenotypes. Here, we addressed these issues by generating multicell-type 3D cardiac microtissues (MTs) starting with just 5,000 cells. We demonstrated previously that hiPSC-ECs derived from cardiac mesoderm affect hiPSC-CMs in 3D MTs (Giacomelli et?al., 2017b) and found here that inclusion of hiPSC-CFs further enhanced structural, electrical, mechanical, and metabolic maturation. CFs mainly originate from the epicardium (Tallquist and Molkentin, 2017), the outer epithelium covering the heart. They play crucial roles in cardiac physiology and pathophysiology MEN2A (Furtado et?al., 2016, Kofron et?al., 2017, Risebro et?al., 2015), contributing to scar tissue formation after myocardial infarction (Rog-Zielinska et?al., 2016). They maintain and remodel the ECM, contributing to the integrity and connectivity of the myocardial architecture (Dostal et?al., 2015). Although non-excitable themselves, CFs modulate active and passive electrical properties of CMs (Klesen et?al., 2018, Kofron et?al., 2017, Mahoney et?al., 2016, Ongstad and Kohl, 2016). CFs have also Z-Ile-Leu-aldehyde been implicated in contractility of hiPSC-CMs in 3D self-assembled (scaffold-free) MTs composed of hiPSC-CMs, primary human cardiac microvasculature ECs, and primary human CFs (Pointon et?al., 2017). MTs have to date only been generated using primary stromal cells, which impacts reproducibility and supply. By replacing primary ECs and CFs Z-Ile-Leu-aldehyde with hiPSC counterparts, we generated thousands of scaffold-free miniaturized cardiac MTs (CMECFs) containing all cellular components in defined ratios and observed enhanced hiPSC-CM maturation. We demonstrated that CFs, expressing connexin 43 (CX43) gap junction protein, had been most reliable in helping hiPSC-CM maturation, which was mediated by cyclic AMP (cAMP). Epidermis fibroblasts (SFs), which usually do not exhibit CX43, and CFs where CX43 was knocked down were not able to few to hiPSC-CMs and didn’t improve maturation. Single-cell (sc) RNA sequencing (RNA-seq) demonstrated that indicators from both cardiac ECs and CFs most likely contributed to raising intracellular cAMP in hiPSC-CMs which was recapitulated with the addition of dibutyryl (db) cAMP, a cell-permeable analog of cAMP. MTs where control CFs had been changed by hiPSC CFs holding a mutation in.