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      A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes

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          Abstract

          We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm 2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

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          Most cited references65

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          3D bioprinting of collagen to rebuild components of the human heart

          Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro–computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.
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            Advanced maturation of human cardiac tissue grown from pluripotent stem cells

            Cardiac tissues generated from human induced pluripotent stem (iPS) cells can serve as platforms for patient-specific studies of physiology and disease 1–6 . The predictive power of these models remains limited by their immature state 1,2,5,6 . We show that this fundamental limitation could be overcome if cardiac tissues are formed from early iPS-derived cardiomyocytes (iPS-CM), soon after the initiation of spontaneous contractions, and subjected to physical conditioning of an increasing intensity. After only 4 weeks of culture, these tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiologic sarcomere length (2.2 μm) and density of mitochondria (30%), the presence of transverse tubules (t-tubules), oxidative metabolism, positive force-frequency relationship, and functional calcium handling for all iPS cell lines studied. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiologic responses to isoproterenol and recapitulating pathological hypertrophy, in support of the utility of this tissue model for studies of cardiac development and disease.
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              Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease

              Summary Cardiomyocytes (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.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                05 April 2024
                2024
                : 12
                : RP87739
                Affiliations
                [1 ] Université de Paris Cité, PARCC, INSERM ( https://ror.org/05f82e368) Paris France
                [2 ] 4Dcell Montreuil France
                University of Toronto ( https://ror.org/03dbr7087) Canada
                University of California, Los Angeles ( https://ror.org/046rm7j60) United States
                University of Toronto Canada
                Paris Cardiovascular Research Center Paris France
                4Dcell Montreuil France
                4Dcell Montreuil France
                Paris Cardiovascular Research Center Paris France
                Paris Cardiovascular Research Center Paris France
                Paris Cardiovascular Research Center Paris France
                Paris Cardiovascular Research Center Paris France
                4Dcell Montreuil France
                4Dcell Montreuil France
                4Dcell Montreuil France
                Paris Cardiovascular Research Center Paris France
                Author notes
                [†]

                Paris Cardiovascular Research Center, Paris, France.

                Author information
                https://orcid.org/0000-0002-3059-3113
                https://orcid.org/0000-0002-3939-3601
                https://orcid.org/0000-0002-5792-4621
                https://orcid.org/0000-0002-4139-7679
                https://orcid.org/0000-0001-5463-6117
                Article
                87739
                10.7554/eLife.87739
                11001295
                38578976
                98f42400-03e8-4e29-aeb6-f5de222443ea
                © 2023, Seguret et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 27 March 2023
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001674, Leducq Foundation;
                Award ID: 18CVD05
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Stem Cells and Regenerative Medicine
                Custom metadata
                A novel hydrogel-based platform uses a limited number of cells for tissue generation and offers unique advantages for high-throughput cardiac tissue culture.
                prc

                Life sciences
                tissue engineering,organoids,heart,cardiac myocytes,induced pluripotent stem cells,drug screening,human

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