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      3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids

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          Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions.

          Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR).

          Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture.

          Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

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          Most cited references 52

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          Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem?

          Cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are increasingly used to model cardiac disease, test drug efficacy and for safety pharmacology. Nevertheless, a major hurdle to more extensive use is their immaturity and similarity to fetal rather than adult cardiomyocytes. Here, we provide an overview of the strategies currently being used to increase maturation in culture, which include prolongation of time in culture, exposure to electrical stimulation, application of mechanical strain, growth in three-dimensional tissue configuration, addition of non-cardiomyocytes, use of hormones and small molecules, and alteration of the extracellular environment. By comparing the outcomes of these studies, we identify the approaches most likely to improve functional maturation of hPSC-CMs in terms of their electrophysiology and excitation-contraction coupling.
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            Mechanisms of myofibroblast activity and phenotypic modulation.

             G Gabbiani,  G Serini (1999)
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              Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics

              Heart pumping is triggered and coordinated by action potentials (APs) originating in and spreading among electrically excitable heart muscle cells (myocytes) via electrotonic coupling. Cardiac nonmyocytes are thought not to participate in AP conduction in situ, although heterocellular electrotonic coupling is common in cell culture. We used optogenetic tools involving cell-specific expression of a voltage-reporting fluorescent protein to monitor electrical activity in myocytes or nonmyocytes of mouse hearts. We confirm the suitability of this technique for measuring cell type-specific voltage signals and show that, when expressed in nonmyocytes, myocyte AP-like signals can be recorded in cryoinjured scar border tissue. This direct evidence of heterocellular electrotonic coupling in the whole heart necessitates a review of current concepts on cardiac electrical connectivity. Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte–nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.

                Author and article information

                Front Mol Biosci
                Front Mol Biosci
                Front. Mol. Biosci.
                Frontiers in Molecular Biosciences
                Frontiers Media S.A.
                14 February 2020
                : 7
                1Cardiology Department, DBMR MEM C812, Bern University Hospital , Bern, Switzerland
                2Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki , Helsinki, Finland
                3IKELOS GmbH , Bern, Switzerland
                4Department of Biomedical Sciences, University of Copenhagen , Copenhagen, Denmark
                5InSphero AG , Schlieren, Switzerland
                6Division of Cardiovascular Medicine, Vanderbilt University Medical School , Nashville, TN, United States
                7Department of Cell Biology and Molecular Biology, Rutgers New Jersey Medical School , Newark, NJ, United States
                8Department of Cardiac Services, Maine Medical Center , Scarborough, ME, United States
                Author notes

                Edited by: William Cho, Queen Elizabeth Hospital (QEH), Hong Kong

                Reviewed by: Michal Mielcarek, Imperial College London, United Kingdom; Zelal Adiguzel, Scientific and Technological Research Council of Turkey, Turkey; RaffaEle Coppini, University of Florence, Italy

                *Correspondence: Christian Zuppinger christian.zuppinger@ 123456dbmr.unibe.ch

                This article was submitted to Molecular Diagnostics and Therapeutics, a section of the journal Frontiers in Molecular Biosciences

                Copyright © 2020 Beauchamp, Jackson, Ozhathil, Agarkova, Galindo, Sawyer, Suter and Zuppinger.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 7, Tables: 1, Equations: 0, References: 72, Pages: 17, Words: 12055
                Funded by: Schweizerische Herzstiftung 10.13039/501100004362
                Molecular Biosciences
                Original Research


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