Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

Austrian Cluster for Tissue Regeneration 1200 Vienna Austria

Center for Translational Medicine St Anne's University Hospital Studentská 812 6 62500 Brno Czech Republic

Department of Biomaterials Science Institute of Dentistry University of Turku 20014 Turku Finland

Department of Chemical Engineering The University of Melbourne Parkville VIC 3010 Australia

Dipartimento di Scienze e Tecnologie Chimiche Università degli Studi di Roma Tor Vergata via della Ricerca Scientifica 1 00133 Rome Italy

Electron Microscopy Facility Fondazione Istituto Italiano Di Tecnologia Via Morego 30 16163 Genoa Italy

Faculty of Medicine Department of Biomedical Sciences Masaryk University 62500 Brno Czech Republic

Faculty of Technical Chemistry Institute of Applied Synthetic Chemistry and Institute of Chemical Technologies and Analytics Vienna University of Technology 1040 Vienna Austria

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology AUVA Research Center 1200 Vienna Austria

Zobrazit více v PubMed

Abbafati C, et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet (London, England) 2020;396(10258):1204. doi: 10.1016/S0140-6736(20)30925-9. PubMed DOI PMC

Namara KM, Alzubaidi H, Jackson JK. Cardiovascular disease as a leading cause of death: How are pharmacists getting involved? Integr. Pharm. Res. Pract. 2019;8:1. doi: 10.2147/IPRP.S133088. PubMed DOI PMC

Roth GA, et al. Global burden of cardiovascular diseases and risk factors, 1990–2019: Update from the GBD 2019 study. J. Am. Coll. Cardiol. 2020;76(25):2982–3021. doi: 10.1016/j.jacc.2020.11.010. PubMed DOI PMC

Heidenreich PA, et al. Forecasting the impact of heart failure in the United States a policy statement from the American Heart Association. Circ. Heart Fail. 2013;6(3):606–619. doi: 10.1161/HHF.0b013e318291329a. PubMed DOI PMC

Seruga B, Ocana A, Amir E, Tannock IF. Failures in phase III: Causes and consequences. Clin. Cancer Res. 2015;21(20):4552–4560. doi: 10.1158/1078-0432.CCR-15-0124. PubMed DOI

McNaughton R, Huet G, Shakir S. An investigation into drug products withdrawn from the EU market between 2002 and 2011 for safety reasons and the evidence used to support the decision-making. BMJ Open. 2014;4(1):e004221. doi: 10.1136/bmjopen-2013-004221. PubMed DOI PMC

Khakoo AY, Yurgin NR, Eisenberg PR, Fonarow GC. Overcoming barriers to development of novel therapies for cardiovascular disease: Insights from the oncology drug development experience. JACC Basic Transl. Sci. 2019;4(2):269–274. doi: 10.1016/j.jacbts.2019.01.011. PubMed DOI PMC

Cho S, Lee C, Skylar-Scott MA, Heilshorn SC, Wu JC. Reconstructing the heart using iPSCs: Engineering strategies and applications. J. Mol. Cell. Cardiol. 2021;157:56–65. doi: 10.1016/j.yjmcc.2021.04.006. PubMed DOI PMC

Kim J, Koo BK, Knoblich JA. Human organoids: Model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol. 2020;21(10):571–584. doi: 10.1038/s41580-020-0259-3. PubMed DOI PMC

Marshall JJ, Mason JO. Mouse vs man: Organoid models of brain development and disease. Brain Res. 2019;1724:146427. doi: 10.1016/j.brainres.2019.146427. PubMed DOI

Gorzalczany SB, Rodriguez Basso AG. Strategies to apply 3Rs in preclinical testing. Pharmacol. Res. Perspect. 2021;9(5):e00863. doi: 10.1002/prp2.863. PubMed DOI PMC

Takahashi K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–872. doi: 10.1016/j.cell.2007.11.019. PubMed DOI

Lian X, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U.S.A. 2012;109(27):E1848–E1857. doi: 10.1073/pnas.1200250109. PubMed DOI PMC

Lian X, et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 2013;8(1):162–175. doi: 10.1038/nprot.2012.150. PubMed DOI PMC

Sadahiro T, Yamanaka S, Ieda M. Direct cardiac reprogramming: Progress and challenges in basic biology and clinical applications. Circ. Res. 2015;116(8):1378–1391. doi: 10.1161/CIRCRESAHA.116.305374. PubMed DOI

Burridge PW, et al. Chemically defined generation of human cardiomyocytes. Nat. Methods. 2014;11(8):855–860. doi: 10.1038/nmeth.2999. PubMed DOI PMC

Burridge PW, Diecke S, Matsa E, Sharma A, Wu H, Wu JC. Modeling cardiovascular diseases with patient-specific human pluripotent stem cell-derived cardiomyocytes. Methods Mol. Biol. 2014;1353:119–130. doi: 10.1007/7651_2015_196. PubMed DOI PMC

Burridge PW, et al. Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat. Med. 2016;22(5):547–556. doi: 10.1038/nm.4087. PubMed DOI PMC

Sharma A, Li G, Rajarajan K, Hamaguchi R, Burridge PW, Wu SM. Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation. J. Vis. Exp. 2015;97:e52628. PubMed PMC

Sharma A, et al. Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity. Nat. Protoc. 2018;13(12):3018–3041. doi: 10.1038/s41596-018-0076-8. PubMed DOI PMC

Lan F, et al. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell. 2013;12(1):101–113. doi: 10.1016/j.stem.2012.10.010. PubMed DOI PMC

Sharma A, et al. Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform. Circ. Res. 2014;115(6):556–566. doi: 10.1161/CIRCRESAHA.115.303810. PubMed DOI PMC

Li T-S, et al. Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair. Stem Cells. 2010;28(11):2088–2098. doi: 10.1002/stem.532. PubMed DOI PMC

Nguyen DC, et al. Microscale generation of cardiospheres promotes robust enrichment of cardiomyocytes derived from human pluripotent stem cells. Stem Cell Rep. 2014;3(2):260–268. doi: 10.1016/j.stemcr.2014.06.002. PubMed DOI PMC

Richards DJ, et al. Inspiration from heart development: Biomimetic development of functional human cardiac organoids. Biomaterials. 2017;142:112–123. doi: 10.1016/j.biomaterials.2017.07.021. PubMed DOI PMC

Zimmermann W-H. Tissue engineering of a differentiated cardiac muscle construct. Circ. Res. 2001;90(2):223–230. doi: 10.1161/hh0202.103644. PubMed DOI

Weinberger F, Mannhardt I, Eschenhagen T. Engineering cardiac muscle tissue: A maturating field of research. Circ. Res. 2017;120(9):1487–1500. doi: 10.1161/CIRCRESAHA.117.310738. PubMed DOI

Dhahri W, Romagnuolo R, Laflamme MA. Training heart tissue to mature. Nat. Biomed. Eng. 2018;2(6):351–352. doi: 10.1038/s41551-018-0253-7. PubMed DOI

Hirt MN, Hansen A, Eschenhagen T. Cardiac tissue engineering. Circ. Res. 2014;114(2):354–367. doi: 10.1161/CIRCRESAHA.114.300522. PubMed DOI

Schaaf S, et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One. 2011;6(10):e26397. doi: 10.1371/journal.pone.0026397. PubMed DOI PMC

Vunjak Novakovic G, Eschenhagen T, Mummery C. Myocardial tissue engineering: In vitro models. Cold Spring Harb. Perspect. Med. 2014;4(3):a014076. doi: 10.1101/cshperspect.a014076. PubMed DOI PMC

Mathur A, et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci. Rep. 2015;5(1):1–7. doi: 10.1038/srep08883. PubMed DOI PMC

Ma Z, et al. Self-organizing human cardiac microchambers mediated by geometric confinement. Nat. Commun. 2015;6:7413. doi: 10.1038/ncomms8413. PubMed DOI PMC

Giacomelli E, et al. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells. Development. 2017;144(6):1008–1017. PubMed PMC

Giacomelli E, et al. Human-iPSC-derived cardiac stromal cells enhance maturation in 3D cardiac microtissues and reveal non-cardiomyocyte contributions to heart disease. Cell Stem Cell. 2020;26(6):862–879.e11. doi: 10.1016/j.stem.2020.05.004. PubMed DOI PMC

Giacomelli E, Sala L, van Oostwaard DW, Bellin M. Cardiac microtissues from human pluripotent stem cells recapitulate the phenotype of long-QT syndrome. Biochem. Biophys. Res. Commun. 2021;572:118–124. doi: 10.1016/j.bbrc.2021.07.068. PubMed DOI

Tsan YC, et al. Physiologic biomechanics enhance reproducible contractile development in a stem cell derived cardiac muscle platform. Nat. Commun. 2021;12(1):1–16. doi: 10.1038/s41467-021-26496-1. PubMed DOI PMC

Grosberg A, Alford PW, McCain ML, Parker KK. Ensembles of engineered cardiac tissues for physiological and pharmacological study: Heart on a chip. Lab Chip. 2011;11(24):4165–4173. doi: 10.1039/c1lc20557a. PubMed DOI PMC

Shim J, Grosberg A, Nawroth JC, Parker KK, Bertoldi K. Modeling of cardiac muscle thin films: Pre-stretch, passive and active behavior. J. Biomech. 2012;45(5):832–841. doi: 10.1016/j.jbiomech.2011.11.024. PubMed DOI PMC

Wang G, et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 2014;20(6):616–623. doi: 10.1038/nm.3545. PubMed DOI PMC

Marsano A, et al. Beating heart on a chip: A novel microfluidic platform to generate functional 3D cardiac microtissues. Lab Chip. 2016;16(3):599–610. doi: 10.1039/C5LC01356A. PubMed DOI

Ugolini GS, et al. On-chip assessment of human primary cardiac fibroblasts proliferative responses to uniaxial cyclic mechanical strain. Biotechnol. Bioeng. 2016;113:859–869. doi: 10.1002/bit.25847. PubMed DOI

Ugolini GS, Visone R, Redaelli A, Moretti M, Rasponi M. Generating multicompartmental 3D biological constructs interfaced through sequential injections in microfluidic devices. Adv. Healthc. Mater. 2017 doi: 10.1002/adhm.201601170. PubMed DOI

Occhetta P, et al. A three-dimensional in vitro dynamic micro-tissue model of cardiac scar formation. Integr. Biol. 2018;10(3):174–183. doi: 10.1039/C7IB00199A. PubMed DOI

Schneider O, Zeifang L, Fuchs S, Sailer C, Loskill P. User-friendly and parallelized generation of human induced pluripotent stem cell-derived microtissues in a centrifugal heart-on-a-chip. Tissue Eng. Part A. 2019;25(9–10):786–798. doi: 10.1089/ten.tea.2019.0002. PubMed DOI PMC

Khademhosseini A, et al. Microfluidic patterning for fabrication of contractile cardiac organoids. Biomed. Microdevices. 2007;9(2):149–157. doi: 10.1007/s10544-006-9013-7. PubMed DOI

Skardal A, et al. Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Sci. Rep. 2017;7(1):8837. doi: 10.1038/s41598-017-08879-x. PubMed DOI PMC

Zhang YS, et al. From cardiac tissue engineering to heart-on-a-chip: Beating challenges. Biomed. Mater. 2015;10(3):034006. doi: 10.1088/1748-6041/10/3/034006. PubMed DOI PMC

Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science. 2014;345(6194):1247125. doi: 10.1126/science.1247125. PubMed DOI

Clevers H. Modeling development and disease with organoids. Cell. 2016;165(7):1586–1597. doi: 10.1016/j.cell.2016.05.082. PubMed DOI

Lancaster MA, Huch M. Disease modelling in human organoids. DMM Dis. Model. Mech. 2019;12(7):dmm039347. doi: 10.1242/dmm.039347. PubMed DOI PMC

Sasai Y. Next-generation regenerative medicine: Organogenesis from stem cells in 3D culture. Cell Stem Cell. 2013;12(5):520–530. doi: 10.1016/j.stem.2013.04.009. PubMed DOI

Sasai Y. Cytosystems dynamics in self-organization of tissue architecture. Nature. 2013;493(7432):318–326. doi: 10.1038/nature11859. PubMed DOI

Schutgens F, Clevers H. Human organoids: Tools for understanding biology and treating diseases. Annu. Rev. Pathol. Mech. Dis. 2020;15:211–234. doi: 10.1146/annurev-pathmechdis-012419-032611. PubMed DOI

Corrò C, Novellasdemunt L, Li VSW. A brief history of organoids. Am. J. Physiol. Cell Physiol. 2020;319(1):C151–C165. doi: 10.1152/ajpcell.00120.2020. PubMed DOI PMC

Hofbauer P, et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell. 2021;184(12):3299–3317.e22. doi: 10.1016/j.cell.2021.04.034. PubMed DOI

Andersen P, et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat. Commun. 2018;9(1):1–13. doi: 10.1038/s41467-018-05604-8. PubMed DOI PMC

Lee J, et al. In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix. Nat. Commun. 2020;11(1):4283. doi: 10.1038/s41467-020-18031-5. PubMed DOI PMC

Rossi G, et al. Capturing cardiogenesis in gastruloids. Cell Stem Cell. 2021;28(2):230–240.e6. doi: 10.1016/j.stem.2020.10.013. PubMed DOI PMC

Voges HK, Mills RJ, Elliott DA, Parton RG, Porrello ER, Hudson JE. Development of a human cardiac organoid injury model reveals innate regenerative potential. Development. 2017;144(6):1118–1127. PubMed

Mills RJ, et al. Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proc. Natl Acad. Sci. U.S.A. 2017;114(40):E8372–E8381. doi: 10.1073/pnas.1707316114. PubMed DOI PMC

Richards DJ, et al. Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity. Nat. Biomed. Eng. 2020;4(4):446–462. doi: 10.1038/s41551-020-0539-4. PubMed DOI PMC

Drakhlis L, et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 2021;39(6):737–746. doi: 10.1038/s41587-021-00815-9. PubMed DOI PMC

Lewis-Israeli YR, et al. Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease. Nat. Commun. 2021;12(1):5142. doi: 10.1038/s41467-021-25329-5. PubMed DOI PMC

Silva AC, et al. Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids. Cell Stem Cell. 2021;28(12):2137–2152.e6. doi: 10.1016/j.stem.2021.11.007. PubMed DOI

Drakhlis L, Devadas SB, Zweigerdt R. Generation of heart-forming organoids from human pluripotent stem cells. Nat. Protoc. 2021;16(12):5652–5672. doi: 10.1038/s41596-021-00629-8. PubMed DOI

Yang X, Pabon L, Murry CE. Engineering adolescence: Maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 2014;114(3):511–523. doi: 10.1161/CIRCRESAHA.114.300558. PubMed DOI PMC

Murphy SA, Chen EZ, Tung L, Boheler KR, Kwon C. Maturing heart muscle cells: Mechanisms and transcriptomic insights. Semin. Cell Dev. Biol. 2021;119:49–60. doi: 10.1016/j.semcdb.2021.04.019. PubMed DOI PMC

Pasqualini FS, Nesmith AP, Horton RE, Sheehy SP, Parker KK. Mechanotransduction and metabolism in cardiomyocyte microdomains. Biomed Res. Int. 2016;2016:4081638. doi: 10.1155/2016/4081638. PubMed DOI PMC

Lian X, Zhang J, Zhu K, Kamp TJ, Palecek SP. Insulin inhibits cardiac mesoderm, not mesendoderm, formation during cardiac differentiation of human pluripotent stem cells and modulation of canonical Wnt signaling can rescue this inhibition. Stem Cells. 2013;31(3):447. doi: 10.1002/stem.1289. PubMed DOI PMC

Zuppinger C. 3D cardiac cell culture: A critical review of current technologies and applications. Front. Cardiovasc. Med. 2019;6:87. doi: 10.3389/fcvm.2019.00087. PubMed DOI PMC

Pinto AR, et al. Revisiting cardiac cellular composition. Circ. Res. 2016;118(3):400. doi: 10.1161/CIRCRESAHA.115.307778. PubMed DOI PMC

Litviňuková M, et al. Cells of the adult human heart. Nature. 2020;588(7838):466–472. doi: 10.1038/s41586-020-2797-4. PubMed DOI PMC

Guo Y, Pu WT. Cardiomyocyte maturation. Circ. Res. 2020;126:1086–1106. doi: 10.1161/CIRCRESAHA.119.315862. PubMed DOI PMC

Ahmed RE, Anzai T, Chanthra N, Uosaki H. A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes. Front. Cell Dev. Biol. 2020;8:178. doi: 10.3389/fcell.2020.00178. PubMed DOI PMC

A novel approach for the diagnosis of dilated cardiomyopathy (DCM), ID 667310—BioProject—NCBI [Online]. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA667310. Accessed 09 Dec 2021.

RNA-seq of heart tissues from healthy individuals and DMD patients, ID 628736—BioProject—NCBI [Online]. https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA628736. Accessed 09 Dec 2021.

Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue, ID 831794—BioProject—NCBI [Online]. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA831794. Accessed 12 Jul 2022. PubMed PMC

Feyen DAM, et al. Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes. Cell Rep. 2020;32(3):107925. doi: 10.1016/j.celrep.2020.107925. PubMed DOI PMC

Lionetti V, Stanley WC, Recchia FA. Modulating fatty acid oxidation in heart failure. Cardiovasc. Res. 2011;90(2):202. doi: 10.1093/cvr/cvr038. PubMed DOI PMC

Drawnel FM, et al. Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells. Cell Rep. 2014;9(3):810–820. doi: 10.1016/j.celrep.2014.09.055. PubMed DOI

Guo L, et al. Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes. Toxicol. Sci. 2011;123(1):281–289. doi: 10.1093/toxsci/kfr158. PubMed DOI

Mannhardt I, et al. Human engineered heart tissue: Analysis of contractile force. Stem Cell Rep. 2016;7(1):29–42. doi: 10.1016/j.stemcr.2016.04.011. PubMed DOI PMC

Vandecasteele G, Eschenhagen T, Scholz H, Stein B, Verde I, Fischmeister R. Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase. Nat. Med. 1999;5(3):331–334. doi: 10.1038/6553. PubMed DOI

Schwinger RHG, Böhm M, Erdmann E. Negative inotropic properties of isradipine, nifedipine, diltiazem, and verapamil in diseased human myocardial tissue. J. Cardiovasc. Pharmacol. 1990;15(6):892–899. doi: 10.1097/00005344-199006000-00006. PubMed DOI

Sala L, et al. Musclemotion: A versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo. Circ. Res. 2018;122(3):e5–e16. doi: 10.1161/CIRCRESAHA.117.312067. PubMed DOI PMC

van Meer BJ, Sala L, Tertoolen LGJ, Smith GL, Burton FL, Mummery CL. Quantification of muscle contraction in vitro and in vivo using MUSCLEMOTION software: From stem cell-derived cardiomyocytes to zebrafish and human hearts. Curr. Protoc. Hum. Genet. 2018;99(1):1–21. PubMed

Thavandiran N, et al. Functional arrays of human pluripotent stem cell-derived cardiac microtissues. Sci. Rep. 2020;10(1):1–13. doi: 10.1038/s41598-020-62955-3. PubMed DOI PMC

Thorn CF, et al. Doxorubicin pathways: Pharmacodynamics and adverse effects. Pharmacogenet. Genom. 2011;21(7):440. doi: 10.1097/FPC.0b013e32833ffb56. PubMed DOI PMC

Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: From molecular mechanisms to therapeutic strategies. J. Mol. Cell. Cardiol. 2012;52(6):1213–1225. doi: 10.1016/j.yjmcc.2012.03.006. PubMed DOI

Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy. From the cardiotoxic mechanisms to management. Prog. Cardiovasc. Dis. 2007;49(5):330–352. doi: 10.1016/j.pcad.2006.10.002. PubMed DOI

Silva, D., Santos, D., Coeli, R. & Goldenberg, S. Doxorubicin-induced cardiotoxicity: From mechanisms to development of efficient therapy. Cardiotoxicity. (ed. Tan, W.) 3 - 24. (IntechOpen, 2018) 10.5772/intechopen.79588 (2018).

Tanaka R, et al. Reactive fibrosis precedes doxorubicin-induced heart failure through sterile inflammation. ESC Heart Fail. 2020;7(2):588–603. doi: 10.1002/ehf2.12616. PubMed DOI PMC

Levick SP, et al. Doxorubicin-induced myocardial fibrosis involves the neurokinin-1 receptor and direct effects on cardiac fibroblasts. Heart Lung Circ. 2019;28(10):1598–1605. doi: 10.1016/j.hlc.2018.08.003. PubMed DOI PMC

Page RL, et al. Drugs that may cause or exacerbate heart failure. Circulation. 2016;134(6):e32–e69. doi: 10.1161/CIR.0000000000000426. PubMed DOI

Gilsbach R, et al. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease. Nat. Commun. 2014;5:5288. doi: 10.1038/ncomms6288. PubMed DOI PMC

Grancharova T, et al. A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes. Sci. Rep. 2021;11(1):1–21. doi: 10.1038/s41598-021-94732-1. PubMed DOI PMC

Meilhac SM, Buckingham ME. The deployment of cell lineages that form the mammalian heart. Nat. Rev. Cardiol. 2018;15(11):705–724. doi: 10.1038/s41569-018-0086-9. PubMed DOI

Zhang XH, Wei H, Šarić T, Hescheler J, Cleemann L, Morad M. Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes. Cell Calcium. 2015;57:321. doi: 10.1016/j.ceca.2015.02.003. PubMed DOI PMC

Karbassi E, et al. Cardiomyocyte maturation: Advances in knowledge and implications for regenerative medicine. Nat. Rev. Cardiol. 2020;17(6):341. doi: 10.1038/s41569-019-0331-x. PubMed DOI PMC

Schaper J, Meiser E, Stammler G. Ultrastructural morphometric analysis of myocardium from dogs, rats, hamsters, mice, and from human hearts. Circ. Res. 1985;56(3):377–391. doi: 10.1161/01.RES.56.3.377. PubMed DOI

Spitkovsky D, et al. Activity of complex III of the mitochondrial electron transport chain is essential for early heart muscle cell differentiation. FASEB J. 2004;18(11):1300–1302. doi: 10.1096/fj.03-0520fje. PubMed DOI

Cadenas S. Mitochondrial uncoupling, ROS generation and cardioprotection. Biochim. Biophys. Acta Bioenerg. 2018;1859(9):940–950. doi: 10.1016/j.bbabio.2018.05.019. PubMed DOI

Perestrelo AR, et al. Multiscale analysis of extracellular matrix remodeling in the failing heart. Circ. Res. 2021;128:24–38. doi: 10.1161/CIRCRESAHA.120.317685. PubMed DOI

Rodrigues PG, et al. Early myocardial changes induced by doxorubicin in the nonfailing dilated ventricle. Am. J. Physiol. Heart Circ. Physiol. 2019;316(3):H459–H475. doi: 10.1152/ajpheart.00401.2018. PubMed DOI

Dolci A, Dominici R, Cardinale D, Sandri MT, Panteghini M. Biochemical markers for prediction of chemotherapy-induced cardiotoxicity systematic review of the literature and recommendations for use. Am. J. Clin. Pathol. 2008;130(5):688–695. doi: 10.1309/AJCPB66LRIIVMQDR. PubMed DOI

Ho BX, Pek NMQ, Soh BS. Disease modeling using 3D organoids derived from human induced pluripotent stem cells. Int. J. Mol. Sci. 2018;19:936. doi: 10.3390/ijms19040936. PubMed DOI PMC

Marchianò S, Bertero A, Murry CE. Learn from your elders: Developmental biology lessons to guide maturation of stem cell-derived cardiomyocytes. Pediatr. Cardiol. 2019;40(7):1367–1387. doi: 10.1007/s00246-019-02165-5. PubMed DOI PMC

Xu T, Wu L, Xia M, Simeonov A, Huang R. Systematic identification of molecular targets and pathways related to human organ level toxicity. Chem. Res. Toxicol. 2021;34(2):412–421. doi: 10.1021/acs.chemrestox.0c00305. PubMed DOI PMC

Hoang P, et al. Engineering spatial-organized cardiac organoids for developmental toxicity testing. Stem Cell Rep. 2021;16(5):1228–1244. doi: 10.1016/j.stemcr.2021.03.013. PubMed DOI PMC

Pagliari S, et al. YAP–TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification. Cell Death Differ. 2020;28(4):1193–1207. doi: 10.1038/s41418-020-00643-5. PubMed DOI PMC

Vrbský J, et al. Evidence for discrete modes of YAP1 signaling via mRNA splice isoforms in development and diseases. Genomics. 2021;113(3):1349–1365. doi: 10.1016/j.ygeno.2021.03.009. PubMed DOI

Chen EY, et al. Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinform. 2013;14:128. doi: 10.1186/1471-2105-14-128. PubMed DOI PMC

Kuleshov MV, et al. Enrichr: A comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44(W1):W90–W97. doi: 10.1093/nar/gkw377. PubMed DOI PMC

Xie Z, et al. Gene set knowledge discovery with Enrichr. Curr. Protoc. 2021;1(3):e90. doi: 10.1002/cpz1.90. PubMed DOI PMC

Shannon P, et al. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–2504. doi: 10.1101/gr.1239303. PubMed DOI PMC

Torre D, Lachmann A, Ma’ayan A. BioJupies: Automated generation of interactive notebooks for RNA-Seq data analysis in the cloud. Cell Syst. 2018;7(5):556–561.e3. doi: 10.1016/j.cels.2018.10.007. PubMed DOI PMC

Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis