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Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

ABSTRACT: Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart ("cardiac spheroids", CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.

SUBMITTER: Polonchuk L

PROVIDER: S-EPMC5539326 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

Polonchuk Liudmila L Chabria Mamta M Badi Laura L Hoflack Jean-Christophe JC Figtree Gemma G Davies Michael J MJ Gentile Carmine C

Scientific reports 20170801 1

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart ("cardiac spheroids", CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to inve ...[more]

PMID: 28765558

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Project description:The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC-CM and single cells of hiPSC-derived EC and MC (hiPSC-EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC-EC+MC survival was promoted by the co-culture with hiPSC-CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC-EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC-CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC-CM, hiPSC-EC+MC, and extracellular matrix induced the maturation of hiPSC-CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.

Project description:Cardiovascular diseases are a leading cause of death worldwide. After an ischemic injury, the myocardium undergoes severe necrosis and apoptosis, leading to a dramatic degradation of function. Numerous studies have reported that cardiac fibroblasts (CFs) play a critical role in heart function even after injury. However, CFs present heterogeneous characteristics according to their development stage (i.e., fetal or adult), and the molecular mechanisms by which they maintain heart function are not fully understood. The aim of this study is to explore the hypothesis that a specific population of CFs can repair the injured myocardium in heart failure following ischemic infarction, and lead to a significant recovery of cardiac function. Flow cytometry analysis of CFs defined two subpopulations according to their relative expression of vascular cell adhesion molecule 1 (VCAM1). Whole-transcriptome analysis described distinct profiles for these groups, with a correlation between VCAM1 expression and lymphangiogenesis-related genes up-regulation. Vascular formation assays showed a significant stimulation of lymphatic cells network complexity by VCFs. Injection of human VCAM1-expressing CFs (VCFs) in postinfarct heart failure rat models (ligation of the left anterior descending artery) led to a significant restoration of the left ventricle contraction. Over the course of the experiment, left ventricular ejection fraction and fractional shortening increased by 16.65% ± 5.64% and 10.43% ± 6.02%, respectively, in VCF-treated rats. Histological examinations revealed that VCFs efficiently mobilized the lymphatic endothelial cells into the infarcted area. In conclusion, human CFs present heterogeneous expression of VCAM1 and lymphangiogenesis-promoting factors. VCFs restore the mechanical properties of ventricular walls by mobilizing lymphatic endothelial cells into the infarct when injected into a rat heart failure model. These results suggest a role of this specific population of CFs in the homeostasis of the lymphatic system in cardiac regeneration, providing new information for the study and therapy of cardiac diseases.

Project description:The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. STATEMENT OF SIGNIFICANCE: With the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.

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Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

Publications

Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

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