Bioreactor-based 3D human myocardial ischemia/reperfusion in vitro model: a novel tool to unveil key paracrine factors upon acute myocardial infarction

Abstract

During acute myocardial infarction (AMI), Ischemia/Reperfusion (I/R) injury causes cardiomyocyte (CM) death and loss of tissue function, making AMI one of the major causes of death worldwide. Cell-based in vitro models of I/R injury have been increasingly used as a complementary approach to preclinical research. However, most approaches use murine cells in 2D culture setups, which are not able to recapitulate human cellular physiology, as well as nutrient and gas gradients occurring in the myocardium. In this work we established a novel human in vitro model of myocardial I/R injury using CMs derived from human induced pluripotent stem cells (hiPSC-CMs), which were cultured as 3D aggregates in stirred tank bioreactors. We were able to recapitulate important hallmarks of AMI, including loss of CM viability with disruption of cellular ultrastructure, increased angiogenic potential, and secretion of key proangiogenic and proinflammatory cytokines. Conditioned medium was further used to probe human cardiac progenitor cells (hCPCs) response to paracrine cues from injured hiPSC-CMs through quantitative whole proteome analysis (SWATH-MS). I/R injury hiPSC-CM conditioned media incubation caused upregulation of hCPC proteins associated with migration, proliferation, paracrine signaling, and stress response-related pathways, when compared to the control media incubation.Our results indicate that the model developed herein can serve as a novel tool to interrogate mechanisms of action of human cardiac populations upon AMI. (Translational Research 2020; 215:57À74) Abbreviations: 2D = two-dimensional; 3D = three-dimensional; AMI = acute myocardial infarction; CMs = cardiomyocytes; CPCs = cardiac progenitor cells; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; FDA = fluorescein diacetate; hCPCs = human cardiac progenitor cells; HGF = hepatocyte growth factor; HIF-1a = hypoxia-inducible factor 1 a; hiPSC-CMs = human induced pluripotent stem cell derived cardiomyocytes; I/R = Ischemia/ Reperfusion; IGF-1 = insulin-like growth factor 1; IMS = ischemic mimetic solution; IPA = ingenuity pathway analysis; MS = mass spectrometry; PFA = paraformaldehyde; PI = propidium iodide; SWATH = sequential window acquisition of all theoretical mass spectra; T3 = triiodothyronine From the iBET,

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features

“…Our findings may be related with paracrine factors produced by CM known to be responsible for maintenance of non-myocyte cells viability during in vivo cardiac development, namely, VEGF-A, or angiopoietin (Hsieh et al, 2006). In fact, a previous study from our group has identified expression of angiogenic factors (including VEGF) in the secretome hiPSC-CM aggregates (Sebastião et al, 2020). A deeper understanding of how hiPSC-CM communicate with hiPSC-EC+MC will be important not only to develop more physiologically relevant cardiac tissue models but also to design advanced therapies for cardiac regeneration.…”

Considerations to Model Heart Disease in Women with Preeclampsia and Cardiovascular Disease

“…Tissue engineering approaches using bioreactors or microfluidics devices allow further maturation of hIPS-CMs into a more adult phenotype, but more progress needs to be made as they cannot recapitulate complex features typical of the in vivo microenvironment, such as adult cardiomyocyte function [9,102]. Bioreactors or microfluidic "organ-on-chips" devices provide precise control and better recapitulate the cellular microenvironment of IRI of the heart compared to other in vitro systems, including the monitoring of critical parameters such as pH and oxygen levels [76,103]. However, these cultures do not fully represent the complexity and tissue architecture of the heart due to the absence of different cell types such as fibroblast, endothelial cells and immune cells [102].…”

Beyond Family: Modeling Non-hereditary Heart Diseases With Human Pluripotent Stem Cell-Derived Cardiomyocytes

“…Modeling septic shock by exposure to bacterial lipopolysaccharides affects hPSC-CM survival, electrophysiology and demonstrates their competence in activating innate immune inflammatory responses (Yucel et al, 2017). Indeed, hPSC-CM display stronger macrophage chemo-attractant properties than purified chemokines (Pallotta et al, 2015) and significant stress-responsive paracrine proinflammatory signaling (Sebastiao et al, 2020) mediating fibrosis in vivo and in vitro (Kumar et al, 2019;Zhang et al, 2019). Furthermore, functional expression of coxsackievirus and adenovirus receptor (Scassa et al, 2011) makes hPSC-CMs a better predictive model than murine cardiac cell lines for therapeutic approaches against viral myocarditis (Sharma et al, 2014).…”

“…A recent hEHT I/R model showed for the first time in human cells the cardioprotective effect of ischemic preconditioning and efficacy of one out of three proposed cardioprotection treatments for reperfusion injury (Chen and Vunjak-Novakovic, 2019). Nevertheless, similar hEHTs generated by pure hPSC-CM populations are limited in their maturation potential (Park et al, 2019) and limit the study of the complex bidirectional crosstalk of multiple cell types, important during ischemic stress via paracrine signaling (Sandstedt et al, 2018;Sebastiao et al, 2019Sebastiao et al, , 2020 and neurohormonal stimulation. The latter can be effectively modeled solely with hEHTs, as 2D cultures lack or display functionally impaired a β-adrenergic signaling cascades (Jung et al, 2016;Uzun et al, 2016;Trieschmann et al, 2019), despite being able to form functional sympathetic neurojunctions (Sakai et al, 2017).…”