Generation of microvascular endothelium in induced pluripotent stem cell-derived myocardium
File(s)
Author(s)
King, Oisín
Type
Thesis
Abstract
Physiologically relevant in vitro models of the human myocardium are required to study basic biological mechanisms, and to further the impact of disease modelling and drug discovery. In vivo, a dense network of capillaries communicates bi-directionally with beating myocardium to regulate heart homeostasis (D. L. Brutsaert, 2003; Segers et al., 2018). However, microvascular architectures subject to the contractile and haemodynamic biomechanics of the beating heart have not yet been replicated in vitro. This project aimed to establish a myocardial – endothelial coculture platform which approximated the biomechanical conditions of the human myocardium, enabling the study of myocardial – endothelial communication in the regulation of cardiac homeostasis.
Here we report the generation of a fibrin hydrogel-based 3D coculture platform, in which beating human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CM) are embedded in direct contact with human cardiac microvascular endothelial cells (hCMVEC) and human left ventricular fibroblasts (hLVFB), thus enabling both biochemical and biomechanical modes of heterocellular communication. We then characterise various aspects of the electrophysiology of hiPSC-CM, and demonstrate distinct regulation of action potential, calcium handling, spontaneous beating rate and arrhythmogenesis, depending on coculture condition. We then report the use of microfluidic perfusion and vasculogenic growth factor supplementation to induce spontaneous assembly of functional myocardial microvasculature in a novel Heart-on-a-chip model, with vascularised myocardium again demonstrating altered calcium handling compared to hiPSC-CM control. Additionally, this model allows observation and interrogation of human myocardial microvascular flow dynamics for the first time. Via perfusion and tracking of red blood cells through myocardial microvasculature, we report biomimetic pulsatile flow profile generated by beating hiPSC-CM.
By reproducing the cellular composition and architecture of human myocardial microvasculature, while approximating cardiac contractile and haemodynamic biomechanics, we have reported novel CM-EC coculture platforms which demonstrate significant regulation of myocardial electrophysiology by a microvascular endothelium, in vitro. These results build on the increasing recognition of the myocardial endothelium as a key mediator of heart homeostasis and offer novel in vitro strategies to further explore the role of the endothelium in cardiac health and disease.
Here we report the generation of a fibrin hydrogel-based 3D coculture platform, in which beating human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CM) are embedded in direct contact with human cardiac microvascular endothelial cells (hCMVEC) and human left ventricular fibroblasts (hLVFB), thus enabling both biochemical and biomechanical modes of heterocellular communication. We then characterise various aspects of the electrophysiology of hiPSC-CM, and demonstrate distinct regulation of action potential, calcium handling, spontaneous beating rate and arrhythmogenesis, depending on coculture condition. We then report the use of microfluidic perfusion and vasculogenic growth factor supplementation to induce spontaneous assembly of functional myocardial microvasculature in a novel Heart-on-a-chip model, with vascularised myocardium again demonstrating altered calcium handling compared to hiPSC-CM control. Additionally, this model allows observation and interrogation of human myocardial microvascular flow dynamics for the first time. Via perfusion and tracking of red blood cells through myocardial microvasculature, we report biomimetic pulsatile flow profile generated by beating hiPSC-CM.
By reproducing the cellular composition and architecture of human myocardial microvasculature, while approximating cardiac contractile and haemodynamic biomechanics, we have reported novel CM-EC coculture platforms which demonstrate significant regulation of myocardial electrophysiology by a microvascular endothelium, in vitro. These results build on the increasing recognition of the myocardial endothelium as a key mediator of heart homeostasis and offer novel in vitro strategies to further explore the role of the endothelium in cardiac health and disease.
Version
Open Access
Date Issued
2021-08
Date Awarded
2022-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Terracciano, Cesare
Randi, Anna
Sponsor
British Heart Foundation
Grant Number
FS/16/56/32732
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)