The Bernard and Joan Marshall Young Investigator Prize

The Bernard and Joan Marshall Young Investigator Prize

The role of the epicardium in cardiac repair

Johannes Bargehr

The Anne McLaren Laboratory, Wellcome Trust –MRC Cambridge Stem Cell Institute, Forvie Site, University of Cambridge, Robinson Way, Cambridge CB2 0SZ, UK

Division of Cardiovascular Medicine, University of Cambridge, ACCI Level 6, Box 110, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK

 

  1. Introduction

At present, around 25 Million people worldwide suffer from chronic heart failure. Following the exhaustion of medical and device therapy, these patients have no other therapeutic option to make up for the loss of contractile working myocardium other than heart transplantation. Approaches in regenerative medicine making use of human pluripotent stem cell (hPSC)-derived cardiomyocytes in order to remuscularise the infarcted heart and restore function have provided promising preclinical data in large animal models but limitations such as cardiomyocyte immaturity and poor vascularisation remain. Concomitantly, over the past decade the epicardium has sparked much interest in regenerative cardiovascular medicine due to its pivotal role in embryonic heart development, serving as a cellular source for coronary smooth muscle cells and interstitial fibroblasts. Here the role of hPSC-derived epicardial cells is discussed in the context of regenerative cardiovascular medicine.

  1. The epicardium in embryonic heart development

It is in early embryonic heart development, that the epicardium displays unique functionality that makes it an interesting candidate for cardiac repair. At around 3.5 weeks of human heart development epicardial cells arise from the proepicardial organ and cover the surface of the embryonic heart tube. The epicardium then starts to undergo epithelial to mesenchymal transition (EMT) and invades the subepicardial space and subjacent compact myocardium [1]. During this process epicardial cells give rise to smooth muscle cells (SMC), required for the formation of the coronary vasculature as well as to interstitial cardiac fibroblasts (CF), which result in cardiac compaction and maturation [2-4] (Figure 1, Panel A). Epicardial outgrowth inhibition in chicken embryos has resulted in malformation of the coronary vasculature and myocardial non-compaction, resulting in early embryonic death [5]. The developmental cues driving the chemically defined differentiation of epicardial cells as well as epicardium-derived smooth muscle cells and fibroblasts from human pluripotent stem cells are shown in Figure 2. In this context, some of our own data have shown that the fate of epicardial cells is system and developmental stage dependent (Figure 1, Panel B). Using an athymic rat model of myocardial infarction, we observed that intramyocardial injection of hPSC-derived epicardial cells into the infarct zone resulted in robust fibroblast graft formation. In contrast, using a developmental chicken embryo transplantation of hPSC-epicardial cells into the extraembryonic vasculature resulted in formation of epicardium-derived smooth muscle cells and integration thereof into pre-existing chicken vasculature. Additionally, pioneering work by Nicola Smart and Paul Riley has shown that if primed with Thymosin beta 4, epicardial cells are even capable of giving rise to cardiomyocytes [6]. Increasing the efficiency of this process could open up another avenue in epicardium-mediated cardiac repair.

  1. Remuscularising the failing heart with human pluripotent stem cell-derived-cardiomyocytes

The only therapeutic option for patients with heart failure that directly addresses the underlying loss of cardiomyocytes is heart transplantation. Despite a huge potentially eligible patient population only as many as 200 heart transplantations are performed in the entire UK every year. Regenerative medicine, using human pluripotent stem cells, including induced pluripotent stem cells (iPSC) or human embryonic stem cells (hESCs), is a promising novel therapeutic domain that has fostered the rise of 3D-engineered heart tissue (3D-EHT)-based approaches as well as those making use of direct intramyocardial cell injection of human pluripotent stem cell (hPSC)-derived cardiomyocytes. The latter of the two approaches has been very successful in rodent models of myocardial infarction where transplantation of hESC-derived cardiomyocytes has led to robust cardiovascular graft formation and rescue of global host heart function [7, 8]. Endeavours to translate this approach to a model that more closely resembles human disease demonstrated that transplantation of 1 Billion hESC-derived cardiomyocytes results in robust graft formation in infarcted non-human primates and more recently this was also shown for hiPSC-derived cardiomyocytes using the same model [9, 10]. While these studies have provided compelling pre-clinical data in a close-to-clinical-environment setting, elegantly using clinical grade heart catheters for percutaneous vascular access and delivery into the left ventricular wall, the field is eagerly awaiting functional data corroborating the concept that robust cardiac grafting also results in improvement of host heart function. While this technology has provided promising evidence for clinical applicability and translational potential a number of shortcomings remain.

  1. Key challenges remaining

Mechanistic insights into embryonic organ development have rendered possible the in vitro generation of a large number of different body tissues from hPSCs. While derived and terminally differentiated cells express all markers of their adult human counterparts and exhibit functionality, given the process and the time required for their derivation, they fall short of reaching sound levels of maturity. For cardiomyocytes, this means that hPSC-derived, beating monolayer cultures at best resemble the phenotype of those found in a third trimester embryo [11, 12]. Transplantation of cardiomyocytes with a relatively immature phenotype means suboptimal structural integrity of cardiac grafts and related function post engraftment. Additionally, cell death following transplantation into ischaemic myocardium is high resulting in optimizable cardiac graft size and vascular supply to grafts is poor compared to physiologic myocardial tissue [13]. Meeting these shortcomings could potentially catalyse critical progress in regenerative cardiovascular repair. While the embryonic identity of hPSC-derived tissues is often seen as a drawback in regenerative medicine, the functionality displayed by the epicardium in early embryonic heart development and reflected by hPSC-derived epicardium could critically aid meeting some of the key limitations currently present in cardiac repair, including vascularisation and maturation.

  1. Potential applications of the epicardium in regenerative medicine

Regenerative medicine has fostered the paradigm that a better understanding of developmental processes can aid organ regeneration. While only deeper molecular insights have allowed for the derivation of specific body tissues in vitro, the generation of more complex tissue-systems could help advance cardiac repair beyond current limitations. The use of the epicardium as an adjuvant cardiovascular therapeutic for tissue engineering application is hence a tantalising approach. HPSC-derived epicardial cells, if added to cardiomyocytes in 3D-EHTs could help to better vascularise transplantable heart patches and provide for structural integrity and maturation [14]. On a different note the capability to generate epicardial cells will also allow for mimicking of developmental processes like embryonic heart tube formation. Such a multi-cellular construct, incorporating epicardium, myocardium and endocardium, if devised, would allow for unprecedented opportunities to move forward our understanding of cardiac developmental and regenerative processes. Furthermore, the field of drug toxicity testing could substantially benefit from more mature hiPSC-derived cardiomyocytes to gain a more high-fidelity readout of calcium traces that more closely resemble the toxic effects seen in patients following drug administration. The functionality of hPSC-derived epicardial cells seen in preclinical studies makes them a potentially interesting adjuvant therapeutic for cardiac repair that promises to address some of the key limitations that the field currently faces. This could usher in a new, more complex era of cardiac regeneration that will move closer to generating bona fide cardiac tissue and more efficient repair.

Figure 1. Fate and function of the epicardium in development and disease. (A) The epicardium during embryonic heart development. At 3.5 weeks of human heart development the epicardium starts to cover the heart surface. It subsequently starts to invade the subepicardial space and undergoes epithelial to mesenchymal transition, giving rise to cardiac fibroblasts (CF) (1) and coronary smooth muscle cells (SMC) (2). It remains controversial whether epicardial cells can also give rise to endothelial cells (EC) (3) and cardiomyocytes (CM) (4). (B) The epicardium during cardiac injury. Top schematic demonstrates that in the adult organism CFs and SMCs are epicardium-derived. The bottom schematic demonstrates reactivation of the embryonic program in the epicardium following cardiac injury. Epicardial cells start to invade the myocardium and the infarct zone (1) mainly giving rise to CFs (3) within the infarct with a small fraction giving rise to the more mature myofibroblasts (MF) (4). In contrast, outside of the infarct zone the epicardium is capable of giving rise to smooth muscle cells (2).

Figure 2. Molecular cues driving germ layer and cardiovascular lineage specification. Activin and FGF pathways maintain pluripotency of hESCs [15]. Activation of FGF and inhibition of Activin/ Nodal signalling direct ectoderm formation, while Wnt-inhibition and activation of Activin and BMP signalling triggers endoderm formation[16]. The main cardiovascular lineages derive from the mesoderm, which forms through activation of Wnt, Activin and BMP signalling [17]. VEGF-A directs the differentiation to endothelial cells [18, 19], PDGF-BB and TGF-β1 to smooth muscle cells [20, 21], Wnt, BMP and RA to epicardium [22], and BMP activation and Wnt inhibition to cardiomyocytes [7, 23]. The epicardium, being a cardiovascular progenitor population, is capable of giving rise to cardiac fibroblasts and smooth muscle cells [22].

 

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