Targeting the epicardium for heart repair
In the aftermath of a myocardial infarction, the millions of cardiomyocytes that have died are not replaced. Instead, the wound is healed by fibrotic tissue that will forever struggle to support the pumping function of the heart. With ischemic heart disease being the number one killer worldwide, finding a way to repair the heart is one of the most important challenges in medicine today. In search of innovative solutions, some scientists have turned their attention to the epicardium, the thin layer of cells covering the surface of the heart. We know that the epicardium responds to injury by secreting signals that recruit inflammatory immune cells and stabilize the surrounding vasculature. But, in an alternative scenario, it could drive tissue regeneration by instructing uninjured cardiomyocytes to self-renew or by producing new blood vessel components and, possibly, new cardiomyocytes. This already happens in some lower vertebrate species such as the salamander and zebrafish, which can fully repair severely injured muscle tissue without scarring thanks to epicardial activation. Similar mechanisms are observed in mammals, but only up until around seven days after birth. One way to replicate this in the adult human heart could be to take inspiration from our own embryonic development: in the early stages of heart formation, the epicardium gives rise to progenitors that differentiate into multiple cell types including fibroblasts and mural cells. It also stimulates the proliferation of cardiomyocytes, which is essential for the formation of a compact zone of myocardium underneath the epicardium. However, we still lack a lot of information on epicardial biology and how it could be harnessed for regeneration.
Imitating human epicardial development in vitro
In the last decade, biomedical research has been revolutionized by a new class of human in vitro models: organoids. Organoids are 3D miniature tissues that replicate the cell composition, structure, and function of a given organ. Crucially, unlike engineered tissues obtained through the co-culture of differentiated cells, organoids are formed by coaxing stem cells into self-organization via mechanisms imitating embryonic development. This makes them highly valuable tools to study normal organ development as well as pathological mechanisms in vitro, bridging the gap between 2D cell culture and animal models. The rapid development of organoid models has notably played a major role in the recent passing of legislation stating that drugs no longer need to be tested in animals before human trials to receive FDA approval1. The catch, for cardiovascular researchers, is how difficult it has been to generate heart organoids compared to other organs such as the brain, intestine, or kidney. To this day, no one has been able to fully recapitulate the architecture of the heart in a self-organizing organoid, though there has been significant progress in the last two years2,3.
In a study published in Nature Biotechnology this week, our lab established a cardiac organoid model showing the co-development of ventricular myocardium and epicardium, which we called epicardioids. This is the first organoid showing a fully functional epicardium: it forms a stable epithelial outer layer, gives rise to multipotent epicardium-derived cells (EPDCs), and stimulates cardiomyocyte proliferation. The latter promotes the morphological, molecular, and functional patterning of the myocardium into compact and trabecular-like layers, a unique feature of epicardioids. To dissect the mechanisms of epicardioid formation, we collected single-cell RNA and ATAC sequencing data at eight time points of differentiation, which we analyzed in collaboration with experts in single-cell genomics at the TUM. By generating multiomic ‘metacells’ containing both transcriptome and chromatin accessibility information, we were able to reconstruct the different lineage trajectories of cells and infer the associated gene regulatory networks. We then validated key findings through genetic lineage tracing. These analyses showed that epicardioids are formed by two main progenitor populations: first heart field (FHF) cells, which differentiate into ventricular cardiomyocytes, and juxta-cardiac field (JCF) cells, which give rise to both cardiomyocytes and epicardial cells. The JCF was only recently discovered as a common progenitor pool of the myocardium and epicardium in mice4,5; we have now successfully generated and characterized human JCF cells both in epicardioids and in a 2D differentiation system6. We could also demonstrate that EPDCs spontaneously differentiate into fibroblasts, mural cells, and cardiomyocytes – providing evidence for the controversial myocytic potential of mammalian epicardial cells.
Applications of epicardioids in cardiovascular medicine
Beyond developmental insights, the complexity of epicardioids in terms of cell composition, organization, and crosstalk makes them a versatile platform for disease modeling. The presence of epicardium-derived fibroblasts notably allowed us to recapitulate the myofibroblast activation driving fibrotic remodeling in stress-induced and congenital left ventricular hypertrophy, a process that has been difficult to study in vitro until now. Going forward, we hope that studies in epicardioids will shed light on the epicardium’s role in development and disease and bring us closer to therapies tapping into its regenerative potential.
- Wadman, M. FDA no longer has to require animal testing for new drugs. Science 379, 127–128 (2023).
- Hofbauer, P. et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell 184, 3299-3317.e22 (2021).
- Drakhlis, L. et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 39, 737–746 (2021).
- Tyser, R. C. V. et al. Characterization of a common progenitor pool of the epicardium and myocardium. Science (80-. ). 371, (2021).
- Zhang, Q. et al. Unveiling Complexity and Multipotentiality of Early Heart Fields. Circ. Res. 129, 474–487 (2021).
- Zawada, D. et al. Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools. Nat. Commun. 14, 1722 (2023).