Proepicardial Cell Differentiation During Cardiac Development

Epicardial cells have an emerging role as cardiac stem cells. These cells and their progenitors, proepicardial (PE) cells, have been recently shown to give rise to all resident cell types in the heart. The contribution of PE-derived cells to the development of atrioventricular (AV) valves and myocardium is not well established. However, our data indicate that PE cell differentiation is affected the surrounding environment. PE cells that were co-cultured with cardiac myocytes (CM) differentiated into CM cells. On the other hand, PE cells that were co-cultured with AV valve progenitor cells of the AV cushion, migrated into the cushion region of the developing heart and became mesenchymal cells. PE cells contribute to the myocardium in that they have the potential of differentiating into CMs. Confocal, flow cytometry, and live cell imaging studies showed that PE cells co-cultured with CMs did not only differentiate into beating CMs but also stimulated the proliferation of existing CMs. CMs are the limiting factor in the healing of the myocardium after a myocardial infarct, therefore finding a progenitor source for CM will provide the possibility for regeneration-based therapies for heart disease. PE cells also contribute to AV valve development by undergoing epithelial-to-mesenchymal transformation (EMT) and stimulating the deposition of fibrous extracellular matrix (ECM) molecules including periostin, tenascin, and collagen. The expression and localization of these ECM proteins is critical to the proper development and function of the heart. These phenomenons were inhibited by the addition of a TGFb family inhibitor (SB 431542). Our studies indicate that members of the TGFb family of proteins, specifically the combination of TGFb1, 2 and 3, are critical regulators of this process. PE cells cultured with the combination of TGFb1, 2 and 3, had a significant increased in EMT compared to PEs alone. Using confocal analysis and real time PCR, we investigated the expression and localization of ECM molecules in these transformed cells. These observations indicate that the AV cushions release TGFb molecules that attract PE cells, which then express fibrous ECM proteins. Since AV valve and sepal birth defects are amongst the most common of all birth defects, delineating molecular mechanisms of their formation is critical. We further suggest that understanding the mechanisms of valve formation, and specifically the role of these multipotent cells, will benefit the long term goal of developing new therapies for these birth defects and pave the way for the in vitro production of replacement valvular and septal tissues