Abstract
Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The limited availability of human CFs from native heart impedes investigations of CF biology and their role in disease. Human pluripotent stem cells (hPSCs) provide a highly renewable and genetically defined cell source, but efficient methods to generate CFs from hPSCs have not been described. Here, we show differentiation of hPSCs using sequential modulation of Wnt and FGF signaling to generate second heart field progenitors that efficiently give rise to hPSC-CFs. The hPSC-CFs resemble native heart CFs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by TGFβ1 and angiotensin II. Furthermore, hPSC-CFs exhibit a more embryonic phenotype when compared to fetal and adult primary human CFs. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration.
Highlights
Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease
Given that high bFGF concentrations can be supportive of maintenance of pluripotency of Human pluripotent stem cells (hPSCs), we examined the expression of the pluripotency gene OCT4 and showed it to be completely downregulated during the CF differentiation (Fig. 3c), similar to the downregulation observed in cardiomyocyte differentiation protocols[7,23,40,41]
In the present study, we demonstrate that stage-specific activation of Wnt and fibroblast growth factor (FGF) signaling promotes the efficient differentiation of hPSCs via second heart field progenitors (SHFPs) to CFs
Summary
Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. Lineage tracing with the epicardial marker, Tbx[18], demonstrated Tbx18-expressing fibroblasts compromised only one-third of the CFs in embryonic and adult heart[13]; a more recent investigation with a Wt1-Cre mouse showed up to 80% of CFs in the adult heart were derived from the epicardium[19]. Another significant population of CFs is derived from the endocardium at the time of endocardial cushion formation by an endothelial-tomesenchymal transition[19,20], and endocardial cells are generated in part from the second heart field progenitors (SHFPs)[21]. The differentiated hPSC-CFs exhibit cell morphology, growth, gene expression, fibroblast markers, ECM production, and myofibroblast transformation similar to native human CFs
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