Functional Cardiac Fibroblasts Derived from Human Pluripotent Stem Cells via Second Heart Field Progenitors

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 (EP) of the cardiomyocytes compared to co-culture with dermal fibroblasts (DFs). The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration. We performed RNA-seq for hPSC-CFs, primary CFs and DFs to compare the gene expression for each type of fibroblasts. We also added the hPSC-CMs and hPSCs as the positive and negativ controls in the RNA-seq to compare the gene expression for cardaic factors. Five cell types (hPSC-CFs, human adult CFs and DFs, hPSC-CMs and hPSCs) with 2 biological replicates for each cell type of a total 10 samples were sequenced.