Abstract 17734: Functional Cardiac Fibroblasts Derived From Human Pluripotent Stem Cells via Second Heart Field Progenitors

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Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The sparse availability of human CFs from the native heart limits studies investigating their biology and applications to regenerative medicine. Whereas the directed differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs), endothelial cells and smooth muscle cells has advanced significantly, efficient differentiation to CFs has not been described. Here, we reveal that hPSCs can be directed to differentiate to CFs via second heart field (SHF) progenitors by the sequential modulation of Wnt and FGF signaling. Confluent monolayer hPSCs were treated with GSK3β inhibitor (CHIR) for 24 hrs, followed by 1 day culture in the differentiation medium, and then treated with bFGF in a defined medium until 20 days differentiation. Flow cytometry and qRT-PCR showed sequential upregulation of markers for mesoderm (T), cardiac mesoderm (MESP1) and SHF progenitors including GATA4, ISL1, TBX1 and HAND2 during day 1-6 differentiation, in contrast to the CMs differentiation protocol (GiWi) in which the first heart field genes, including HCN4, TBX5 and HAND1 were upregulated in the early differentiation. Continuous treatment with bFGF after day 6 further promoted fibroblasts differentiation. Flow cytometry for fibroblast markers showed ~77% of the cells were fibroblasts after 20 days of differentiation. The hPSC-derived CFs resemble native heart CFs in overall gene expression demonstrated by RNA-seq. Moreover, the hPSC-CFs express key cardiac transcription factors including GATA4, ISL1, HAND2, HEY1 and SOX17. The hPSC-CFs produced abundant extracellular matrix (ECM) when seeded at a high density and cultured for 10 days, forming a unique 3D ECM scaffold composed of collagen and fibronectin, which was comparable to the native CFs ECM scaffold, but distinct from dermal fibroblast ECM scaffold. Optical mapping showed the co-culture of hPSC-CFs coupled with hPSC-CMs and modulated the electrophysiological properties of hPSC-CMs. We conclude that CFs can be efficiently differentiated from hPSCs via SHF progenitors in high yield and purity, thereby providing an unlimited cell source for research and therapeutic applications in cardiac regeneration.

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