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

Jianhua Zhang,Ran Tao,Katherine Campbell,Juliana Carvalho,Edward Ruiz,Gina Kim,Eric Schmuck,Amish Raval,James Thomson,Todd Herron,Jose Jalife,Timothy Kamp

doi:10.1016/j.yjmcc.2018.07.009

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 applications in cardiac regeneration. Human pluripotent stem cells (hPSCs) provide an unlimited cell source, but effective 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. Confluent monolayer hPSCs were treated with GSK3β inhibitor (CHIR) followed by treatment with bFGF in a defined medium for 20 days of 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. 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 BMP4, GATA4, HAND2, HEY1, ISL1, NKX2-5, SOX17 and WT1. The hPSC-CFs produced abundant extracellular matrix (ECM) when seeded at a high density, forming a unique 3D ECM scaffold composed of collagen and fibronectin. Furthermore, treatment of hPSC-CFs with TGF β1 caused myofibroblast differentiation demonstrated by upregulation of α-smooth muscle actin (SMA) measured by flow cytometry and immunolabeling. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes alters the electrophysiological properties of the cardiomyocytes based on optical mapping of membrane potential. We conclude that CFs can be efficiently differentiated from hPSCs via SHF progenitors in high yield and purity. The hPSC-CFs provide a powerful cell source for tissue engineering, disease modeling, drug discovery, 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

Introduction

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

Methods

Results

Conclusion