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

Introduction

The mesenchymal cell populations present in the myocardium are often referred to as cardiac fibroblasts (CFs) and are distinct from fibroblasts present in other organs. CFs play many essential roles in the developing and adult heart by contributing to homeostasis of the organ. In developing hearts, CFs provide key signals to promote the proliferation of cardiomyocytes (CMs) and contribute to endocardial cushion formation and genesis of the valves. ^, Abnormal CF function can contribute to certain forms of congenital heart disease. CFs are largely responsible for the generation and remodeling of extracellular matrix (ECM), which is essential to maintaining the structure and function of the heart. CFs secrete and respond to a variety of soluble signaling molecules. CFs can also regulate the electrophysiologic properties of the heart. In disease, CFs can be key drivers of pathology, leading to pathologic fibrosis and promoting arrhythmogenesis. Given the many roles of CFs in cardiac biology and pathology, increasing efforts have been made to characterize and target CFs for therapeutic intervention.

CFs are heterogeneous populations of cells in the myocardium. The heterogeneity has been revealed by genetic lineage tracing studies that define the distinct developmental origins of CF populations. In addition, single-cell RNA sequencing approaches have revealed at least seven distinct populations of CFs in the adult human heart. Likewise, CFs can be transformed in disease into populations of cells with distinct functions; these populations of cells are known as myofibroblasts . Studying defined populations of CFs holds promise to advance the field, and emerging evidence suggests different populations of CFs contribute to different functional and pathologic effects in the heart. Human pluripotent stem cells (hPSCs) provide an opportunity to generate distinct populations of human CFs for investigation and various applications. In this chapter, we describe the distinct developmental origins of CFs, identify the progress that has been made in generating a population of second heart field (SHF)-derived CFs from hPSCs, and highlight the applications for these hPSC-CFs.

Developmental Origins of Cardiac Fibroblasts

The heart develops from mesodermal progenitors in a highly orchestrated series of events with sequential contributions from first heart field (FHF) and second heart field (SHF) progenitors to form the linear heart tube composed of an inner endocardial layer and an outer myocardial layer. The developing heart undergoes complex looping and morphogenesis with the proepicardial organ subsequently generating the surrounding epicardium and a subset of craniofacial neural crest contributing to the outflow track formation. In this dynamic developmental process, CFs arise from at least four distinct sources: epicardium, endocardium, SHF, and cardiac neural crest ( Fig. 58.1 ). Each of these CF populations plays crucial roles in heart development and cardiac biology.

Epicardial-Derived Cardiac Fibroblasts

The epicardium is considered the major source of CFs in the adult heart. Clonal analysis based on the expression of the transcription factor Mesp1 in mice has revealed that most epicardial cells come from an independent population of unipotent Mesp1 progenitors that give rise to the epicardium lineage and a small fraction of bipotent Mesp1 progenitors differentiating to cardiomyocytes and epicardial-derived cells (EPDCs). At the molecular level, the developing epicardium can be distinguished from the myocardium and endocardium by expression of the transcription factors Wt1 and Tbx18 ^, and the aldehyde dehydrogenase enzyme retinaldehyde dehydrogenase 2 (Aldh1a2). Cells from the early epicardium undergo an epithelial-mesenchymal transition (EMT) in response to various signals, including transforming growth factor beta-1 (TGFβ1), ^, Wnt, retinoic acid (RA), FGF, and PDGF, to give rise to CFs and coronary vascular smooth muscle cells that invade the myocardial layer and contribute to the fibroblast and vascular populations of the developing heart.

Endocardial-Derived Cardiac Fibroblasts

Endocardial-derived CFs contribute to the genesis of the cardiac valves and are present in the interventricular septum. ^, Although endocardial and endothelial cells share substantial molecular and functional characteristics, molecular and lineage distinctions between these two cell types suggest that the endocardium and myocardium derive from a common precursor distinct from endothelial cells. The initially acellular atrioventricular (AV) cushions become mesenchymalized as a result of EMT of the endocardial cells that are lining the cushions. ^, ^, Several AV-expressed growth factors have been identified as playing an important role in AV cushion formation. Studies using mice expressing Cre-recombinase driven by the promoter of the endothelial specific receptor tyrosine kinase (Tie2) have shown that almost all CFs in the AV cushions, and the cells of the mesenchymal cap on the primary atrial septum, are endocardially derived. These studies also demonstrated that the postnatal fate of endocardial-derived cells is limited largely to the fibrous tissues that constitute the leaflets of the AV valves.

Cardiac Neural Crest–Derived Cardiac Fibroblasts

The neural crest is a transient structure that forms from the lateral borders of the neural plate as they join in the midline during closure of the neural tube. The neural crest cells (NCCs) undergo an epithelial-to-mesenchymal transition, and a subset of NCCs migrate into the cardiac outflow tract (OFT) where they contribute to the septation of the OFT into the pulmonary artery and aorta and to the development of the semilunar valves. In adult hearts, it is debatable the degree of contribution of neural crest CFs to the semilunar valve cells, ^, but a significant population of neural crest CFs persists in the right atria.

Second Heart Field–Derived Cardiac Fibroblasts

The heart tube first forms from FHF progenitors, and then the SHF progenitors add to both outflow and inflow portions of the heart tube, with the anterior SHF progenitors contributing to the genesis of the right ventricle and outflow tract, and posterior SHF progenitors contributing to the atria and the dorsal mesenchymal protrusion. ^, ^, In addition, an early population diverging from the Isl-1 labeled SHF also contributes to the genesis of a portion of endocardial cells. ^, Although most focus on SHF progenitors has evaluated their potency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells, evidence suggests SHF progenitors also generate CFs. The most studied example of a CF population derived from SHF progenitors is in the genesis of the dorsal mesenchymal protrusion at the venous pole of the heart, which is closely associated with the dorsal mesocardium and is an integral part of the AV mesenchymal complex. ^, ^, The dorsal mesenchymal protrusion is largely myocardialized, contributing to the base of the atrial septum with some resident CFs remaining. ^, Less studied are the CFs that emerge from SHF in the forming outflow tract and right ventricle. Studies using mouse and human PSCs have likewise shown that CFs can be derived directly from the SHF progenitors. ^,

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