A Defined and Cost-Efficient Strategy for Generating Functionally Quiescent Human iPSC-Derived Cardiac Fibroblasts

Cardiac fibroblasts (CFs) are central regulators of myocardial structure, extracellular matrix homeostasis, and pathological remodeling, yet robust ex vivo experimental access to naïve human CFs remains limited. Primary human CFs exhibit restricted proliferative capacity, donor-to-donor variability, and rapid phenotypic drift in culture, constraining their utility for mechanistic and translational studies. Although human induced pluripotent stem cells (iPSCs) provide a renewable source for cardiovascular cell types, robust and developmentally faithful differentiation strategies for generating human CFs remain comparatively underdeveloped. Here, we describe a defined and cost-effective differentiation strategy for generating quiescent human CFs from iPSCs through sequential recapitulation of embryonic lineage specification. Differentiation is guided through cardiac progenitor, proepicardial, and epicardial intermediates using temporally controlled modulation of Wnt, retinoic acid, TGF-β, and FGF signaling pathways. This developmentally constrained approach enforces lineage fidelity while minimizing heterogeneous mesenchymal conversion commonly observed in direct fibroblast induction protocols. The resulting iPSC-derived CFs display canonical fibroblast morphology, marker expression, and functional behavior. Cells remain quiescent with intact nuclear (telomere) integrity under basal conditions while retaining the capacity for coordinated activation in response to profibrotic stimuli, including enhanced migration and contractile responses. Importantly, these fibroblasts exhibit preserved mitochondrial respiratory capacity and stable intracellular ATP levels across passages, indicating maintenance of metabolic programs necessary for extracellular matrix synthesis and fibroblast lineage stability. This platform generates scalable populations of phenotypically stable fibroblasts using chemically defined and cost-efficient culture conditions, enabling reproducible expansion and experimental manipulation. By embedding epicardial lineage history into fibroblast specification while preserving functional plasticity and metabolic competence, this method provides a developmentally grounded system for studying cardiac fibroblast biology. Together, this strategy establishes a robust, accessible and cost-effective approach for producing human cardiac fibroblasts suitable for disease modeling, tissue engineering, and pharmacological screening, offering a versatile resource for investigating the mechanisms that govern fibrotic remodeling and cardiometabolic disease.