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

A Defined and Cost-Efficient Strategy for Generating Functionally Quiescent Human iPSC-Derived Cardiac Fibroblasts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article A Defined and Cost-Efficient Strategy for Generating Functionally Quiescent Human iPSC-Derived Cardiac Fibroblasts Somaya Ibrahim, Rushita Bagchi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9098586/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract 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. iPSC cell lineage quiescent cardiac fibroblasts metabolic competence nuclear stability Figures Figure 1 Figure 2 Figure 2 Figure 3 Figure 4 Figure 4 Figure 4 Introduction Cardiac fibroblasts (CFs) constitute the majority of non-myocyte cell population in the mammalian heart and are indispensable regulators of myocardial structure, extracellular matrix (ECM) homeostasis, and intercellular signaling ( 1 – 3 ). In physiological conditions, CFs maintain tissue integrity through controlled ECM synthesis and turnover. In disease states such as myocardial infarction, cardiometabolic stress, and pressure overload, CFs undergo phenotypic activation characterized by enhanced proliferation, migration, and excessive ECM deposition, driving maladaptive remodeling and fibrosis that contribute directly to heart failure progression ( 4 – 6 ). Despite their central role in cardiac pathophysiology, access to human CFs for mechanistic and translational studies remains limited. Primary human CFs are commercially available but exhibit restricted proliferative capacity, pronounced donor-to-donor variability, and rapid senescence in vitro , limiting experimental reproducibility and scalability. Moreover, primary cells often reflect end-stage disease phenotypes, complicating the study of early pathogenic mechanisms and therapeutic intervention windows. Human induced pluripotent stem cells (iPSCs) provide a renewable source of patient-specific platform for modeling cardiovascular development and disease ( 7 ). Robust protocols exist for generating cardiomyocytes, endothelial cells, and smooth muscle cells; however, reliable and developmentally faithful differentiation methods for human CFs have lagged behind. Direct fibroblast differentiation strategies frequently yield heterogeneous populations with incomplete lineage specification and limited functional fidelity. In contrast, stepwise differentiation through defined embryonic intermediates enables controlled lineage commitment and improved reproducibility. Here, we present a chemically defined, modified ( 8 ) stepwise protocol for differentiating human iPSCs into quiescent CFs via cardiac progenitor, proepicardial, and epicardial intermediates. Sequential modulation of Wnt, retinoic acid, TGF-β, and FGF signaling recapitulates key embryonic patterning events that govern fibroblast lineage specification ( 9 , 10 ). Importantly, the protocol yields quiescent fibroblasts with reproducible morphology, transcriptomic fidelity, scalability, and cost-effectiveness. Validation of lineage identity at each stage is achieved through immunostaining, qPCR, and functional assays for stage-specific markers. The resulting iPSC-derived CFs exhibit canonical fibroblast marker expression, contractile and migratory responses, metabolic competence, and responsiveness to profibrotic stimuli, supporting their broad utility in disease modeling, tissue engineering, and pharmacological screening. Methods Maintenance of Human iPSCs Human induced pluripotent stem cells (iPSCs) were maintained under feeder-free conditions in six-well plates coated with hESC-qualified Matrigel and cultured in mTeSR Plus medium at 37°C in a humidified atmosphere containing 5% CO 2 . Medium was exchanged every other day using 2 mL per well. Cells were passaged every 3–4 days using Gentle Cell Dissociation Solution (GCDS; 1 mL per well) following a 5–6 min incubation at 37°C and reseeded onto freshly coated Matrigel plates. The human iPSC line was provided by Greenstone Biosciences Inc. and cultured as described previously ( 7 ). Only cultures exhibiting compact colony morphology, high nuclear-to-cytoplasmic ratios, and minimal spontaneous differentiation were used for differentiation experiments. Differentiation was initiated at 80–90% confluence using iPSCs between passages 20 and 30 to ensure consistency across experiments. Stage 1: Differentiation to Cardiac Progenitors (Day 0–5) To initiate mesodermal differentiation and cardiac progenitor specification, iPSC maintenance medium was replaced on Day 0 with RPMI-1640 supplemented with B27 minus insulin and 5 µM CHIR99021 (2 mL per well). Cultures were incubated for 24 h at 37°C with 5% CO 2 to activate canonical Wnt signaling and promote mesodermal lineage entry. On Day 2, medium was replaced with fresh RPMI/B27 minus insulin without small-molecule supplementation to support cell viability and stabilize early lineage commitment. On Day 3, Wnt signaling was inhibited by replacing the medium with RPMI/B27 minus insulin supplemented with 2 µM IWP-2 (2 mL per well), thereby directing differentiation toward cardiac progenitor fate. For individual iPSC lines, IWP-2 concentrations were empirically titrated within the range of 3–5 µM to minimize cytotoxicity while maintaining effective pathway inhibition. Troubleshooting Inadequate CHIR99021 exposure results in poor mesoderm specification. If early morphology does not transition uniformly from compact colonies to a spread epithelial sheet by Day 2–3, CHIR concentration may require titration. Sensitivity to CHIR varies between iPSC lines; optimal concentrations ranged between 4–6 µM. Wnt inhibition is highly dose sensitive. Cytotoxicity during Days 3–5 typically reflects excessive IWP-2 concentration. Empirical titration (3–5 µM) is recommended for each line to balance effective cardiac specification with cell viability. On Day 5, medium was exchanged for fresh RPMI/B27 minus insulin. At this stage, cells exhibited a transition from compact pluripotent colonies to a more spread epithelial-like morphology, consistent with cardiac progenitor specification. Stage 2: Differentiation to Proepicardial and Epicardial Cells (Day 6–12) To promote epicardial lineage specification, cardiac progenitor cultures were dissociated on Day 6 using TrypLE Express (1 mL per well, 5 min at 37°C). Cells were gently triturated to single-cell suspension, collected in RPMI medium, and centrifuged at 200 × g for 3 min at room temperature. Pelleted cells were resuspended in Advanced DMEM/F12 supplemented with GlutaMAX, 5 µM CHIR99021, 2 µM retinoic acid, 5 µM Y-27632, and 1% fetal bovine serum (FBS). Cells were reseeded onto Matrigel-coated six-well plates at a density of 20,000 cells/cm² (approximately a 1:12 split ratio) in 2 mL per well and cultured overnight. Troubleshooting Single-cell dissociation at this stage induces stress. Inclusion of Y-27632 during initial replating is essential to support survival. If attachment remains inefficient, increasing seeding density to 25,000 cells/cm² can improve recovery. On Day 7, medium was replaced with Advanced DMEM/F12 supplemented with GlutaMAX, 5 µM CHIR99021, and 2 µM retinoic acid (2 mL per well) to reinforce epicardial specification. On Day 9, cultures were transitioned to basal Advanced DMEM/F12 with GlutaMAX only, allowing stabilization of the epicardial program in the absence of exogenous signaling cues. On Day 11, cells were again dissociated using TrypLE Express, pelleted, and resuspended in Advanced DMEM/F12 supplemented with 2 µM SB431542 to inhibit TGF-β signaling. Cells were seeded onto Matrigel-coated six-well plates at a 1:6 split ratio in 2 mL per well. Troubleshooting Failure to achieve uniform cobblestone morphology by Day 11–12 typically reflects suboptimal retinoic acid exposure or uneven CHIR signaling. Retinoic acid should be freshly prepared and protected from light. If cells acquire spindle-like morphology during Stage 2, TGF-β signaling may be insufficiently suppressed. Ensure SB431542 is freshly prepared and added at the correct concentration during stabilization. On Day 12, medium was refreshed with Advanced DMEM/F12 containing 2 µM SB431542. Cultures were maintained under same conditions with medium changes every other day until reaching confluence. At this stage, cells exhibited a cobblestone-like epithelial morphology characteristic of proepicardial and epicardial intermediates. Troubleshooting An inefficient transition may occur if epicardial cultures are not fully confluent by Day 14. Initiating fibroblast induction at suboptimal density reduces uniformity of mesenchymal conversion. Cultures should exhibit robust epithelial morphology prior to induction. If cells display excessive αSMA organization or stress fiber formation under basal conditions, mechanical stress or overconfluence may be contributing factors. Maintaining consistent seeding density and avoiding prolonged culture without passaging helps preserve a quiescent phenotype. Excessive exposure to exogenous growth factors may also promote activation; strict adherence to hFGF-B and SB431542 concentrations is recommended. Passaging stress can induce transient growth arrest. Gentle dissociation and avoidance of over-trypsinization are critical. Cells should not exceed 80–90% confluence prior to passaging to prevent contact-mediated activation. Stage 3: Differentiation into Quiescent Cardiac Fibroblasts (Day 14–20) To induce epicardial-to-fibroblast transition and stabilize a quiescent fibroblast phenotype, epicardial cells were dissociated on Day 14 using TrypLE Express (1 mL per well), pelleted, and resuspended in fibroblast growth medium (FGM; DME/F12 supplemented with hFGF-B, human insulin, FBS, and GA-1000) supplemented with 10 µM SB431542 and 2 ng/mL hFGF-B. Cells were seeded onto Matrigel-coated six-well plates at a density of 8000-10,000 cells/cm² in 2 mL per well. On Day 16, the medium was refreshed with FGM supplemented with SB431542. On Day 18, cells were passaged as described above and reseeded at 10,000 cells/cm² in FGM containing SB431542. On Day 20, cultures were transitioned to FGM supplemented with 10 µM SB431542 alone, with medium changes performed every other day thereafter. By this stage, cells exhibited elongated, spindle-shaped morphology characteristic of quiescent cardiac fibroblasts and demonstrated robust proliferative capacity. Cells were either cryopreserved in DME/F12 medium supplemented with 20% FBS and 10% DMSO or expanded and seeded for downstream experimental applications. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) using the Verso cDNA Synthesis Kit (Thermo Scientific™). Quantitative PCR was performed on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) using TaqMan™ Fast Advanced Master Mix (Applied Biosystems) in combination with gene-specific TaqMan™ expression assays. Relative mRNA expression levels were calculated using the 2 −ΔΔCt method and normalized to 18S ribosomal RNA as an internal control ( 7 ). TaqMan™ assay identifiers are provided in Supplemental Table 1. Immunoblotting Total protein lysates were prepared from iPSCs, proepicardial, epicardial, and iPSC-derived cardiac fibroblasts treated with either TGF-β1 or vehicle (control) using RIPA Lysis and Extraction Buffer (Thermo Scientific) supplemented with Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit following the manufacturer’s protocol. Equal amounts of protein (10–15 µg) were resolved by SDS–PAGE using precast tris-glycine gradient gels (4–20%; Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad) for 30 minutes at room temperature, then incubated overnight at 4°C with primary antibodies against α–smooth muscle actin (αSMA; mouse monoclonal, Sigma) or OxPhos cocktail (Thermo Scientific). After washing, membranes were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Protein bands were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged on a ChemiDoc™ MP Imaging System (Bio-Rad). Band intensities were quantified using Quantity One software (Bio-Rad), and αSMA and OxPhos signals were normalized to GAPDH or Calnexin as a loading control. Antibody, reagent, and software details are provided in Supplemental Tables 2–4. Immunofluorescence iPSCs, proepicardial, epicardial, and iPSC-derived cardiac fibroblasts were cultured on glass coverslips (Fisherbrand 18CIR-1) in their respective growth media. Following treatment, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature. Cells were permeabilized with 0.3% Triton X-100 and blocked for 1 hour in PBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA; Sigma). Coverslips were incubated overnight at 4°C with primary antibodies. After washing, samples were incubated for 1 hour at room temperature in the dark with species-specific Alexa Fluor 488– and Alexa Fluor 594-conjugated secondary antibodies (Thermo Scientific), where indicated. Nuclei were counterstained using ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen). Coverslips were cured overnight and imaged using a Keyence BZ-X810 imaging system. Antibody and reagent information is listed in Supplemental Tables 2 and 4. Mitochondrial respiration (Seahorse) assay and total ATP detection Fibroblast mitochondrial function was assessed using a Seahorse XFe96 analyzer (Agilent). Cells were seeded onto cell culture plates and incubated overnight at 37°C in 5% CO 2 humidified incubator. Mitochondrial stress test was performed as per manufacturer’s instructions (Seahorse XF Cell Mito Stress Test Kit, Agilent) to evaluate the oxygen consumption rate (OCR), using the following injections: oligomycin (1 µM) to measure ATP production, FCCP (1 µM) to assess maximal respiration, and rotenone/antimycin A (0.5 µM) to determine non-mitochondrial oxygen consumption. Cells were stained with Hoechst 33342 to determine the nuclei count using a Cytation 10 high content imaging system (Agilent Biotek), and OCR values were normalized to nuclei per well. Total ATP was determined using commercially available reagents (Abcam) as described previously ( 11 ). Flash-frozen cell pellets were lysed in ATP assay buffer using a Dounce homogenizer. The lysate was centrifuged at 13000g at 4 4oC, and the supernatant was incubated with the necessary reaction components for 30 minutes at room temperature, protected from light. An ATP standard calibration curve was generated using serial dilutions of ATP. Fluorescence signals from standards and samples were then measured on a multimode reader at Ex/Em = 535/587 nm. ATP concentrations were calculated and normalized to the corresponding protein content of the cells. 3D collagen gel contraction assay Fibroblasts-incorporated collagen hydrogel matrices were prepared in 48-well plates using PureCol EZ Gel solution (Advanced BioMatrix). These gels were incubated in serum-supplemented medium 24 hours prior to overnight serum deprivation for equilibration. Gels were then released from the wells, and cells were treated with vehicle, TGF-β1 (10 ng/mL), or Angiotensin II (1 µM). Well images were acquired every 24 h for up to 120 h. Gel area for each well was calculated using ImageJ software and data are reported as percent contraction. Wound healing assay using electric cell-substrate impedance sensing (ECIS) ECIS wound-healing assays were performed using the 8-well ECIS arrays (8W1E, PET) on the ECIS-Zθ equipment (Applied Biophysics). The arrays were treated with 10 mM L-cysteine (Sigma-Aldrich) prior to array stabilization and cell seeding. iPSC-CFs and HCFs were seeded at equal densities (50,000/well) and allowed to form a uniform monolayer before the wounding protocol. Wounding was performed twice (total 60s) using a current of 3000 µA at a frequency of 60kHz. After wounding, medium was exchanged to remove cell debris. The healing process was then observed for ~ 20h, with capacitance and impedance recorded with a single frequency of 60kHz at 4s intervals. Normalized data was computed using the built-in default script in ECIS software. Peptide Nucleic Acid Quantitative Fluorescence in-situ Hybridization (PNA-QFISH) and Analysis To understand nuclear stability and integrity of iPSC-derived CFs, a modified Q-FISH method was used as described elsewhere ( 12 ). Cells on glass coverslips were fixed with 4% paraformaldehyde-fixed cells, followed by treatment with 50 µg/mL proteinase K (Gold Biotechnology) at 37°C for 15 minutes. The coverslips were then dehydrated in ethanol gradient (100%, 90%, and 70%). Cells were hybridized with 2ng/ml of PNA probe (PNA Bio) followed by denaturation at 72 o C for 2min. Coverslips were incubated at 30 o C for 3h in the dark in a humidified chamber. After hybridization, coverslips were washed twice in 70% formamide (Thermo Scientific) containing 10 mM Tris for 10min, followed by one wash with saline-sodium citrate (SSC) at 55°C, and twice with 2x SSC buffer containing 0.05% Tween 20. Coverslips were mounted onto slides using Prolong Glass mounting media containing DAPI. To visualize 3D telomere and nuclear structures, all z-stack images from 12–30 nuclei per condition were captured using Cy3 and DAPI filters (Zeiss AxioImager Z1 fluorescence microscope), with constant exposure times of 400 ± 50ms and 200 ± 50ms, respectively, during sample acquisition. Using Zen3.11.1 analysis software, interphase nuclei were deconvolved at a constrained iterative algorithm (strength 7 for Cy3 and 5 for DAPI) and converted to TIFF images. Semi-automated quantitative image analysis was performed using DIPImage MATLAB software (The MathWorks, Natick, MA, USA) to analyze mean telomere number and intensity for 12–30 interphase nuclei from each sample. Statistical analysis All statistical analyses were performed using GraphPad Prism 10 (Supplemental Table 3). Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests with Welch’s correction. Comparisons among multiple groups were analyzed using one-way or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc multiple-comparison test. A p-value < 0.05 was considered statistically significant. Results Validation of Differentiation Derivation of cardiac fibroblasts from pluripotent stem cells presents two fundamental challenges: (i) enforcing developmental restriction toward the epicardial–fibroblast lineage, and (ii) distinguishing bona fide quiescent fibroblasts from phenotypically overlapping mesenchymal or partially activated cell states ( 13 ). To address these challenges, we evaluated differentiation fidelity across three orthogonal axes: lineage trajectory, cell identity, and functional competence. Developmental Routing of Fibroblast Fate Cardiac fibroblasts arise predominantly from the embryonic epicardium through a temporally regulated sequence of lineage restriction, epithelial organization, and mesenchymal transition ( 14 ) (Fig. 1 A). Undifferentiated iPSCs expressed canonical pluripotency markers SSEA4, OCT4 (POU5F1), SOX2, and NANOG ( 15 , 16 ), confirming an intact pluripotent state prior to induction (Fig. 1 B and 2 A). Critically, differentiation proceeded through a morphologically and transcriptionally defined pro-epicardial/epicardial state. Cells at this stage adopted cohesive epithelial morphology and robustly expressed WT1 and TCF21 (Fig. 1 C-D, Fig. 2 B middle panel, Fig. 2 C left panel), transcriptional regulators that delineate fibroblast-competent epicardial populations in vivo ( 17 ). Maintenance of epithelial junctional organization was evidenced by zona occludens-1 (ZO-1) localization (Fig. 1 C-D) and its gene TJP1 (Fig. 2 B), together with cardiac-specific GATA 4 expression in pro-epicardial as well as in epicardial stages (Fig. 2 B). This further indicated that lineage specification preceded mesenchymal transition, rather than arising from nonspecific mesodermal outgrowth ( 18 ). Distinguishing Cardiac Fibroblasts from Other Fibroblast States Fibroblasts derived from different tissues share overlapping morphological and molecular features, raising the concern that pluripotent stem cell–derived fibroblast-like cells may represent a generic stromal state rather than a lineage-restricted cardiac fibroblast population ( 19 ). Addressing this distinction requires more than identifying fibroblast markers; it requires evidence of cardiac-specific lineage history, exclusion of alternative mesodermal fates, and functional behaviors aligned with the myocardial context. iPSC-derived fibroblasts generated using this protocol exhibited expression of core fibroblast markers, including DDR2, vimentin, and TCF21 (Fig. 1 E), confirming the acquisition of a mesenchymal fibroblast identity. However, DDR2 expression is not merely confirmatory but also discriminatory, as it is selectively enriched in cardiac fibroblasts and mediates collagen-dependent signaling, which is critical for myocardial extracellular matrix (ECM) remodeling ( 20 ). Its expression therefore supports cardiac-relevant fibroblast specialization rather than generic stromal identity as demonstrated by suppression of pluripotency markers POU5F1 , SOX2 , and NANOG (Fig. 1 E, 2 A) with TJP1 (Fig. 2 B). Engagement of a cardiac-appropriate extracellular matrix (ECM) program was further evidenced by expression of TCF21 , FN1, and COL1A1 (Fig. 2 C). COL1A1 and FN1 are matrix components that are central structural elements of the myocardial interstitium and represent core functional outputs of cardiac fibroblasts. Equally critical was the absence of alternative lineage programs. Differentiated cells lacked cardiomyocyte-associated genes (MYH6), endothelial markers (PECAM1), and smooth muscle markers (MYH11) under basal conditions, excluding contamination by parallel mesodermal derivatives (Fig. 2 D). This negative selection is essential, as partial expression of fibroblast markers alone is insufficient to establish lineage identity in pluripotent stem cell–derived cultures, where partial marker overlap can otherwise confound identity assignment. Functional Hallmarks of Cardiac Fibroblasts Cardiac fibroblasts are distinguished from generic stromal populations by their integration of mechanical sensing, matrix remodeling, and context-dependent activation within the myocardial environment. These behaviors are not universal fibroblast traits but emerge from a cardiac-specific developmental history and are essential for heart morphogenesis, repair, and pathological remodeling ( 21 ). We therefore evaluated whether iPSC-derived fibroblasts exhibit functional behaviors that reflect cardiac lineage competence rather than generic mesenchymal activity. Migratory Competence, Cytoskeletal Organization, and Matrix Remodeling During cardiac development and post-injury repair, fibroblasts migrate directionally within a mechanically dynamic extracellular matrix to populate emerging interstitial niches ( 22 ). In response to pro-fibrotic TGF-β stimulation, iPSC-derived fibroblasts exhibited robust migratory behavior, indicating appropriate cytoskeletal organization and integrin (vinculin)-mediated matrix engagement (Fig. 2 E). This behavior is not trivial, as incompletely specified mesenchymal cells frequently display either impaired motility or unregulated migration ( 23 ), which is inconsistent with cardiac fibroblast behavior as shown in HCFs treated with TGF-β (Fig. 2 E). A defining feature of cardiac fibroblasts is their ability to physically remodel fibrillar collagen networks through controlled force generation ( 21 ). In three-dimensional collagen matrices, iPSC-derived CFs generated measurable contractile forces, demonstrating functional engagement with native-like extracellular matrix substrates (Fig. 2 F-G). This behavior reflects active-matrix sensing and force transduction, processes mediated by fibroblast-specific cytoskeletal and adhesion complexes. Importantly, matrix contraction was limited under basal conditions, consistent with a quiescent fibroblast state (Fig. 2 F upper panel, 2G). This contrasts with generic fibroblasts or constitutively activated myofibroblasts, which often exhibit elevated baseline contractility independent of environmental cues ( 24 ). The restrained basal contractility observed here suggests that force generation is tightly regulated rather than constitutively engaged. Context-Dependent Activation and Phenotypic Plasticity Cardiac fibroblasts are characterized by their capacity to remain functionally quiescent under homeostatic conditions while rapidly transitioning to an activated, matrix-producing phenotype in response to injury-associated signals ( 25 ). iPSC-derived CFs preserved this phenotypic plasticity, as evidenced by a marked increase in matrix contraction following stimulation with angiotensin II or TGF-β (Fig. 2 F-G). Functional Discrimination of Fibroblast States The migratory, contractile, and stimulus-responsive behaviors observed in iPSC-derived fibroblasts define a functional profile consistent with cardiac fibroblast identity. These behaviors are tightly linked to the mechanical and signaling environment of the heart and are not uniformly shared by fibroblasts derived from non-cardiac tissues. Wound healing is an intrinsic characteristic of fibroblasts. iPS-CFs, similar to HCFs, showed recovery of cell migration and reestablishment of cell-cell adhesion after irreversible electroporation of cells seeded on electrode arrays. In fact, iPS-CFs demonstrated a faster, more robust re-establishment of the cell monolayer, as evidenced by decreased capacitance, increased resistance, and increased impedance, in contrast to HCFs (Fig. 2 H-J). Quiescence and Regulated Activation In the heart, fibroblasts must dynamically adapt their metabolic state to support both quiescent maintenance and rapid activation following developmental cues or injury ( 26 ). Consequently, preservation of metabolic competence is not merely a measure of cell health, but a defining feature of lineage stability and functional identity. To further understand mitochondrial performance, extracellular flux analysis was performed using sequential administration of oligomycin (Complex V inhibition), FCCP (protonophore-mediated uncoupling), and rotenone/antimycin A (Complex I and III inhibition) (Fig. 3 A). Basal oxygen consumption rates were comparable between iPSC-CFs and primary HCFs, indicating equivalent steady-state electron flux through the ETC (Fig. 3 B, upper left panel). Basal respiration integrates NADH- and FADH 2 -driven input into Complexes I and II and reflects constitutive oxidative metabolism. The preservation of basal OCR demonstrates that iPSC-CFs have transitioned away from the glycolytic metabolic program characteristic of pluripotency and into an oxidative phenotype consistent with differentiated cardiac mesenchyme ( 27 ). ATP-linked respiration, defined by the oligomycin-sensitive fraction of oxygen consumption, was similarly preserved between groups (Fig. 3 B, upper middle panel). Because oligomycin blocks ATP synthase (Complex V), this parameter reflects oxygen consumption directly coupled to phosphorylation. Proton leak, representing oxygen consumption independent of ATP production, was modest but significantly reduced in iPSC-CFs relative to HCFs (Fig. 3 B, upper right panel). Maximal respiration following FCCP-mediated uncoupling was comparable between groups (Fig. 3 B, lower left panel), indicating intact ETC capacity and preserved substrate oxidation potential. Notably, iPSC-CFs exhibited significantly increased spare respiratory capacity compared to HCFs (Fig. 3 B, lower middle panel). Spare capacity, defined as the difference between basal and maximal respiration, reflects the bioenergetic reserve available to meet sudden increases in ATP demand. Non-mitochondrial oxygen consumption, measured following complete ETC inhibition with rotenone and antimycin A, was similar between groups (Fig. 3 B, lower right panel), indicating that extramitochondrial oxidase activity does not account for the observed respiratory profile. iPSC-CFs generated using this protocol exhibited intact expression of mitochondrial respiratory chain complexes I–V, indicating preservation of oxidative phosphorylation capacity rather than reliance on glycolytic compensation (Fig. 3 C). This is a critical distinction, as pluripotent cells and incompletely differentiated mesenchymal populations typically exhibit suppressed mitochondrial maturation, altered respiratory complex expression, and reduced electron transport chain (ETC) protein abundance. Maintenance of a fully assembled ETC therefore supports completion of mitochondrial maturation concomitant with fibroblast specification. Consistent with preserved mitochondrial function, intracellular ATP content remained stable across early passages in iPSC-CFs and was comparable to HCFs (Fig. 3 D). Importantly, ATP levels did not decline with expansion, arguing against cumulative metabolic stress, replicative exhaustion, or dedifferentiation during passaging. Together, these findings indicate that the differentiation strategy not only enforces transcriptional and structural fibroblast identity but also stabilizes the mitochondrial programs required to sustain it. Cellular senescence is a major experimental limitation of cardiac fibroblasts, which makes the source of these cells critical for in vitro studies. Nuclear instability and structure of iPSC-CFs assessed by Q-FISH showed increased mean telomere intensity per telomere in iPSC-CFs which were significantly higher than HCFs (Fig. 3 E), suggesting that iPSC-CFs possess younger and healthier nuclei in comparison to commercially available primary HCFs. Compared to iPSC-CFs, generic fibroblasts (HCFs) exhibited altered telomeric DNA morphology and structure (Fig. 3 F-G), reflecting impaired cellular function from serial passaging and leading to replicative senescence. As shown previously by our group ( 7 ), iPSC-CFs generated using this protocol respond robustly to TGF-β 1 stimulation and transition to an activated fibroblast state, as evidenced by increase in ACTA2 ( α-SMA ) gene expression (Fig. 3 H). Of note, this response is equivalent to commercially available HCFs. When subjected to serial passaging in vitro, the iPSC-CFs showed increased protein expression of α-SMA (Fig. 3 I). Additionally, these cells also showed distinguishable patterns of α-SMA expression when grown on tissue culture dishes with different substrate chemistry (Fig. 3 J). This is critical as variable substrates can pre-activate these cells and therefore hinder their responsiveness to pathologic stimuli in vitro , therefore producing confounding observations. Discussion and Conclusion The ability to generate human cardiac fibroblasts with defined lineage history, functional competence, and phenotypic stability has remained a persistent challenge in cardiovascular and developmental biology. Here, we describe a developmentally constrained differentiation strategy that yields quiescent cardiac fibroblasts from human iPSCs by explicitly recapitulating the epicardial lineage trajectory that gives rise to fibroblasts in the embryonic heart. Rather than relying on direct mesenchymal induction or marker-based enrichment, this protocol enforces fibroblast identity through sequential restriction of developmental potential, thereby reducing heterogeneity and preserving lineage fidelity. A central feature of this approach is its routing of differentiation through proepicardial and epicardial intermediates, reflecting the predominant embryonic origin of cardiac fibroblasts in vivo. Transient activation and subsequent resolution of epicardial transcriptional programs, including WT1 and TCF21 (Fig. 1 ), distinguishes this system from generic fibroblast differentiation strategies and argues against nonspecific mesenchymal conversion. By embedding epicardial lineage history into fibroblast specification, the protocol establishes a developmental context that shapes subsequent functional and metabolic behaviors. Importantly, the resulting fibroblasts occupy a quiescent yet plastic state that mirrors cardiac fibroblasts in the uninjured heart. These cells remain non-contractile under basal conditions while retaining the capacity for coordinated activation in response to profibrotic cues (Fig. 2 ). This balance between quiescence and inducible activation is a defining feature of cardiac fibroblast biology and is frequently lost in primary cell cultures or direct conversion systems, which often yield constitutively activated myofibroblast-like populations. Preservation of this regulated phenotype enables interrogation of early fibrotic events that precede irreversible remodeling. Beyond transcriptional and functional identity, this protocol stabilizes metabolic programs essential for fibroblast lineage maintenance (Fig. 3 ). iPSC-derived CFs exhibit preserved oxidative phosphorylation capacity and stable ATP levels across passages, supporting the energetic demands of extracellular matrix synthesis and remodeling. Increasing evidence indicates that metabolic state is not merely permissive but instructive in fibroblast activation and fate decisions. Equivalent ATP-coupled respiration confirms intact proton motive force utilization and efficient conversion of electrochemical gradient into ATP. This is particularly relevant given that collagen biosynthesis, vesicular trafficking, cytoskeletal turnover, and myosin ATPase–dependent force generation are energetically intensive processes intrinsic to fibroblast identity ( 28 , 29 ). Reduced leaks suggest tighter coupling and preserved membrane integrity, consistent with a metabolically restrained quiescent state. This tighter coupling efficiency may reflect reduced unnecessary proton cycling, thereby optimizing ATP yield while limiting oxidative stress which are features compatible with stable lineage maintenance. By maintaining metabolic competence alongside lineage restriction, this platform minimizes culture-induced drift and supports longitudinal studies of fibroblast plasticity and disease progression. The developmental grounding of this system distinguishes it from existing fibroblast differentiation approaches and enhances its translational relevance. Patient-specific iPSC-derived cardiac fibroblasts generated using this method provide a tractable platform for modeling cardiometabolic disease, fibrosis, and gene–environment interactions in a human context (Fig. 4 ). In tissue engineering applications, these cells can supply developmentally appropriate extracellular matrix and paracrine signaling to support cardiomyocyte maturation and tissue organization (Fig. 4 ). Furthermore, their scalability and phenotypic stability make them well suited for pharmacological screening and precision medicine efforts (Fig. 4 ). It is critical to note here that with FGM made in house without the use of commercially available pre-assembled media kits, the cost of this iPSC-CF differentiation and propagation protocol is reduced by almost thirty percent, which makes this an affordable avenue for several researchers globally. Several limitations warrant consideration. Differentiation efficiency requires modest line-specific optimization of Wnt pathway modulation, reflecting intrinsic variability among iPSC lines. In addition, the use of Matrigel introduces potential batch-to-batch variability and limits full chemical definition of the system. Future refinements may incorporate fully defined extracellular matrix substrates, mechanical conditioning, and single-cell transcriptomic benchmarking against adult human cardiac fibroblast subtypes to further refine maturation state and subtype specification. In summary, this work establishes a developmentally informed framework for generating human cardiac fibroblasts that integrates lineage history, functional plasticity, and metabolic stability. By recapitulating epicardial specification and resolution, this protocol yields fibroblasts that more closely reflect their in vivo counterparts than existing approaches and is rapid and budget friendly. Robust quality control including reproducibility and repeatability of this platform provides a foundation for dissecting the developmental and disease mechanisms governing cardiac fibroblast behavior and offers a versatile and cost-effective resource for basic, translational, and precision cardiovascular research. Abbreviations ACTA2 – actin alpha 2 (a.k.a. alpha smooth muscle actin αSMA) ATP – adenosine triphosphate BCA – bicinchoninic acid assay cDNA – complementary DNA CF – cardiac fibroblasts CHIR99021 – GSK-3α/β inhibitor COL1A1 – collagen 1 alpha1 Cy3 – cyanine dye 3 DAPI – 4′,6-diamidino-2-phenylindole DDR2 – discoidin domain receptor tyrosine kinase 2 DMEM/F12 – Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 ECIS – electric cell-substrate impedance sensing ECM – extracellular matrix ETC – electron transport chain FBS – fetal bovine serum FCCP – carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FGF – fibroblast growth factor FN1 – fibronectin 1 GCDS – gentle cell dissociation solution hESC – human embryonic stem cells iPSC –induced pluripotent stem cell iPS-CF/ iPSC-CF – iPSC-derived cardiac fibroblasts IWP-2 – inhibitor of WNT processing-2 mRNA – messenger ribonucleic acid MYH6 – myosin heavy chain 6 NANOG – homeobox transcription factor Nanog (on ch12) OCR – oxygen consumption rate OCT4 (POU5F1) – Octamer-binding transcription factor 4A (a.k.a. POU-type homeodomain – containing DNA binding protein (on ch6) OxPhos – oxidative phosphorylation PBS – phosphate-buffered saline PECAM1 – platelet endothelial cell adhesion molecule 1 Q-FISH – quantitative Fluorescence in-situ Hybridization qPCR - quantitative polymerase chain reaction RIPA – radioimmunoprecipitation assay RPMI-1640 – Roswell Park Memorial Institute 1640 medium SB431542 – TGF- β inhibitor SDS-PAGE – sodium dodecyl sulfate–polyacrylamide gel electrophoresis SOX2 – SRY (sex determining region Y)-box 2 (on ch3) SSEA4 – Stage-specific embryonic antigen 4 TCF21 – transcription factor 21 TGF-β – transforming growth factor TJP1 – tight junction protein 1 WT1 – Wilms tumor 1 ZO-1 – zona occludens1 Declarations Competing interests: The authors have no financial and non-financial competing interests to declare. Author Contribution S.Y.I. and R.A.B. conceived and designed the study; S.Y.I. and R.A.B. performed the experiments and analyzed the data; S.Y.I. drafted the manuscript and prepared the figures; all authors edited and approved the final version of the manuscript. Acknowledgement We thank Dr. Andrew Morris for access to equipment for this study, and Dr. Ashim Bagchi for valuable input on the manuscript and assistance with Q-FISH studies for this project. Fluorescence imaging and seahorse assays were performed at the Cellular Imaging Core of the Center for Microbial Pathogenesis and Host Inflammatory Responses (CMPHIR) at UAMS (NIGMS COBRE Grant 5P30GM145393-05). We acknowledge Dr. Joseph Wu and Greenstone Biosciences Inc. for providing the human iPSC line GSB-L2053 used in this study. R.A.B. received support through a career development award from the American Heart Association (23CDA1048663), funding from the Vice Chancellor for Research and Innovation (VCRI) and the Arkansas Biosciences Institute, the Sturgis Grant for Diabetes Research from the College of Medicine, and the Medical Research Endowment Grant from the VCRI, University of Arkansas for Medical Sciences. Schematics for figures 1 and 4 were generated using a licensed version of Biorender.com. Data Availability The data that support the findings of this study are available from the authors upon reasonable request. References Butt RP, Laurent GJ, Bishop JE. Collagen production and replication by cardiac fibroblasts is enhanced in response to diverse classes of growth factors. Eur J Cell Biol. 1995;68(3):330–5. Mouton AJ, Ma Y, Rivera Gonzalez OJ, Daseke MJ 2nd, Flynn ER, Freeman TC, et al. Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis. Basic Res Cardiol. 2019;114(2):6. Fu X, Khalil H, Kanisicak O, Boyer JG, Vagnozzi RJ, Maliken BD, et al. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest. 2018;128(5):2127–43. Bahr J, Poschmann G, Jungmann A, Busch M, Ding Z, Vogt J, et al. A secretome atlas of cardiac fibroblasts from healthy and infarcted mouse hearts. Commun Biol. 2025;8(1):675. Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM, et al. Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell. 2009;16(2):233–44. Wang J, Yang Bennett DS, Echard EJ, Chen B, Ciampa G, Zhao W, et al. Junctophilin-2 Regulates Store-Operated Calcium Entry to Drive Cardiac Fibroblast Activation, Fibrotic Repair, and Angiogenesis After Myocardial Infarction. Circulation. 2025;152(10):699–716. Ibrahim SY, Holdiness R, Thadisena A, Boyle KE, Jun SR, Bagchi RA. 3-D biomechanics and epigenomics reveal atypical fibroblast responses in cardiometabolic disease. Am J Physiol Heart Circ Physiol. 2025;329(5):H1267–77. Zhang H, Shen M, Wu JC. Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells. Methods Mol Biol. 2022;2454:109–15. Wiesinger A, Boink GJJ, Christoffels VM, Devalla HD. Retinoic acid signaling in heart development: Application in the differentiation of cardiovascular lineages from human pluripotent stem cells. Stem Cell Rep. 2021;16(11):2589–606. Mossahebi-Mohammadi M, Quan M, Zhang JS, Li X. FGF Signaling Pathway: A Key Regulator of Stem Cell Pluripotency. Front Cell Dev Biol. 2020;8:79. Fox BM, Gil HW, Kirkbride-Romeo L, Bagchi RA, Wennersten SA, Haefner KR, et al. Metabolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice. Kidney Int. 2019;95(3):590–610. Poon SSS, Lansdorp PM. Quantitative fluorescence in situ hybridization (Q-FISH). Curr Protoc Cell Biol 2001;Chap 18:18 4 1–4 21. Floy ME, Givens SE, Matthys OB, Mateyka TD, Kerr CM, Steinberg AB, et al. Developmental lineage of human pluripotent stem cell-derived cardiac fibroblasts affects their functional phenotype. FASEB J. 2021;35(9):e21799. Quijada P, Trembley MA, Small EM. The Role of the Epicardium During Heart Development and Repair. Circ Res. 2020;126(3):377–94. Carter AC, Davis-Dusenbery BN, Koszka K, Ichida JK, Eggan K. Nanog-independent reprogramming to iPSCs with canonical factors. Stem Cell Rep. 2014;2(2):119–26. Dorota A, Maryniak N, Mariankowska A, Milczarek C, Dorota M, Zywiec W, et al. Induced Pluripotent Stem Cells (iPSC) and Their Use in Disease Modeling. Cureus. 2025;17(10):e93999. Braitsch CM, Yutzey KE. Transcriptional Control of Cell Lineage Development in Epicardium-Derived Cells. J Dev Biol. 2013;1(2):92–111. Sun X, Malandraki-Miller S, Kennedy T, Bassat E, Klaourakis K, Zhao J et al. The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development. Development. 2021;148(9). Kobayashi T, Yamashita A, Tsumaki N, Watanabe H. Subpopulations of fibroblasts derived from human iPS cells. Commun Biol. 2024;7(1):736. LeBleu VS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34(3):3519–36. Tallquist MD, Molkentin JD. Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol. 2017;14(8):484–91. Cao Z, Ball JK, Lateef AH, Virgile CP, Corbin EA. Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation. ACS Omega. 2023;8(6):5406–14. Manso AM, Kang SM, Plotnikov SV, Thievessen I, Oh J, Beggs HE, et al. Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration. Am J Physiol Heart Circ Physiol. 2009;296(3):H627–38. Bekedam FT, Smal R, Smit MC, Aman J, Vonk-Noordegraaf A, Bogaard HJ, et al. Author Correction: Mechanical stimulation of induced pluripotent stem cell derived cardiac fibroblasts. Sci Rep. 2024;14(1):13264. Li L, Fan D, Wang C, Wang JY, Cui XB, Wu D, et al. Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-beta1 pathways in cardiac fibroblasts. Cardiovasc Res. 2011;91(1):80–9. Bretherton R, Bugg D, Olszewski E, Davis J. Regulators of cardiac fibroblast cell state. Matrix Biol. 2020;91–2:117–35. Nagalingam RS, Jayousi F, Hamledari H, Dababneh S, Hosseini D, Lindsay C, et al. Molecular and metabolomic characterization of hiPSC-derived cardiac fibroblasts transitioning to myofibroblasts. Front Cell Dev Biol. 2024;12:1496884. Childers RC, Sunyecz I, West TA, Cismowski MJ, Lucchesi PA, Gooch KJ. Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure. Am J Physiol Heart Circ Physiol. 2019;316(3):H596–608. Barrick SK, Greenberg MJ. Cardiac myosin contraction and mechanotransduction in health and disease. J Biol Chem. 2021;297(5):101297. Additional Declarations No competing interests reported. Supplementary Files IbrahimandBagchiSuppTables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 26 Mar, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 11 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9098586","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":604718807,"identity":"397ccc3f-cc42-4ce5-af4b-29b918395cd9","order_by":0,"name":"Somaya Ibrahim","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Somaya","middleName":"","lastName":"Ibrahim","suffix":""},{"id":604718808,"identity":"ee773a3c-433d-4b68-b05f-3ec5d4ca665c","order_by":1,"name":"Rushita Bagchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBAC+/mNjR9+ttXJGRzmMf7A+OdPYgMhLQY3Dh+W7G2TMDY43lcmwdhwjBgtaWnMrG0SiRvOnP/GwNhwmBgtZ8yYGdsk0jfcyN1GnBb7+T1ALf8kcoFaNn9g/EGEFjuJM2bWQFtAWjZIEGWLsUT+N2mQwwzuvzEgTovhjBwzaaD3Ewxu5BCpBeh9Y1AgG84E6pVIbDhmTEgHg8H9HkNQVMrzS+QYf/j4548cQS2oIIFE9aNgFIyCUTAKcAAAV5NSAlL9IXgAAAAASUVORK5CYII=","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Rushita","middleName":"","lastName":"Bagchi","suffix":""}],"badges":[],"createdAt":"2026-03-12 00:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9098586/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9098586/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104535705,"identity":"ad8393b9-c229-46cf-acb1-3919a92b587e","added_by":"auto","created_at":"2026-03-13 03:45:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":959706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentiation of human iPSCs into cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Overview of the workflow of differentiation of iPSCs to cardiac fibroblasts. (B) Representative immunofluorescence images of pluripotency markers in iPSCs, stained for SSEA4 (red), OCT4 (green), phalloidin (grey), and nuclei (DAPI, blue). (C-E) Representative immunofluorescence overlay images of positive and negative markers for (C) pro-epicardial (NANOG, WT1, ZO-1, TCF21; red), (D) epicardial (NANOG, WT1, ZO-1, TCF21; red), and (E) fibroblast (NANOG, DDR2, Vimentin, TCF21; red) cells respectively. Scale bar= 50µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/63a648442016a8d0a1f9b782.png"},{"id":104781669,"identity":"cea52a7a-408f-4bfb-9985-d887e590a41c","added_by":"auto","created_at":"2026-03-17 07:56:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":897584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional and functional validation of iPSC-derived cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Gene expression of markers of (A) pluripotency (\u003cem\u003ePOU5F1, SOX2, NANOG\u003c/em\u003e), (B) pro-epicardial and epicardial cells (\u003cem\u003eGATA4, WT1, TJP1\u003c/em\u003e), and (C) cardiac fibroblasts (\u003cem\u003eTCF21, FN1, COL1A1\u003c/em\u003e); n= 3-5, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001 via ANOVA. (D) Gene expression of endothelial cells (\u003cem\u003ePECAM1\u003c/em\u003e), vascular smooth muscle cells (\u003cem\u003eMYH11\u003c/em\u003e) and cardiomyocytes (\u003cem\u003eMYH6\u003c/em\u003e) in iPS-CFs; n=3, *p\u0026lt;0.05,**p\u0026lt;0.01 via unpaired t-test. (E) Representative immunofluorescence images of HCFs and iPS-CFs treated with vehicle or TGF-b, stained for vinculin (green), phalloidin (red) and nuclei (DAPI, blue). Scale bar= 50µm. (F-G) Representative images (F) and quantification (G) of collagen gel contraction by iPS-CF with vehicle (control), TGF-b1, and Ang II; n=3-5, ****p\u0026lt;0.0001 vs control via ANOVA.\u0026nbsp; (H-J) ECIS wound healing parameter measurements of HCFs and iPS-CFs over 20h timecourse.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/3ca9d909ba675cad826df9e5.png"},{"id":104589900,"identity":"be237d12-ddec-492b-af61-3477120bbd8e","added_by":"auto","created_at":"2026-03-13 16:50:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":897584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional and functional validation of iPSC-derived cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Gene expression of markers of (A) pluripotency (\u003cem\u003ePOU5F1, SOX2, NANOG\u003c/em\u003e), (B) pro-epicardial and epicardial cells (\u003cem\u003eGATA4, WT1, TJP1\u003c/em\u003e), and (C) cardiac fibroblasts (\u003cem\u003eTCF21, FN1, COL1A1\u003c/em\u003e); n= 3-5, *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001 via ANOVA. (D) Gene expression of endothelial cells (\u003cem\u003ePECAM1\u003c/em\u003e), vascular smooth muscle cells (\u003cem\u003eMYH11\u003c/em\u003e) and cardiomyocytes (\u003cem\u003eMYH6\u003c/em\u003e) in iPS-CFs; n=3, *p\u0026lt;0.05,**p\u0026lt;0.01 via unpaired t-test. (E) Representative immunofluorescence images of HCFs and iPS-CFs treated with vehicle or TGF-b, stained for vinculin (green), phalloidin (red) and nuclei (DAPI, blue). Scale bar= 50µm. (F-G) Representative images (F) and quantification (G) of collagen gel contraction by iPS-CF with vehicle (control), TGF-b1, and Ang II; n=3-5, ****p\u0026lt;0.0001 vs control via ANOVA.\u0026nbsp; (H-J) ECIS wound healing parameter measurements of HCFs and iPS-CFs over 20h timecourse.\u003c/p\u003e","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/8101b1bdc85374157a663c51.png"},{"id":104535707,"identity":"57c985aa-c350-4d0e-9634-d356a74471e0","added_by":"auto","created_at":"2026-03-13 03:45:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":439636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional comparison of HCFs and iPSC-derived cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mitochondrial respiration (oxygen consumption rate, OCR) measured using seahorse analyzer in HCFs and iPS-CFs. (B) Quantification of parameters of mitochondrial function; n=14-22, *p\u0026lt;0.05 via unpaired t-test. (C) Representative immunoblot of subunits of the OxPhos complexes in HCFs and iPS-CFs. (D) Total ATP content measurements in HCFs and P0-P2 iPS-CFs; n=3-5, ***p\u0026lt;0.001, ***p\u0026lt;0.0001 via ANOVA. (E-G) Quantification (E) and representative 2D and 3D Q-FISH images of telomeres in nuclei of HCF (F) and iPS-CFs (G); n=12-30 from three independent experiments, \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05 or less vs corresponding HCF data point via multiple unpaired t-test; (inset) ****p\u0026lt;0.0001 via unpaired t-test. (H) \u003cem\u003eACTA2\u003c/em\u003e gene expression of HCFs and iPS-CFs treated with vehicle or TGFB; n=4-5, *p\u0026lt;0.05, **p\u0026lt;0.01 via unpaired t-test. (I) Representative immunoblot image and quantification of aSMA expression of P0-P2 iPS-CFs; n=3, *p\u0026lt;0.05, ns=not significant via ANOVA. (J) Representative immunoblot image and quantification of aSMA expression of iPS-CFs grown on different substrates; n=3, *p\u0026lt;0.05, ns=not significant via ANOVA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/00a9b995b5df5fae4b3a1ad6.png"},{"id":104808606,"identity":"59935a4a-eb10-45aa-a4dd-43631d7d5c5e","added_by":"auto","created_at":"2026-03-17 12:38:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":897584,"visible":true,"origin":"","legend":"","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/091328899f863da63d2c955f.png"},{"id":104781697,"identity":"c8bfc680-193a-45e7-9fa1-92c1092a1f3c","added_by":"auto","created_at":"2026-03-17 07:56:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":421638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtocol overview and applications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSummary of the stepwise differentiation of human iPSCs into quiescent cardiac fibroblasts via proepicardial and epicardial intermediates, and their potential applications in cardiovascular research and therapeutic development.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/34e24c66d968c367ddfd0615.png"},{"id":104589853,"identity":"21d72ba1-6cdc-4709-8929-909427662278","added_by":"auto","created_at":"2026-03-13 16:49:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":421638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtocol overview and applications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSummary of the stepwise differentiation of human iPSCs into quiescent cardiac fibroblasts via proepicardial and epicardial intermediates, and their potential applications in cardiovascular research and therapeutic development.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/aec34789a0a25719c95d3ada.png"},{"id":107868411,"identity":"c135faa7-4d7a-4758-9a15-fad1ed2a409d","added_by":"auto","created_at":"2026-04-27 07:14:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4326173,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/fc92f8ac-211c-47d3-a985-e42978685c44.pdf"},{"id":104781450,"identity":"bc0d602c-64d2-42c3-a254-08a8a0c15243","added_by":"auto","created_at":"2026-03-17 07:55:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25650,"visible":true,"origin":"","legend":"","description":"","filename":"IbrahimandBagchiSuppTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9098586/v1/3154b77ac7459cd6c62e68a9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Defined and Cost-Efficient Strategy for Generating Functionally Quiescent Human iPSC-Derived Cardiac Fibroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiac fibroblasts (CFs) constitute the majority of non-myocyte cell population in the mammalian heart and are indispensable regulators of myocardial structure, extracellular matrix (ECM) homeostasis, and intercellular signaling (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In physiological conditions, CFs maintain tissue integrity through controlled ECM synthesis and turnover. In disease states such as myocardial infarction, cardiometabolic stress, and pressure overload, CFs undergo phenotypic activation characterized by enhanced proliferation, migration, and excessive ECM deposition, driving maladaptive remodeling and fibrosis that contribute directly to heart failure progression (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite their central role in cardiac pathophysiology, access to human CFs for mechanistic and translational studies remains limited. Primary human CFs are commercially available but exhibit restricted proliferative capacity, pronounced donor-to-donor variability, and rapid senescence \u003cem\u003ein vitro\u003c/em\u003e, limiting experimental reproducibility and scalability. Moreover, primary cells often reflect end-stage disease phenotypes, complicating the study of early pathogenic mechanisms and therapeutic intervention windows.\u003c/p\u003e \u003cp\u003eHuman induced pluripotent stem cells (iPSCs) provide a renewable source of patient-specific platform for modeling cardiovascular development and disease (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Robust protocols exist for generating cardiomyocytes, endothelial cells, and smooth muscle cells; however, reliable and developmentally faithful differentiation methods for human CFs have lagged behind. Direct fibroblast differentiation strategies frequently yield heterogeneous populations with incomplete lineage specification and limited functional fidelity. In contrast, stepwise differentiation through defined embryonic intermediates enables controlled lineage commitment and improved reproducibility.\u003c/p\u003e \u003cp\u003eHere, we present a chemically defined, modified (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) stepwise protocol for differentiating human iPSCs into quiescent CFs via cardiac progenitor, proepicardial, and epicardial intermediates. Sequential modulation of Wnt, retinoic acid, TGF-β, and FGF signaling recapitulates key embryonic patterning events that govern fibroblast lineage specification (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Importantly, the protocol yields quiescent fibroblasts with reproducible morphology, transcriptomic fidelity, scalability, and cost-effectiveness. Validation of lineage identity at each stage is achieved through immunostaining, qPCR, and functional assays for stage-specific markers. The resulting iPSC-derived CFs exhibit canonical fibroblast marker expression, contractile and migratory responses, metabolic competence, and responsiveness to profibrotic stimuli, supporting their broad utility in disease modeling, tissue engineering, and pharmacological screening.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of Human iPSCs\u003c/h2\u003e \u003cp\u003eHuman induced pluripotent stem cells (iPSCs) were maintained under feeder-free conditions in six-well plates coated with hESC-qualified Matrigel and cultured in mTeSR Plus medium at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Medium was exchanged every other day using 2 mL per well. Cells were passaged every 3\u0026ndash;4 days using Gentle Cell Dissociation Solution (GCDS; 1 mL per well) following a 5\u0026ndash;6 min incubation at 37\u0026deg;C and reseeded onto freshly coated Matrigel plates. The human iPSC line was provided by Greenstone Biosciences Inc. and cultured as described previously (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnly cultures exhibiting compact colony morphology, high nuclear-to-cytoplasmic ratios, and minimal spontaneous differentiation were used for differentiation experiments. Differentiation was initiated at 80\u0026ndash;90% confluence using iPSCs between passages 20 and 30 to ensure consistency across experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStage 1: Differentiation to Cardiac Progenitors (Day 0–5)\u003c/h3\u003e\n\u003cp\u003eTo initiate mesodermal differentiation and cardiac progenitor specification, iPSC maintenance medium was replaced on Day 0 with RPMI-1640 supplemented with B27 minus insulin and 5 \u0026micro;M CHIR99021 (2 mL per well). Cultures were incubated for 24 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e to activate canonical Wnt signaling and promote mesodermal lineage entry.\u003c/p\u003e \u003cp\u003eOn Day 2, medium was replaced with fresh RPMI/B27 minus insulin without small-molecule supplementation to support cell viability and stabilize early lineage commitment.\u003c/p\u003e \u003cp\u003eOn Day 3, Wnt signaling was inhibited by replacing the medium with RPMI/B27 minus insulin supplemented with 2 \u0026micro;M IWP-2 (2 mL per well), thereby directing differentiation toward cardiac progenitor fate. For individual iPSC lines, IWP-2 concentrations were empirically titrated within the range of 3\u0026ndash;5 \u0026micro;M to minimize cytotoxicity while maintaining effective pathway inhibition.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTroubleshooting\u003c/strong\u003e \u003cp\u003eInadequate CHIR99021 exposure results in poor mesoderm specification. If early morphology does not transition uniformly from compact colonies to a spread epithelial sheet by Day 2\u0026ndash;3, CHIR concentration may require titration. Sensitivity to CHIR varies between iPSC lines; optimal concentrations ranged between 4\u0026ndash;6 \u0026micro;M.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWnt inhibition is highly dose sensitive. Cytotoxicity during Days 3\u0026ndash;5 typically reflects excessive IWP-2 concentration. Empirical titration (3\u0026ndash;5 \u0026micro;M) is recommended for each line to balance effective cardiac specification with cell viability.\u003c/p\u003e \u003cp\u003eOn Day 5, medium was exchanged for fresh RPMI/B27 minus insulin. At this stage, cells exhibited a transition from compact pluripotent colonies to a more spread epithelial-like morphology, consistent with cardiac progenitor specification.\u003c/p\u003e\n\u003ch3\u003eStage 2: Differentiation to Proepicardial and Epicardial Cells (Day 6–12)\u003c/h3\u003e\n\u003cp\u003eTo promote epicardial lineage specification, cardiac progenitor cultures were dissociated on Day 6 using TrypLE Express (1 mL per well, 5 min at 37\u0026deg;C). Cells were gently triturated to single-cell suspension, collected in RPMI medium, and centrifuged at 200 \u0026times; g for 3 min at room temperature. Pelleted cells were resuspended in Advanced DMEM/F12 supplemented with GlutaMAX, 5 \u0026micro;M CHIR99021, 2 \u0026micro;M retinoic acid, 5 \u0026micro;M Y-27632, and 1% fetal bovine serum (FBS).\u003c/p\u003e \u003cp\u003eCells were reseeded onto Matrigel-coated six-well plates at a density of 20,000 cells/cm\u0026sup2; (approximately a 1:12 split ratio) in 2 mL per well and cultured overnight.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTroubleshooting\u003c/strong\u003e \u003cp\u003eSingle-cell dissociation at this stage induces stress. Inclusion of Y-27632 during initial replating is essential to support survival. If attachment remains inefficient, increasing seeding density to 25,000 cells/cm\u0026sup2; can improve recovery.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eOn Day 7, medium was replaced with Advanced DMEM/F12 supplemented with GlutaMAX, 5 \u0026micro;M CHIR99021, and 2 \u0026micro;M retinoic acid (2 mL per well) to reinforce epicardial specification.\u003c/p\u003e \u003cp\u003eOn Day 9, cultures were transitioned to basal Advanced DMEM/F12 with GlutaMAX only, allowing stabilization of the epicardial program in the absence of exogenous signaling cues.\u003c/p\u003e \u003cp\u003eOn Day 11, cells were again dissociated using TrypLE Express, pelleted, and resuspended in Advanced DMEM/F12 supplemented with 2 \u0026micro;M SB431542 to inhibit TGF-β signaling. Cells were seeded onto Matrigel-coated six-well plates at a 1:6 split ratio in 2 mL per well.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTroubleshooting\u003c/strong\u003e \u003cp\u003eFailure to achieve uniform cobblestone morphology by Day 11\u0026ndash;12 typically reflects suboptimal retinoic acid exposure or uneven CHIR signaling. Retinoic acid should be freshly prepared and protected from light.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eIf cells acquire spindle-like morphology during Stage 2, TGF-β signaling may be insufficiently suppressed. Ensure SB431542 is freshly prepared and added at the correct concentration during stabilization.\u003c/p\u003e \u003cp\u003eOn Day 12, medium was refreshed with Advanced DMEM/F12 containing 2 \u0026micro;M SB431542. Cultures were maintained under same conditions with medium changes every other day until reaching confluence. At this stage, cells exhibited a cobblestone-like epithelial morphology characteristic of proepicardial and epicardial intermediates.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTroubleshooting\u003c/strong\u003e \u003cp\u003eAn inefficient transition may occur if epicardial cultures are not fully confluent by Day 14. Initiating fibroblast induction at suboptimal density reduces uniformity of mesenchymal conversion. Cultures should exhibit robust epithelial morphology prior to induction.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eIf cells display excessive αSMA organization or stress fiber formation under basal conditions, mechanical stress or overconfluence may be contributing factors. Maintaining consistent seeding density and avoiding prolonged culture without passaging helps preserve a quiescent phenotype. Excessive exposure to exogenous growth factors may also promote activation; strict adherence to hFGF-B and SB431542 concentrations is recommended.\u003c/p\u003e \u003cp\u003ePassaging stress can induce transient growth arrest. Gentle dissociation and avoidance of over-trypsinization are critical. Cells should not exceed 80\u0026ndash;90% confluence prior to passaging to prevent contact-mediated activation.\u003c/p\u003e\n\u003ch3\u003eStage 3: Differentiation into Quiescent Cardiac Fibroblasts (Day 14–20)\u003c/h3\u003e\n\u003cp\u003eTo induce epicardial-to-fibroblast transition and stabilize a quiescent fibroblast phenotype, epicardial cells were dissociated on Day 14 using TrypLE Express (1 mL per well), pelleted, and resuspended in fibroblast growth medium (FGM; DME/F12 supplemented with hFGF-B, human insulin, FBS, and GA-1000) supplemented with 10 \u0026micro;M SB431542 and 2 ng/mL hFGF-B.\u003c/p\u003e \u003cp\u003eCells were seeded onto Matrigel-coated six-well plates at a density of 8000-10,000 cells/cm\u0026sup2; in 2 mL per well.\u003c/p\u003e \u003cp\u003eOn Day 16, the medium was refreshed with FGM supplemented with SB431542. On Day 18, cells were passaged as described above and reseeded at 10,000 cells/cm\u0026sup2; in FGM containing SB431542.\u003c/p\u003e \u003cp\u003eOn Day 20, cultures were transitioned to FGM supplemented with 10 \u0026micro;M SB431542 alone, with medium changes performed every other day thereafter. By this stage, cells exhibited elongated, spindle-shaped morphology characteristic of quiescent cardiac fibroblasts and demonstrated robust proliferative capacity. Cells were either cryopreserved in DME/F12 medium supplemented with 20% FBS and 10% DMSO or expanded and seeded for downstream experimental applications.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer\u0026rsquo;s instructions. Equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) using the Verso cDNA Synthesis Kit (Thermo Scientific\u0026trade;). Quantitative PCR was performed on a QuantStudio\u0026trade; 7 Flex Real-Time PCR System (Applied Biosystems) using TaqMan\u0026trade; Fast Advanced Master Mix (Applied Biosystems) in combination with gene-specific TaqMan\u0026trade; expression assays. Relative mRNA expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and normalized to 18S ribosomal RNA as an internal control (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). TaqMan\u0026trade; assay identifiers are provided in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eTotal protein lysates were prepared from iPSCs, proepicardial, epicardial, and iPSC-derived cardiac fibroblasts treated with either TGF-β1 or vehicle (control) using RIPA Lysis and Extraction Buffer (Thermo Scientific) supplemented with Halt\u0026trade; protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were quantified using the Pierce\u0026trade; BCA Protein Assay Kit following the manufacturer\u0026rsquo;s protocol. Equal amounts of protein (10\u0026ndash;15 \u0026micro;g) were resolved by SDS\u0026ndash;PAGE using precast tris-glycine gradient gels (4\u0026ndash;20%; Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad).\u003c/p\u003e \u003cp\u003eMembranes were blocked with EveryBlot Blocking Buffer (Bio-Rad) for 30 minutes at room temperature, then incubated overnight at 4\u0026deg;C with primary antibodies against α\u0026ndash;smooth muscle actin (αSMA; mouse monoclonal, Sigma) or OxPhos cocktail (Thermo Scientific). After washing, membranes were incubated with appropriate horseradish peroxidase\u0026ndash;conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Protein bands were visualized using SuperSignal\u0026trade; West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged on a ChemiDoc\u0026trade; MP Imaging System (Bio-Rad). Band intensities were quantified using Quantity One software (Bio-Rad), and αSMA and OxPhos signals were normalized to GAPDH or Calnexin as a loading control. Antibody, reagent, and software details are provided in Supplemental Tables\u0026nbsp;2\u0026ndash;4.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eiPSCs, proepicardial, epicardial, and iPSC-derived cardiac fibroblasts were cultured on glass coverslips (Fisherbrand 18CIR-1) in their respective growth media. Following treatment, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature. Cells were permeabilized with 0.3% Triton X-100 and blocked for 1 hour in PBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA; Sigma). Coverslips were incubated overnight at 4\u0026deg;C with primary antibodies. After washing, samples were incubated for 1 hour at room temperature in the dark with species-specific Alexa Fluor 488\u0026ndash; and Alexa Fluor 594-conjugated secondary antibodies (Thermo Scientific), where indicated. Nuclei were counterstained using ProLong\u0026trade; Diamond Antifade Mountant with DAPI (Invitrogen). Coverslips were cured overnight and imaged using a Keyence BZ-X810 imaging system. Antibody and reagent information is listed in Supplemental Tables\u0026nbsp;2 and 4.\u003c/p\u003e\n\u003ch3\u003eMitochondrial respiration (Seahorse) assay and total ATP detection\u003c/h3\u003e\n\u003cp\u003eFibroblast mitochondrial function was assessed using a Seahorse XFe96 analyzer (Agilent). Cells were seeded onto cell culture plates and incubated overnight at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. Mitochondrial stress test was performed as per manufacturer\u0026rsquo;s instructions (Seahorse XF Cell Mito Stress Test Kit, Agilent) to evaluate the oxygen consumption rate (OCR), using the following injections: oligomycin (1 \u0026micro;M) to measure ATP production, FCCP (1 \u0026micro;M) to assess maximal respiration, and rotenone/antimycin A (0.5 \u0026micro;M) to determine non-mitochondrial oxygen consumption. Cells were stained with Hoechst 33342 to determine the nuclei count using a Cytation 10 high content imaging system (Agilent Biotek), and OCR values were normalized to nuclei per well.\u003c/p\u003e \u003cp\u003eTotal ATP was determined using commercially available reagents (Abcam) as described previously (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Flash-frozen cell pellets were lysed in ATP assay buffer using a Dounce homogenizer. The lysate was centrifuged at 13000g at 4 4oC, and the supernatant was incubated with the necessary reaction components for 30 minutes at room temperature, protected from light. An ATP standard calibration curve was generated using serial dilutions of ATP. Fluorescence signals from standards and samples were then measured on a multimode reader at Ex/Em\u0026thinsp;=\u0026thinsp;535/587 nm. ATP concentrations were calculated and normalized to the corresponding protein content of the cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D collagen gel contraction assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFibroblasts-incorporated collagen hydrogel matrices were prepared in 48-well plates using PureCol EZ Gel solution (Advanced BioMatrix). These gels were incubated in serum-supplemented medium 24 hours prior to overnight serum deprivation for equilibration. Gels were then released from the wells, and cells were treated with vehicle, TGF-β1 (10 ng/mL), or Angiotensin II (1 \u0026micro;M). Well images were acquired every 24 h for up to 120 h. Gel area for each well was calculated using ImageJ software and data are reported as percent contraction.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay using electric cell-substrate impedance sensing (ECIS)\u003c/h2\u003e \u003cp\u003eECIS wound-healing assays were performed using the 8-well ECIS arrays (8W1E, PET) on the ECIS-Zθ equipment (Applied Biophysics). The arrays were treated with 10 mM L-cysteine (Sigma-Aldrich) prior to array stabilization and cell seeding. iPSC-CFs and HCFs were seeded at equal densities (50,000/well) and allowed to form a uniform monolayer before the wounding protocol. Wounding was performed twice (total 60s) using a current of 3000 \u0026micro;A at a frequency of 60kHz. After wounding, medium was exchanged to remove cell debris. The healing process was then observed for ~\u0026thinsp;20h, with capacitance and impedance recorded with a single frequency of 60kHz at 4s intervals. Normalized data was computed using the built-in default script in ECIS software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePeptide Nucleic Acid Quantitative Fluorescence in-situ Hybridization (PNA-QFISH) and Analysis\u003c/h2\u003e \u003cp\u003eTo understand nuclear stability and integrity of iPSC-derived CFs, a modified Q-FISH method was used as described elsewhere (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Cells on glass coverslips were fixed with 4% paraformaldehyde-fixed cells, followed by treatment with 50 \u0026micro;g/mL proteinase K (Gold Biotechnology) at 37\u0026deg;C for 15 minutes. The coverslips were then dehydrated in ethanol gradient (100%, 90%, and 70%). Cells were hybridized with 2ng/ml of PNA probe (PNA Bio) followed by denaturation at 72\u003csup\u003eo\u003c/sup\u003eC for 2min. Coverslips were incubated at 30\u003csup\u003eo\u003c/sup\u003eC for 3h in the dark in a humidified chamber. After hybridization, coverslips were washed twice in 70% formamide (Thermo Scientific) containing 10 mM Tris for 10min, followed by one wash with saline-sodium citrate (SSC) at 55\u0026deg;C, and twice with 2x SSC buffer containing 0.05% Tween 20. Coverslips were mounted onto slides using Prolong Glass mounting media containing DAPI. To visualize 3D telomere and nuclear structures, all z-stack images from 12\u0026ndash;30 nuclei per condition were captured using Cy3 and DAPI filters (Zeiss AxioImager Z1 fluorescence microscope), with constant exposure times of 400\u0026thinsp;\u0026plusmn;\u0026thinsp;50ms and 200\u0026thinsp;\u0026plusmn;\u0026thinsp;50ms, respectively, during sample acquisition. Using Zen3.11.1 analysis software, interphase nuclei were deconvolved at a constrained iterative algorithm (strength 7 for Cy3 and 5 for DAPI) and converted to TIFF images. Semi-automated quantitative image analysis was performed using DIPImage MATLAB software (The MathWorks, Natick, MA, USA) to analyze mean telomere number and intensity for 12\u0026ndash;30 interphase nuclei from each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 10 (Supplemental Table\u0026nbsp;3). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparisons between two groups were conducted using unpaired two-tailed Student\u0026rsquo;s t-tests with Welch\u0026rsquo;s correction. Comparisons among multiple groups were analyzed using one-way or two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc multiple-comparison test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eValidation of Differentiation\u003c/h2\u003e \u003cp\u003eDerivation of cardiac fibroblasts from pluripotent stem cells presents two fundamental challenges: (i) enforcing developmental restriction toward the epicardial\u0026ndash;fibroblast lineage, and (ii) distinguishing bona fide quiescent fibroblasts from phenotypically overlapping mesenchymal or partially activated cell states (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). To address these challenges, we evaluated differentiation fidelity across three orthogonal axes: lineage trajectory, cell identity, and functional competence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDevelopmental Routing of Fibroblast Fate\u003c/h2\u003e \u003cp\u003eCardiac fibroblasts arise predominantly from the embryonic epicardium through a temporally regulated sequence of lineage restriction, epithelial organization, and mesenchymal transition (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Undifferentiated iPSCs expressed canonical pluripotency markers SSEA4, OCT4 (POU5F1), SOX2, and NANOG (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), confirming an intact pluripotent state prior to induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Critically, differentiation proceeded through a morphologically and transcriptionally defined pro-epicardial/epicardial state. Cells at this stage adopted cohesive epithelial morphology and robustly expressed WT1 and TCF21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB middle panel, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC left panel), transcriptional regulators that delineate fibroblast-competent epicardial populations \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Maintenance of epithelial junctional organization was evidenced by zona occludens-1 (ZO-1) localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D) and its gene \u003cem\u003eTJP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), together with cardiac-specific GATA 4 expression in pro-epicardial as well as in epicardial stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This further indicated that lineage specification preceded mesenchymal transition, rather than arising from nonspecific mesodermal outgrowth (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDistinguishing Cardiac Fibroblasts from Other Fibroblast States\u003c/h2\u003e \u003cp\u003eFibroblasts derived from different tissues share overlapping morphological and molecular features, raising the concern that pluripotent stem cell\u0026ndash;derived fibroblast-like cells may represent a generic stromal state rather than a lineage-restricted cardiac fibroblast population (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Addressing this distinction requires more than identifying fibroblast markers; it requires evidence of cardiac-specific lineage history, exclusion of alternative mesodermal fates, and functional behaviors aligned with the myocardial context. iPSC-derived fibroblasts generated using this protocol exhibited expression of core fibroblast markers, including DDR2, vimentin, and TCF21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), confirming the acquisition of a mesenchymal fibroblast identity. However, DDR2 expression is not merely confirmatory but also discriminatory, as it is selectively enriched in cardiac fibroblasts and mediates collagen-dependent signaling, which is critical for myocardial extracellular matrix (ECM) remodeling (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Its expression therefore supports cardiac-relevant fibroblast specialization rather than generic stromal identity as demonstrated by suppression of pluripotency markers \u003cem\u003ePOU5F1\u003c/em\u003e, \u003cem\u003eSOX2\u003c/em\u003e, and \u003cem\u003eNANOG\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) with \u003cem\u003eTJP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eEngagement of a cardiac-appropriate extracellular matrix (ECM) program was further evidenced by expression of \u003cem\u003eTCF21\u003c/em\u003e, \u003cem\u003eFN1, and COL1A1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). COL1A1 and FN1 are matrix components that are central structural elements of the myocardial interstitium and represent core functional outputs of cardiac fibroblasts. Equally critical was the absence of alternative lineage programs. Differentiated cells lacked cardiomyocyte-associated genes (MYH6), endothelial markers (PECAM1), and smooth muscle markers (MYH11) under basal conditions, excluding contamination by parallel mesodermal derivatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This negative selection is essential, as partial expression of fibroblast markers alone is insufficient to establish lineage identity in pluripotent stem cell\u0026ndash;derived cultures, where partial marker overlap can otherwise confound identity assignment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Hallmarks of Cardiac Fibroblasts\u003c/h2\u003e \u003cp\u003eCardiac fibroblasts are distinguished from generic stromal populations by their integration of mechanical sensing, matrix remodeling, and context-dependent activation within the myocardial environment. These behaviors are not universal fibroblast traits but emerge from a cardiac-specific developmental history and are essential for heart morphogenesis, repair, and pathological remodeling (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). We therefore evaluated whether iPSC-derived fibroblasts exhibit functional behaviors that reflect cardiac lineage competence rather than generic mesenchymal activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMigratory Competence, Cytoskeletal Organization, and Matrix Remodeling\u003c/h2\u003e \u003cp\u003eDuring cardiac development and post-injury repair, fibroblasts migrate directionally within a mechanically dynamic extracellular matrix to populate emerging interstitial niches (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In response to pro-fibrotic TGF-β stimulation, iPSC-derived fibroblasts exhibited robust migratory behavior, indicating appropriate cytoskeletal organization and integrin (vinculin)-mediated matrix engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This behavior is not trivial, as incompletely specified mesenchymal cells frequently display either impaired motility or unregulated migration (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), which is inconsistent with cardiac fibroblast behavior as shown in HCFs treated with TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eA defining feature of cardiac fibroblasts is their ability to physically remodel fibrillar collagen networks through controlled force generation (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In three-dimensional collagen matrices, iPSC-derived CFs generated measurable contractile forces, demonstrating functional engagement with native-like extracellular matrix substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G). This behavior reflects active-matrix sensing and force transduction, processes mediated by fibroblast-specific cytoskeletal and adhesion complexes.\u003c/p\u003e \u003cp\u003eImportantly, matrix contraction was limited under basal conditions, consistent with a quiescent fibroblast state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF upper panel, 2G). This contrasts with generic fibroblasts or constitutively activated myofibroblasts, which often exhibit elevated baseline contractility independent of environmental cues (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The restrained basal contractility observed here suggests that force generation is tightly regulated rather than constitutively engaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eContext-Dependent Activation and Phenotypic Plasticity\u003c/h2\u003e \u003cp\u003eCardiac fibroblasts are characterized by their capacity to remain functionally quiescent under homeostatic conditions while rapidly transitioning to an activated, matrix-producing phenotype in response to injury-associated signals (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). iPSC-derived CFs preserved this phenotypic plasticity, as evidenced by a marked increase in matrix contraction following stimulation with angiotensin II or TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Discrimination of Fibroblast States\u003c/h2\u003e \u003cp\u003eThe migratory, contractile, and stimulus-responsive behaviors observed in iPSC-derived fibroblasts define a functional profile consistent with cardiac fibroblast identity. These behaviors are tightly linked to the mechanical and signaling environment of the heart and are not uniformly shared by fibroblasts derived from non-cardiac tissues.\u003c/p\u003e \u003cp\u003eWound healing is an intrinsic characteristic of fibroblasts. iPS-CFs, similar to HCFs, showed recovery of cell migration and reestablishment of cell-cell adhesion after irreversible electroporation of cells seeded on electrode arrays. In fact, iPS-CFs demonstrated a faster, more robust re-establishment of the cell monolayer, as evidenced by decreased capacitance, increased resistance, and increased impedance, in contrast to HCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eQuiescence and Regulated Activation\u003c/h2\u003e \u003cp\u003eIn the heart, fibroblasts must dynamically adapt their metabolic state to support both quiescent maintenance and rapid activation following developmental cues or injury (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Consequently, preservation of metabolic competence is not merely a measure of cell health, but a defining feature of lineage stability and functional identity.\u003c/p\u003e \u003cp\u003eTo further understand mitochondrial performance, extracellular flux analysis was performed using sequential administration of oligomycin (Complex V inhibition), FCCP (protonophore-mediated uncoupling), and rotenone/antimycin A (Complex I and III inhibition) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Basal oxygen consumption rates were comparable between iPSC-CFs and primary HCFs, indicating equivalent steady-state electron flux through the ETC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, upper left panel). Basal respiration integrates NADH- and FADH\u003csub\u003e2\u003c/sub\u003e-driven input into Complexes I and II and reflects constitutive oxidative metabolism. The preservation of basal OCR demonstrates that iPSC-CFs have transitioned away from the glycolytic metabolic program characteristic of pluripotency and into an oxidative phenotype consistent with differentiated cardiac mesenchyme (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eATP-linked respiration, defined by the oligomycin-sensitive fraction of oxygen consumption, was similarly preserved between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, upper middle panel). Because oligomycin blocks ATP synthase (Complex V), this parameter reflects oxygen consumption directly coupled to phosphorylation. Proton leak, representing oxygen consumption independent of ATP production, was modest but significantly reduced in iPSC-CFs relative to HCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, upper right panel). Maximal respiration following FCCP-mediated uncoupling was comparable between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lower left panel), indicating intact ETC capacity and preserved substrate oxidation potential. Notably, iPSC-CFs exhibited significantly increased spare respiratory capacity compared to HCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lower middle panel). Spare capacity, defined as the difference between basal and maximal respiration, reflects the bioenergetic reserve available to meet sudden increases in ATP demand. Non-mitochondrial oxygen consumption, measured following complete ETC inhibition with rotenone and antimycin A, was similar between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lower right panel), indicating that extramitochondrial oxidase activity does not account for the observed respiratory profile. iPSC-CFs generated using this protocol exhibited intact expression of mitochondrial respiratory chain complexes I\u0026ndash;V, indicating preservation of oxidative phosphorylation capacity rather than reliance on glycolytic compensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This is a critical distinction, as pluripotent cells and incompletely differentiated mesenchymal populations typically exhibit suppressed mitochondrial maturation, altered respiratory complex expression, and reduced electron transport chain (ETC) protein abundance. Maintenance of a fully assembled ETC therefore supports completion of mitochondrial maturation concomitant with fibroblast specification. Consistent with preserved mitochondrial function, intracellular ATP content remained stable across early passages in iPSC-CFs and was comparable to HCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Importantly, ATP levels did not decline with expansion, arguing against cumulative metabolic stress, replicative exhaustion, or dedifferentiation during passaging. Together, these findings indicate that the differentiation strategy not only enforces transcriptional and structural fibroblast identity but also stabilizes the mitochondrial programs required to sustain it.\u003c/p\u003e \u003cp\u003eCellular senescence is a major experimental limitation of cardiac fibroblasts, which makes the source of these cells critical for in vitro studies. Nuclear instability and structure of iPSC-CFs assessed by Q-FISH showed increased mean telomere intensity per telomere in iPSC-CFs which were significantly higher than HCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), suggesting that iPSC-CFs possess younger and healthier nuclei in comparison to commercially available primary HCFs. Compared to iPSC-CFs, generic fibroblasts (HCFs) exhibited altered telomeric DNA morphology and structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G), reflecting impaired cellular function from serial passaging and leading to replicative senescence.\u003c/p\u003e \u003cp\u003eAs shown previously by our group (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), iPSC-CFs generated using this protocol respond robustly to TGF-β\u003csub\u003e1\u003c/sub\u003e stimulation and transition to an activated fibroblast state, as evidenced by increase in \u003cem\u003eACTA2\u003c/em\u003e (\u003cem\u003eα-SMA\u003c/em\u003e) gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Of note, this response is equivalent to commercially available HCFs. When subjected to serial passaging in vitro, the iPSC-CFs showed increased protein expression of α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Additionally, these cells also showed distinguishable patterns of α-SMA expression when grown on tissue culture dishes with different substrate chemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). This is critical as variable substrates can pre-activate these cells and therefore hinder their responsiveness to pathologic stimuli \u003cem\u003ein vitro\u003c/em\u003e, therefore producing confounding observations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion and Conclusion","content":"\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003cp\u003eThe ability to generate human cardiac fibroblasts with defined lineage history, functional competence, and phenotypic stability has remained a persistent challenge in cardiovascular and developmental biology. Here, we describe a developmentally constrained differentiation strategy that yields quiescent cardiac fibroblasts from human iPSCs by explicitly recapitulating the epicardial lineage trajectory that gives rise to fibroblasts in the embryonic heart. Rather than relying on direct mesenchymal induction or marker-based enrichment, this protocol enforces fibroblast identity through sequential restriction of developmental potential, thereby reducing heterogeneity and preserving lineage fidelity.\u003c/p\u003e \u003cp\u003eA central feature of this approach is its routing of differentiation through proepicardial and epicardial intermediates, reflecting the predominant embryonic origin of cardiac fibroblasts in vivo. Transient activation and subsequent resolution of epicardial transcriptional programs, including WT1 and TCF21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), distinguishes this system from generic fibroblast differentiation strategies and argues against nonspecific mesenchymal conversion. By embedding epicardial lineage history into fibroblast specification, the protocol establishes a developmental context that shapes subsequent functional and metabolic behaviors.\u003c/p\u003e \u003cp\u003eImportantly, the resulting fibroblasts occupy a quiescent yet plastic state that mirrors cardiac fibroblasts in the uninjured heart. These cells remain non-contractile under basal conditions while retaining the capacity for coordinated activation in response to profibrotic cues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This balance between quiescence and inducible activation is a defining feature of cardiac fibroblast biology and is frequently lost in primary cell cultures or direct conversion systems, which often yield constitutively activated myofibroblast-like populations. Preservation of this regulated phenotype enables interrogation of early fibrotic events that precede irreversible remodeling.\u003c/p\u003e \u003cp\u003eBeyond transcriptional and functional identity, this protocol stabilizes metabolic programs essential for fibroblast lineage maintenance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). iPSC-derived CFs exhibit preserved oxidative phosphorylation capacity and stable ATP levels across passages, supporting the energetic demands of extracellular matrix synthesis and remodeling. Increasing evidence indicates that metabolic state is not merely permissive but instructive in fibroblast activation and fate decisions. Equivalent ATP-coupled respiration confirms intact proton motive force utilization and efficient conversion of electrochemical gradient into ATP. This is particularly relevant given that collagen biosynthesis, vesicular trafficking, cytoskeletal turnover, and myosin ATPase\u0026ndash;dependent force generation are energetically intensive processes intrinsic to fibroblast identity (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Reduced leaks suggest tighter coupling and preserved membrane integrity, consistent with a metabolically restrained quiescent state. This tighter coupling efficiency may reflect reduced unnecessary proton cycling, thereby optimizing ATP yield while limiting oxidative stress which are features compatible with stable lineage maintenance. By maintaining metabolic competence alongside lineage restriction, this platform minimizes culture-induced drift and supports longitudinal studies of fibroblast plasticity and disease progression.\u003c/p\u003e \u003cp\u003eThe developmental grounding of this system distinguishes it from existing fibroblast differentiation approaches and enhances its translational relevance. Patient-specific iPSC-derived cardiac fibroblasts generated using this method provide a tractable platform for modeling cardiometabolic disease, fibrosis, and gene\u0026ndash;environment interactions in a human context (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In tissue engineering applications, these cells can supply developmentally appropriate extracellular matrix and paracrine signaling to support cardiomyocyte maturation and tissue organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, their scalability and phenotypic stability make them well suited for pharmacological screening and precision medicine efforts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is critical to note here that with FGM made in house without the use of commercially available pre-assembled media kits, the cost of this iPSC-CF differentiation and propagation protocol is reduced by almost thirty percent, which makes this an affordable avenue for several researchers globally.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral limitations warrant consideration. Differentiation efficiency requires modest line-specific optimization of Wnt pathway modulation, reflecting intrinsic variability among iPSC lines. In addition, the use of Matrigel introduces potential batch-to-batch variability and limits full chemical definition of the system. Future refinements may incorporate fully defined extracellular matrix substrates, mechanical conditioning, and single-cell transcriptomic benchmarking against adult human cardiac fibroblast subtypes to further refine maturation state and subtype specification.\u003c/p\u003e \u003cp\u003eIn summary, this work establishes a developmentally informed framework for generating human cardiac fibroblasts that integrates lineage history, functional plasticity, and metabolic stability. By recapitulating epicardial specification and resolution, this protocol yields fibroblasts that more closely reflect their \u003cem\u003ein vivo\u003c/em\u003e counterparts than existing approaches and is rapid and budget friendly. Robust quality control including reproducibility and repeatability of this platform provides a foundation for dissecting the developmental and disease mechanisms governing cardiac fibroblast behavior and offers a versatile and cost-effective resource for basic, translational, and precision cardiovascular research.\u003c/p\u003e \u003c/div\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eACTA2 \u0026ndash; actin alpha 2 (a.k.a. alpha smooth muscle actin \u0026alpha;SMA)\u003c/p\u003e\n\u003cp\u003eATP \u0026ndash; adenosine triphosphate\u003c/p\u003e\n\u003cp\u003eBCA \u0026ndash; bicinchoninic acid assay\u003c/p\u003e\n\u003cp\u003ecDNA \u0026ndash; complementary DNA\u003c/p\u003e\n\u003cp\u003eCF \u0026ndash; cardiac fibroblasts\u003c/p\u003e\n\u003cp\u003eCHIR99021 \u0026ndash; GSK-3\u0026alpha;/\u0026beta; inhibitor\u003c/p\u003e\n\u003cp\u003eCOL1A1 \u0026ndash; collagen 1 alpha1\u003c/p\u003e\n\u003cp\u003eCy3 \u0026ndash; cyanine dye 3\u003c/p\u003e\n\u003cp\u003eDAPI \u0026ndash; 4\u0026prime;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eDDR2 \u0026ndash; discoidin domain receptor tyrosine kinase 2\u003c/p\u003e\n\u003cp\u003eDMEM/F12 \u0026ndash; Dulbecco\u0026rsquo;s Modified Eagle Medium/Nutrient Mixture F-12\u003c/p\u003e\n\u003cp\u003eECIS \u0026ndash; electric cell-substrate impedance sensing\u003c/p\u003e\n\u003cp\u003eECM \u0026ndash; extracellular matrix\u003c/p\u003e\n\u003cp\u003eETC \u0026ndash; electron transport chain\u003c/p\u003e\n\u003cp\u003eFBS \u0026ndash; fetal bovine serum\u003c/p\u003e\n\u003cp\u003eFCCP \u0026ndash; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone\u003c/p\u003e\n\u003cp\u003eFGF \u0026ndash; fibroblast growth factor\u003c/p\u003e\n\u003cp\u003eFN1 \u0026ndash; fibronectin 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGCDS \u0026ndash; gentle cell dissociation solution\u003c/p\u003e\n\u003cp\u003ehESC \u0026ndash; human embryonic stem cells\u003c/p\u003e\n\u003cp\u003eiPSC \u0026ndash;induced pluripotent stem cell\u003c/p\u003e\n\u003cp\u003eiPS-CF/ iPSC-CF \u0026ndash; iPSC-derived cardiac fibroblasts\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIWP-2 \u0026ndash; inhibitor of WNT processing-2\u003c/p\u003e\n\u003cp\u003emRNA \u0026ndash; messenger ribonucleic acid\u003c/p\u003e\n\u003cp\u003eMYH6 \u0026ndash; myosin heavy chain 6\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNANOG \u0026ndash; homeobox transcription factor Nanog (on ch12)\u003c/p\u003e\n\u003cp\u003eOCR \u0026ndash; oxygen consumption rate\u003c/p\u003e\n\u003cp\u003eOCT4 (POU5F1) \u0026ndash; \u0026nbsp;Octamer-binding transcription factor 4A (a.k.a. POU-type homeodomain \u0026ndash; containing DNA binding protein (on ch6)\u003c/p\u003e\n\u003cp\u003eOxPhos \u0026ndash; oxidative phosphorylation\u003c/p\u003e\n\u003cp\u003ePBS \u0026ndash; phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePECAM1 \u0026ndash; \u0026nbsp;platelet endothelial cell adhesion molecule 1\u003c/p\u003e\n\u003cp\u003eQ-FISH \u0026ndash; quantitative Fluorescence in-situ Hybridization\u003c/p\u003e\n\u003cp\u003eqPCR - quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRIPA \u0026ndash; radioimmunoprecipitation assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRPMI-1640 \u0026ndash; Roswell Park Memorial Institute 1640 medium\u003c/p\u003e\n\u003cp\u003eSB431542 \u0026ndash; TGF- \u0026beta; inhibitor\u003c/p\u003e\n\u003cp\u003eSDS-PAGE \u0026ndash; sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003eSOX2 \u0026ndash; SRY (sex determining region Y)-box 2 (on ch3)\u003c/p\u003e\n\u003cp\u003eSSEA4 \u0026ndash; Stage-specific embryonic antigen 4\u003c/p\u003e\n\u003cp\u003eTCF21 \u0026ndash; transcription factor 21\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta; \u0026ndash; transforming growth factor\u003c/p\u003e\n\u003cp\u003eTJP1 \u0026ndash; tight junction protein 1\u003c/p\u003e\n\u003cp\u003eWT1 \u0026ndash; Wilms tumor 1\u003c/p\u003e\n\u003cp\u003eZO-1 \u0026ndash; zona occludens1\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors have no financial and non-financial competing interests to declare.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Y.I. and R.A.B. conceived and designed the study; S.Y.I. and R.A.B. performed the experiments and analyzed the data; S.Y.I. drafted the manuscript and prepared the figures; all authors edited and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Andrew Morris for access to equipment for this study, and Dr. Ashim Bagchi for valuable input on the manuscript and assistance with Q-FISH studies for this project. Fluorescence imaging and seahorse assays were performed at the Cellular Imaging Core of the Center for Microbial Pathogenesis and Host Inflammatory Responses (CMPHIR) at UAMS (NIGMS COBRE Grant 5P30GM145393-05). We acknowledge Dr. Joseph Wu and Greenstone Biosciences Inc. for providing the human iPSC line GSB-L2053 used in this study. R.A.B. received support through a career development award from the American Heart Association (23CDA1048663), funding from the Vice Chancellor for Research and Innovation (VCRI) and the Arkansas Biosciences Institute, the Sturgis Grant for Diabetes Research from the College of Medicine, and the Medical Research Endowment Grant from the VCRI, University of Arkansas for Medical Sciences. Schematics for figures 1 and 4 were generated using a licensed version of Biorender.com.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eButt RP, Laurent GJ, Bishop JE. Collagen production and replication by cardiac fibroblasts is enhanced in response to diverse classes of growth factors. Eur J Cell Biol. 1995;68(3):330\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMouton AJ, Ma Y, Rivera Gonzalez OJ, Daseke MJ 2nd, Flynn ER, Freeman TC, et al. Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis. Basic Res Cardiol. 2019;114(2):6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu X, Khalil H, Kanisicak O, Boyer JG, Vagnozzi RJ, Maliken BD, et al. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest. 2018;128(5):2127\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahr J, Poschmann G, Jungmann A, Busch M, Ding Z, Vogt J, et al. A secretome atlas of cardiac fibroblasts from healthy and infarcted mouse hearts. Commun Biol. 2025;8(1):675.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIeda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM, et al. Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell. 2009;16(2):233\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Yang Bennett DS, Echard EJ, Chen B, Ciampa G, Zhao W, et al. Junctophilin-2 Regulates Store-Operated Calcium Entry to Drive Cardiac Fibroblast Activation, Fibrotic Repair, and Angiogenesis After Myocardial Infarction. Circulation. 2025;152(10):699\u0026ndash;716.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbrahim SY, Holdiness R, Thadisena A, Boyle KE, Jun SR, Bagchi RA. 3-D biomechanics and epigenomics reveal atypical fibroblast responses in cardiometabolic disease. Am J Physiol Heart Circ Physiol. 2025;329(5):H1267\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Shen M, Wu JC. Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells. Methods Mol Biol. 2022;2454:109\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiesinger A, Boink GJJ, Christoffels VM, Devalla HD. Retinoic acid signaling in heart development: Application in the differentiation of cardiovascular lineages from human pluripotent stem cells. Stem Cell Rep. 2021;16(11):2589\u0026ndash;606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMossahebi-Mohammadi M, Quan M, Zhang JS, Li X. FGF Signaling Pathway: A Key Regulator of Stem Cell Pluripotency. Front Cell Dev Biol. 2020;8:79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFox BM, Gil HW, Kirkbride-Romeo L, Bagchi RA, Wennersten SA, Haefner KR, et al. Metabolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice. Kidney Int. 2019;95(3):590\u0026ndash;610.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoon SSS, Lansdorp PM. Quantitative fluorescence in situ hybridization (Q-FISH). Curr Protoc Cell Biol 2001;Chap 18:18 4 1\u0026ndash;4 21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFloy ME, Givens SE, Matthys OB, Mateyka TD, Kerr CM, Steinberg AB, et al. Developmental lineage of human pluripotent stem cell-derived cardiac fibroblasts affects their functional phenotype. FASEB J. 2021;35(9):e21799.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuijada P, Trembley MA, Small EM. The Role of the Epicardium During Heart Development and Repair. Circ Res. 2020;126(3):377\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarter AC, Davis-Dusenbery BN, Koszka K, Ichida JK, Eggan K. Nanog-independent reprogramming to iPSCs with canonical factors. Stem Cell Rep. 2014;2(2):119\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorota A, Maryniak N, Mariankowska A, Milczarek C, Dorota M, Zywiec W, et al. Induced Pluripotent Stem Cells (iPSC) and Their Use in Disease Modeling. Cureus. 2025;17(10):e93999.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraitsch CM, Yutzey KE. Transcriptional Control of Cell Lineage Development in Epicardium-Derived Cells. J Dev Biol. 2013;1(2):92\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Malandraki-Miller S, Kennedy T, Bassat E, Klaourakis K, Zhao J et al. The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development. Development. 2021;148(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi T, Yamashita A, Tsumaki N, Watanabe H. Subpopulations of fibroblasts derived from human iPS cells. Commun Biol. 2024;7(1):736.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeBleu VS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34(3):3519\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTallquist MD, Molkentin JD. Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol. 2017;14(8):484\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Z, Ball JK, Lateef AH, Virgile CP, Corbin EA. Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation. ACS Omega. 2023;8(6):5406\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManso AM, Kang SM, Plotnikov SV, Thievessen I, Oh J, Beggs HE, et al. Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration. Am J Physiol Heart Circ Physiol. 2009;296(3):H627\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBekedam FT, Smal R, Smit MC, Aman J, Vonk-Noordegraaf A, Bogaard HJ, et al. Author Correction: Mechanical stimulation of induced pluripotent stem cell derived cardiac fibroblasts. Sci Rep. 2024;14(1):13264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L, Fan D, Wang C, Wang JY, Cui XB, Wu D, et al. Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-beta1 pathways in cardiac fibroblasts. Cardiovasc Res. 2011;91(1):80\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBretherton R, Bugg D, Olszewski E, Davis J. Regulators of cardiac fibroblast cell state. Matrix Biol. 2020;91\u0026ndash;2:117\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagalingam RS, Jayousi F, Hamledari H, Dababneh S, Hosseini D, Lindsay C, et al. Molecular and metabolomic characterization of hiPSC-derived cardiac fibroblasts transitioning to myofibroblasts. Front Cell Dev Biol. 2024;12:1496884.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChilders RC, Sunyecz I, West TA, Cismowski MJ, Lucchesi PA, Gooch KJ. Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure. Am J Physiol Heart Circ Physiol. 2019;316(3):H596\u0026ndash;608.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrick SK, Greenberg MJ. Cardiac myosin contraction and mechanotransduction in health and disease. J Biol Chem. 2021;297(5):101297.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"iPSC, cell lineage, quiescent cardiac fibroblasts, metabolic competence, nuclear stability","lastPublishedDoi":"10.21203/rs.3.rs-9098586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9098586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCardiac fibroblasts (CFs) are central regulators of myocardial structure, extracellular matrix homeostasis, and pathological remodeling, yet robust \u003cem\u003eex vivo\u003c/em\u003e experimental access to na\u0026iuml;ve 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.\u003c/p\u003e \u003cp\u003eHere, 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.\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eThis 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.\u003c/p\u003e \u003cp\u003eTogether, 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.\u003c/p\u003e","manuscriptTitle":"A Defined and Cost-Efficient Strategy for Generating Functionally Quiescent Human iPSC-Derived Cardiac Fibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 03:45:41","doi":"10.21203/rs.3.rs-9098586/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"81287309554781556220604353692947619716","date":"2026-04-02T07:38:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338576223695982472686346420295576345717","date":"2026-04-02T06:27:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T06:19:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T05:56:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T05:56:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2026-03-11T23:52:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d12d561-c601-4514-ac5e-1ea1e3c5bc06","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T06:24:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 03:45:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9098586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9098586","identity":"rs-9098586","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}