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Fibroblast

Fibroblasts are mesenchymal cells that synthesize and maintain the extracellular matrix (ECM), providing structural support to tissues and organs throughout the body.^[1] These cells, which constitute a major component of connective tissue, are characterized by their elongated, spindle-shaped morphology and ability to produce key ECM components such as collagens, elastin, and fibronectin.^[2] Originating primarily from mesodermal precursors during embryonic development, fibroblasts can also arise through epithelial-to-mesenchymal transition (EMT) or endothelial-to-mesenchymal transition (EndMT) in specific contexts, such as cardiac tissue formation.^[2] In healthy tissues, fibroblasts play essential roles in maintaining homeostasis by regulating ECM turnover and serving as signaling niche cells that influence neighboring cell behaviors through secreted factors like growth factors and cytokines.^[2] During wound healing, they proliferate, migrate to injury sites, and differentiate into contractile myofibroblasts expressing alpha-smooth muscle actin (α-SMA), which facilitate tissue contraction and repair by remodeling the ECM.^[2] However, in pathological conditions, dysregulated fibroblast activation leads to excessive ECM deposition, driving fibrosis—a process implicated in approximately 45% of deaths in developed countries, affecting organs like the lungs, liver, and heart.^[2] Fibroblasts exhibit remarkable heterogeneity, with transcriptional profiles varying significantly across organs; for instance, less than 20% of fibroblast genes overlap between tissues like the heart, skeletal muscle, intestine, and bladder in mice.^[2] This diversity is encoded by positional cues such as HOX genes and contributes to their specialized functions, including adipogenesis, osteogenesis, and immune modulation.^[2] In cancer, fibroblasts within the tumor microenvironment, often termed cancer-associated fibroblasts (CAFs), promote tumor progression by altering ECM stiffness, secreting pro-angiogenic factors, and suppressing anti-tumor immunity.^[3] Their plasticity and context-dependent roles make fibroblasts critical targets for regenerative medicine and anti-fibrotic therapies.^[2]

Structure and Morphology

Cellular Features

Fibroblasts are characterized by an elongated, spindle-shaped or fusiform morphology, often with stellate features and multiple thin cytoplasmic projections that facilitate interactions with the surrounding extracellular matrix. These cells' size and shape can vary based on the specific tissue environment and functional state. Unlike epithelial cells, which exhibit distinct apical-basal polarity, fibroblasts lack such organized polarity, presenting instead a more isotropic distribution of organelles and a front-back orientation primarily during migration.^[4]^[5] Ultrastructural analysis via transmission electron microscopy reveals a cytoplasm rich in organelles adapted for synthetic activity, including abundant cisternae of rough endoplasmic reticulum and a well-developed Golgi apparatus, which occupy significant portions of the perinuclear region. These features underscore the cell's role in protein processing and secretion, with the rough endoplasmic reticulum often appearing dilated in active fibroblasts. Mitochondria and other organelles are distributed throughout the cytoplasm, supported by a network of cytoskeletal elements. Thin, elongated cytoplasmic extensions extend from the cell body, enabling close apposition to adjacent matrix fibers without forming tight junctions.^[6]^[7]^[8] A prominent cytoskeletal feature of fibroblasts is the presence of vimentin intermediate filaments, which provide structural integrity and mechanical resilience to the cell. These filaments, approximately 10 nm in diameter, form a dynamic network that interconnects organelles and adheres to the plasma membrane, contributing to the maintenance of the cell's elongated form. Vimentin expression is a hallmark mesenchymal marker, distinguishing fibroblasts from other cell types.^[9]^[10] Fibroblasts reside predominantly in connective tissues, where they form the stromal framework of organs; common locations include the dermis of the skin, the paratenon and endotenon of tendons, and the interstitial stroma of visceral organs such as the lungs, heart, and kidneys. In these sites, their morphology adapts to the local matrix density, appearing more flattened in dense tissues like tendons and more irregular in loose areolar connective tissue. These structural attributes equip fibroblasts to synthesize and organize extracellular matrix components, influencing tissue architecture and resilience.^[4]^[7]

Distinction from Fibrocytes

Fibrocytes represent a distinct population of circulating cells that serve as precursors to fibroblasts, originating from bone marrow-derived monocytes rather than the resident tissue populations that give rise to mature fibroblasts.^[11] These cells are recruited to sites of injury or inflammation in the peripheral tissues, where they differentiate into fibroblast-like cells capable of contributing to extracellular matrix production.^[12] Unlike resident fibroblasts, which are primarily tissue-embedded and derived from embryonic mesenchyme or local progenitors, fibrocytes maintain a hematopoietic lineage throughout their circulation phase.^[13] The concept of fibrocytes was first described in 1994 by Bucala et al., who identified them as a novel leukocyte subpopulation in human peripheral blood that expresses both hematopoietic and mesenchymal markers, mediating tissue repair processes.^[14] In healthy individuals, fibrocytes constitute approximately 0.1-0.5% of circulating non-erythrocytic leukocytes, though their numbers can increase under inflammatory conditions.^[15] This low baseline prevalence underscores their role as a responsive, rather than constitutive, component of the immune and repair systems. A key distinction lies in their molecular markers: fibrocytes co-express the pan-leukocyte antigen CD45, the hematopoietic stem cell marker CD34, and early collagen I, reflecting their dual hematopoietic-mesenchymal identity, whereas mature fibroblasts typically downregulate CD45 and CD34 upon tissue integration and full differentiation.^[16] This marker profile enables fibrocytes to retain immune functions, such as antigen presentation via MHC class II, which is less prominent in differentiated fibroblasts.^[17] Morphologically, fibrocytes appear as smaller, more rounded or oval-shaped cells with spindle-like extensions suited for migration, often exhibiting integrin expression (e.g., α4β1) that facilitates their extravasation into tissues; in contrast, mature fibroblasts display an elongated, branched cytoplasm with an elliptical nucleus optimized for matrix synthesis in situ.^[18] Upon recruitment, fibrocytes undergo a functional transition, producing collagen and other matrix components to support fibrosis, but they remain transient progenitors compared to the long-lived, resident fibroblasts that dominate sustained tissue remodeling.^[19] This precursor relationship highlights fibrocytes' role in bridging hematopoiesis and fibrogenesis, particularly in pathological contexts like wound healing.^[20]

Origin and Development

Embryonic and Postnatal Origins

Fibroblasts primarily originate from mesodermal precursors during embryogenesis, with the majority deriving from the paraxial mesoderm and lateral plate mesoderm, which give rise to connective tissue frameworks in various organs.^[21] In specific regions, such as craniofacial tissues, fibroblasts arise from neural crest-derived mesenchyme, contributing to the structural diversity of head and neck connective tissues.^[21] This mesodermal specification is regulated by key transcription factors, including Twist1, Snai1, and Foxc2, which drive the transition to mesenchymal lineages and ensure proper fibroblast differentiation.^[21] Fibroblasts first appear in human embryos during early gestation, around weeks 7–8, coinciding with the onset of organogenesis when mesenchymal cells proliferate to support tissue patterning and extracellular matrix deposition.^[22] Throughout this period, from weeks 7 to 8 post-conception, fibroblasts play a critical role in organogenesis by synthesizing provisional matrices that guide epithelial branching and vascular invasion in developing organs like the lungs, heart, and skin.^[22] Postnatally, fibroblast populations are maintained through self-renewal of resident pools within tissues, facilitated by signaling pathways such as PDGF, which promotes proliferation and homeostasis in organs like the lung and skin.^[21] Additionally, recruitment from bone marrow-derived mesenchymal progenitors contributes to replenishment, particularly during tissue turnover or mild injury, although this source is secondary to local self-renewal in steady-state conditions.^[21] These mechanisms ensure sustained fibroblast function across adulthood. The developmental origins of fibroblasts show strong conservation across mammalian species, as evidenced by similar mesodermal and neural crest contributions in mice and humans, enabling the use of rodent models for studying human fibroblast biology.^[21] In contrast, avian models, such as chick embryos, reveal variations in timing and migratory patterns of mesenchymal precursors, which have been instrumental in elucidating early fibroblast progenitor dynamics despite differences in yolk-dependent development.^[23]

Subtypes and Heterogeneity

Fibroblasts exhibit significant heterogeneity, manifesting as distinct subtypes with specialized functions that contribute to tissue homeostasis and repair. Major subtypes include matrix fibroblasts, also known as matrifibrocytes, which specialize in extracellular matrix (ECM) production and remodeling by expressing genes such as Chad, Cilp2, and Comp, particularly during scar maturation phases.^[24]^[25] Another key subtype is inflammatory fibroblasts, which secrete proinflammatory cytokines and chemokines like IL-6, IL-1, and MCP-1 in response to immune signals, thereby modulating local immune environments.^[26]^[27] These subtypes arise from shared progenitor pools but diverge through transcriptional and epigenetic programs, enabling context-specific roles across tissues. Recent advances in single-cell RNA sequencing (scRNA-seq) have illuminated this diversity through comprehensive atlases. A 2025 study on human skin fibroblasts identified six major subtypes in healthy tissue: superficial (papillary) fibroblasts expressing collagens like COL13A1 for epithelial support; universal (reticular) fibroblasts marked by PI16 and CD34 as potential precursors; FRC-like subtypes with immune genes such as CCL19 for niche maintenance; perivascular fibroblasts enriched in PPARG; hair follicle-associated subtypes (e.g., DPEP1+); and Schwann-like nerve-interfacing subtypes (e.g., NGFR+).^[28] Cross-tissue analyses from 517 human samples across 11 organs revealed nine universal subtypes, including PI16+ progenitors and LRRC15+ matrix fibroblasts, alongside six tissue-specific clusters, underscoring conserved and organ-adapted profiles.^[29] Tissue-specific variations further highlight fibroblast heterogeneity, with dermal fibroblasts showing layered distributions (e.g., papillary vs. reticular) tied to skin architecture, while cardiac fibroblasts express distinct markers like Tcf21 in resident populations to regulate epicardial-mesenchymal transitions and maintain homeostasis.^[30] Epigenetic mechanisms, particularly DNA methylation patterns, drive this diversity by establishing stable, heritable marks that differentiate fibroblast phenotypes, such as hypermethylation of loci associated with ECM genes in lung-derived subtypes.^[31]^[32] Functionally, these subtypes display differential responses to stimuli like transforming growth factor-β (TGF-β), with matrix fibroblasts showing robust activation of ECM synthesis pathways via TGF-β1, whereas inflammatory subtypes exhibit varied sensitivity to TGF-β isoforms, influencing cytokine output and immune modulation without uniform myofibroblast differentiation.^[33]^[34] This subtype-specific responsiveness ensures adaptive tissue responses while linking back to developmental lineages established embryonically.

Physiological Functions

Extracellular Matrix Synthesis

Fibroblasts are the primary cellular source of extracellular matrix (ECM) components in connective tissues, synthesizing fibrillar collagens such as types I, III, and V to provide structural integrity during tissue homeostasis. Type I collagen, the most abundant, forms strong fibrils that confer tensile strength, while types III and V contribute to the elasticity and nucleation of fibrils, respectively. The synthesis begins intracellularly in the rough endoplasmic reticulum, where procollagen chains undergo post-translational modifications including hydroxylation of proline and lysine residues, glycosylation, and assembly into a triple helical procollagen molecule. Upon secretion, procollagen is processed extracellularly by procollagen peptidases, which cleave N- and C-terminal propeptides to yield mature tropocollagen, the basic unit of collagen fibrils.^[35] The stability of these collagen fibrils is enhanced through cross-linking mediated by lysyl oxidase (LOX), a copper-dependent enzyme secreted by fibroblasts that oxidizes specific lysine and hydroxylysine residues, forming covalent bonds between adjacent tropocollagen molecules. This enzymatic cross-linking is essential for the mechanical properties of the ECM, preventing fibril slippage and ensuring long-term tissue resilience in steady-state conditions. In addition to collagens, fibroblasts produce proteoglycans like decorin, a small leucine-rich proteoglycan that binds to collagen fibrils to regulate their diameter and assembly, and glycoproteins such as fibronectin, which facilitates cell adhesion and organizes the provisional matrix by linking collagens to integrins on the cell surface. These components interact to form a functional ECM network that supports tissue architecture.^[36]^[37]^[38] ECM synthesis in fibroblasts is tightly regulated by growth factors, including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), which modulate gene expression and protein production to maintain homeostasis. PDGF stimulates fibroblast proliferation and enhances the transcription of collagen and proteoglycan genes via receptor tyrosine kinase signaling, ensuring balanced matrix deposition. Similarly, FGF isoforms, such as FGF-2, promote ECM assembly by influencing collagen expression and inhibiting excessive degradation, thereby supporting steady-state tissue maintenance. Enzymatic remodeling of the ECM is achieved through matrix metalloproteinases (MMPs), zinc-dependent endopeptidases produced by fibroblasts that selectively degrade collagen and other matrix components to facilitate turnover and prevent accumulation. For instance, MMP-1 (collagenase-1) initiates the cleavage of fibrillar collagens, while MMP-2 and MMP-3 target non-fibrillar elements, with their activity balanced by tissue inhibitors of metalloproteinases (TIMPs) to preserve ECM integrity.^[39]^[40]^[41] In steady-state conditions, fibroblasts dedicate a significant portion of their protein synthetic capacity to ECM production, underscoring their role in maintaining connective tissue volume and function. Variations in ECM output among fibroblast subtypes, such as dermal versus tendon-derived cells, reflect tissue-specific demands but do not alter the core biosynthetic pathways.^[42]

Wound Healing and Tissue Remodeling

Fibroblasts play a pivotal role in the proliferative phase of wound healing, where they migrate into the injury site and contribute to the formation of granulation tissue, a provisional matrix that supports tissue regeneration. During this phase, fibroblasts proliferate and deposit extracellular matrix components, such as collagen and fibronectin, to stabilize the wound bed and facilitate angiogenesis. This process begins shortly after the inflammatory phase, with fibroblasts originating from adjacent dermis or circulating precursors responding to chemotactic signals to fill the defect.^[43]^[44]^[45] A key aspect of fibroblast function in wound closure involves their differentiation into myofibroblasts, specialized cells that express α-smooth muscle actin (α-SMA) and generate contractile forces through actin-myosin interactions. This differentiation is triggered by mechanical tension and growth factors, enabling myofibroblasts to pull wound edges together via stress fibers, thereby accelerating closure and reducing the size of the open wound. The de novo expression of α-SMA enhances the contractile activity of these cells, which is essential for efficient repair in the proliferative and remodeling phases. Studies have shown that preventing wound closure mechanically increases α-SMA expression, underscoring the interplay between tension and differentiation.^[46]^[47]^[48]^[49] Cytokines such as interleukin-4 (IL-4) and transforming growth factor-β1 (TGF-β1) orchestrate fibroblast migration and proliferation during healing. TGF-β1 promotes fibroblast infiltration into the wound, stimulates their proliferation, and induces expression of genes for extracellular matrix production, thereby supporting granulation tissue development. Similarly, IL-4 enhances fibroblast proliferation, migration, and collagen synthesis, often in coordination with TGF-β signaling pathways to amplify repair responses. These cytokines are released by immune cells and keratinocytes, creating a microenvironment conducive to fibroblast activation.^[50]^[51]^[52] In the resolution phase of wound healing, myofibroblasts undergo apoptosis to prevent excessive matrix deposition and scarring, allowing the tissue to regain normal architecture. This programmed cell death is induced when mechanical tension decreases and the wound achieves sufficient tensile strength, typically after closure. Failure of this apoptotic process can lead to persistent myofibroblasts and abnormal repair, but in normal healing, it ensures timely termination of contraction and remodeling. Research demonstrates that factors like fibromodulin can selectively promote myofibroblast apoptosis post-closure, reducing scar formation.^[53] Experimental models, such as in vitro scratch assays, have been instrumental in quantifying fibroblast migration dynamics during wound healing. In these assays, a confluent monolayer is scratched to simulate a wound, and fibroblast closure rates are measured, typically ranging from 10-20 μm/hour under standard conditions, reflecting their chemotactic response to growth factors. These models highlight how environmental cues influence migration speed and provide insights into therapeutic modulation of repair processes.^[54]^[55]^[56]

Pathological Roles

Inflammation and Fibrosis

Fibroblasts become activated in response to pro-fibrotic signals, particularly through the transforming growth factor-β (TGF-β) signaling pathway, which drives their differentiation into myofibroblasts and sustains excessive extracellular matrix (ECM) deposition.^[57] This pathway involves TGF-β binding to its receptors, leading to Smad-dependent transcription that upregulates genes for ECM components like collagen and fibronectin, resulting in persistent fibrotic remodeling in chronic inflammatory conditions. Persistent TGF-β activation prevents myofibroblast apoptosis, thereby maintaining a cycle of ECM accumulation that contributes to tissue stiffness and organ dysfunction.^[58] In fibrosis, mechanisms extend beyond collagen I overproduction to include disruptions in basement membrane integrity and dysregulation of matricellular proteins, which modulate cell-ECM interactions and exacerbate pathological remodeling.^[59] Basement membrane alterations, such as fragmentation of collagen IV and laminin networks, impair epithelial barrier function and facilitate fibroblast invasion into alveolar spaces, as observed in lung fibrosis models.^[60] Matricellular proteins like periostin and osteopontin, secreted by activated fibroblasts, further promote ECM cross-linking and inflammatory signaling, amplifying fibrotic progression in a feed-forward manner.^[59] Fibroblasts engage in bidirectional crosstalk with immune cells during inflammation, secreting chemokines to recruit immune cells and enhance pro-fibrotic responses.^[61] This recruitment sustains a local inflammatory milieu, where macrophages in turn release additional TGF-β to reinforce fibroblast activation.^[62] Additionally, fibroblast-derived interleukin-6 (IL-6) amplifies inflammation by promoting T-cell differentiation and macrophage survival, creating a self-perpetuating loop that bridges acute responses to chronic fibrosis.^[63] A hallmark example of fibroblast involvement in pathological fibrosis is idiopathic pulmonary fibrosis (IPF), where aggregates of proliferating fibroblasts known as fibroblast foci represent sites of active ECM deposition and alveolar collapse.^[64] These foci correlate with disease severity and progression, serving as histological markers of dysregulated repair in the lung interstitium.^[65] Therapeutic strategies targeting fibroblast activation in fibrosis increasingly focus on anti-TGF-β inhibitors, with several agents advancing in clinical trials post-2023 to mitigate ECM overproduction.^[66] For instance, bexotegrast, an integrin-based TGF-β inhibitor, showed promising safety and reductions in fibrotic biomarkers in IPF patients during phase IIa trials in 2024, but development was discontinued in 2025 due to safety concerns in phase 2b.^[66]^[67] Isoform-selective inhibitors, such as those targeting TGF-β3, are also under evaluation for systemic sclerosis-associated fibrosis, aiming to block pro-fibrotic signaling while sparing anti-inflammatory TGF-β isoforms.^[68]

Cancer and Tumor Microenvironment

Cancer-associated fibroblasts (CAFs) primarily originate from resident fibroblasts within the tumor stroma, which become activated in response to tumor-derived signals such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), epidermal growth factor (EGF), and hypoxia-induced factors.^[69] These resident cells, often quiescent, transdifferentiate into activated CAFs through pathways involving PDGF signaling, particularly in pancreatic and hepatic stellate cells converting to myofibroblast-like states.^[69] Additional origins include epithelial cells via epithelial-mesenchymal transition (EMT), endothelial cells via endothelial-mesenchymal transition (EndMT), bone marrow-derived mesenchymal stem cells, adipocytes, and monocytes, though local precursors predominate over circulating sources.^[69] In the tumor microenvironment, CAFs promote cancer progression by stiffening the extracellular matrix (ECM) through excessive synthesis of collagens (types I, III, IV, and V) and hyaluronic acid, which fosters a fibrotic barrier that enhances tumor cell invasion and metastasis while impeding drug penetration and immune cell infiltration.^[69] They further support tumor growth by secreting hepatocyte growth factor (HGF), which stimulates tumor cell proliferation and motility, and vascular endothelial growth factor (VEGF), which induces angiogenesis to sustain nutrient supply to hypoxic tumor regions.^[69] These functions are mediated by signaling cascades like IL-17a/JAK2/STAT3, contributing to stromal remodeling that favors malignancy.^[69] CAFs exhibit functional diversity, with prominent subtypes including inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), as delineated in recent reviews.^[70] iCAFs, characterized by high interleukin-1 receptor expression and low α-smooth muscle actin (α-SMA), are typically located distant from tumor cells and secrete pro-inflammatory cytokines such as CXCL-1 and IL-6 via JAK/STAT and NF-κB pathways, recruiting myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells to suppress antitumor immunity.^[70] In contrast, myCAFs express high α-SMA, reside proximal to tumor cells, and respond to TGF-β by producing dense ECM components like collagen, forming physical barriers that promote therapeutic resistance and correlate with poor prognosis, though their depletion can sometimes accelerate metastasis.^[70] Single-cell RNA sequencing (scRNA-seq) studies have revealed extensive CAF heterogeneity and plasticity across cancer types, identifying up to nine subtypes such as matrix CAFs (mCAFs), inflammatory CAFs (iCAFs), and tumor-like CAFs (tCAFs) in breast, lung, pancreatic, and other tumors, with phenotypes forming a continuum rather than discrete states.^[71] This plasticity enables CAFs to transition between subtypes in response to microenvironmental cues, influencing tumor progression and therapy response.^[71] Prognostic markers like fibroblast activation protein (FAP) expression, which labels myofibroblast-like CAFs involved in ECM remodeling, are associated with aggressive disease and chemoresistance, while CD10 (MME) in tCAFs predicts poor outcomes in non-small cell lung cancer.^[71] Therapeutic strategies targeting CAFs, particularly FAP-expressing populations, include inhibitors, antibodies, CAR-T cells, and radioligands, with several in ongoing clinical trials as of 2025.^[72] For instance, 177Lu-FAPI radioligand therapy has demonstrated an 83% disease control rate in phase I trials for thyroid and other adenocarcinomas, showing safety and efficacy in reducing tumor burden.^[72] Other agents like talabostat and BXCL701 are being evaluated in phase II trials for colorectal, pancreatic, and prostate cancers, often combined with immunotherapy such as pembrolizumab to overcome resistance, though challenges like CAF heterogeneity and suboptimal monotherapy efficacy persist.^[72] No FAP inhibitor has achieved full approval, but combination approaches hold promise for modulating the tumor stroma.^[72]

Applications in Research and Medicine

Feeder Cells in Cell Culture

The use of fibroblasts as feeder cells in cell culture originated with the establishment of mouse embryonic stem (ES) cell lines, where mouse embryonic fibroblasts (MEFs) were employed as supportive layers to maintain pluripotency. In 1981, Evans and Kaufman derived pluripotential cells from mouse embryos by culturing them on MEF feeder layers, enabling indefinite propagation without differentiation.^[73] This approach has since become foundational for ES cell and induced pluripotent stem (iPS) cell maintenance, particularly in protocols involving reprogramming.^[74] MEFs support ES cell pluripotency through both soluble and contact-dependent mechanisms. They secrete extracellular factors, including leukemia inhibitory factor (LIF), which activates the JAK-STAT3 pathway to promote self-renewal and inhibit differentiation.^[75] Additional secreted factors such as fibroblast growth factor 2 (FGF2), Gremlin 1, and activins contribute to this maintenance.^[76] Contact-dependent signaling involves membrane-bound ligands that facilitate juxtacrine interactions, such as those mediated by integrins and Notch receptors, alongside extracellular matrix components like hyaluronic acid that enhance cell adhesion and prevent differentiation.^[76] To prepare MEF feeder layers, primary fibroblasts are isolated from day 13-14 mouse embryos and expanded in culture before inactivation to halt their proliferation while preserving supportive functions. Mitomycin C treatment is a standard method, typically at 10 μg/mL for 2-3 hours, which cross-links DNA and arrests cell division without disrupting secretory or adhesive capabilities.^[77] Gamma-irradiation serves as an alternative inactivation approach. Inactivated MEFs are then plated at densities of approximately 2-5 × 10^4 cells/cm² on gelatin-coated surfaces to form a confluent monolayer for seeding ES cells.^[77] A key advantage of MEF feeder layers is their cost-effectiveness in providing a natural microenvironment that sustains ES cell pluripotency over multiple passages, often outperforming feeder-free conditions in maintaining undifferentiated morphology and colony formation.^[78] They eliminate the need for complex synthetic matrices or high concentrations of recombinant growth factors, making them accessible for laboratory-scale research.^[77] However, MEFs pose limitations due to their xenogeneic origin, raising risks of immunogenic responses, viral contamination, and species-specific pathogen transmission in human applications.^[78] These concerns have driven the development of human-derived alternatives, such as foreskin or endometrial fibroblasts, which support ES and iPS cell growth under xeno-free conditions while minimizing contamination risks.^[79]

Therapeutic Targeting and Engineering

Fibroblasts have emerged as key targets for therapeutic interventions due to their central roles in tissue homeostasis, fibrosis, and cancer progression. Strategies to engineer or modulate fibroblast function aim to restore regenerative capacity, reverse pathological states, and enhance treatment outcomes in various diseases. These approaches leverage genetic editing, chemical modulation, and biomaterial scaffolds to reprogram or deplete aberrant fibroblasts, offering potential for personalized medicine. Recent advances emphasize precision targeting to minimize off-target effects while maximizing therapeutic efficacy. CRISPR-based editing has facilitated the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) or other lineages, bypassing traditional viral methods and reducing integration risks. For instance, genome-scale CRISPR-Cas9 screens have identified barriers to human pluripotency, enabling efficient derivation of iPSCs from somatic cells like fibroblasts by targeting chromatin regulators such as USP22.^[80] Advances in scaffold-based CRISPR delivery, including non-viral systems using biocompatible hydrogels and nanofibers, have improved gene editing efficiency in three-dimensional environments that mimic extracellular matrix cues.^[81] These engineered fibroblasts hold promise for autologous cell therapies in regenerative medicine, with applications in generating patient-specific tissues for transplantation. Rejuvenation of senescent fibroblasts represents a promising anti-aging strategy, particularly through small-molecule interventions that reverse age-related dysfunction without full dedifferentiation. Partial chemical reprogramming using cocktails of small molecules, such as the 7c formulation, has rejuvenated mouse fibroblasts by improving mitochondrial function, reducing aging metabolites, and resetting transcriptomic and epigenomic clocks, distinct from transcription factor-based methods.^[82] In human dermal fibroblasts, partial reprogramming using Yamanaka factors has reversed mesenchymal drift associated with aging, restoring youthful gene expression profiles and enhancing proliferative capacity, as demonstrated in 2025 research.^[83] These interventions, which avoid genetic alterations, offer safer in vivo applicability for treating age-related tissue decline and improving wound healing outcomes. In cancer, targeting cancer-associated fibroblasts (CAFs) via depletion therapies seeks to disrupt the tumor-supportive microenvironment. Bispecific antibodies directed against CAF markers like fibroblast activation protein (FAP) have entered early clinical trials by 2025, aiming to selectively eliminate CAFs while sparing healthy fibroblasts and enhancing immune infiltration. For example, FAP-targeted immunotherapies, including bispecific constructs linking tumor antigens to immune effectors, have shown preclinical efficacy in reducing CAF-mediated drug resistance in solid tumors.^[84] Ongoing phase I/II trials evaluate these agents in combination with checkpoint inhibitors, reporting preliminary tolerability and tumor regression in subsets of patients with various solid tumors, including pancreatic and breast cancers, as of mid-2025. Regenerative applications utilize fibroblast sheets to accelerate wound closure and tissue integration, particularly in burn injuries where donor sites are limited. Autologous fibroblast sheets, expanded via micrografting techniques like modified MEEK, have been clinically applied to cover extensive burns, promoting vascularization and reducing contraction compared to traditional split-thickness grafts. Cell transfer-generated skin grafts incorporating fibroblasts have demonstrated improved healing in porcine burn models and early human trials.^[85] These sheet-based therapies, often combined with dermal scaffolds, have achieved over 80% graft take rates in severe burn patients, minimizing infection risks and hospitalization duration. Recent advances in anti-fibrotic therapies focus on inhibiting mechanotransduction pathways in activated fibroblasts to halt excessive extracellular matrix deposition. According to a 2023 comprehensive review, targeting YAP/TAZ signaling or Piezo1 channels disrupts fibroblast-to-myofibroblast differentiation in response to stiff matrices, as seen in idiopathic pulmonary fibrosis where ECM stiffness can reach 10-30 kPa or higher.^[86] Small-molecule inhibitors of RhoA/ROCK and TGFβ/Smad pathways, such as losartan, have shown efficacy in preclinical models of renal and hepatic fibrosis, with phase II trials confirming reduced collagen synthesis. These mechanosensitive targets offer disease-modifying potential, with emerging combinations integrating inhibitors to enhance resolution in chronic fibrotic conditions.

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### Summary of MEF Feeder Cell Preparation and Inactivation
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