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Journal of Visualized Experiments : JoVE Oct 2013The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While...
The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.
Topics: Actins; Animals; Colon; Cytological Techniques; Humans; Mice; Myofibroblasts; Vimentin
PubMed: 24145735
DOI: 10.3791/50611 -
Monographs in Clinical Cytology 2017
Topics: Cell Proliferation; Diagnosis, Differential; Humans; Immunophenotyping; Myofibroblasts; Neoplasm Grading; Neoplasms, Fibrous Tissue
PubMed: 28750378
DOI: 10.1159/000475094 -
Biology Open Mar 2020Mechanical force is a fundamental regulator of cell phenotype. Myofibroblasts are central mediators of fibrosis, a major unmet clinical need characterised by the...
Mechanical force is a fundamental regulator of cell phenotype. Myofibroblasts are central mediators of fibrosis, a major unmet clinical need characterised by the deposition of excessive matrix proteins. Traction forces of myofibroblasts play a key role in remodelling the matrix and modulate the activities of embedded stromal cells. Here, we employ a combination of unsupervised computational analysis, cytoskeletal profiling and single cell traction force microscopy as a functional readout to uncover how the complex spatiotemporal dynamics and mechanics of living human myofibroblast shape sub-cellular profiling of traction forces in fibrosis. We resolve distinct biophysical communities of myofibroblasts, and our results provide a new paradigm for studying functional heterogeneity in human stromal cells.
Topics: Biomarkers; Biomechanical Phenomena; Biophysical Phenomena; Cells, Cultured; Cytoskeleton; Fluorescent Antibody Technique; Humans; Molecular Imaging; Myofibroblasts; Single-Cell Analysis
PubMed: 32139395
DOI: 10.1242/bio.049809 -
The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.PloS One 2013Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development...
Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.
Topics: Actins; Animals; Biomechanical Phenomena; Calcium Signaling; Cell Adhesion; Extracellular Matrix; Intracellular Space; Mechanical Phenomena; Myofibroblasts; Rats; Stress, Mechanical
PubMed: 23691248
DOI: 10.1371/journal.pone.0064560 -
Biochimica Et Biophysica Acta Jul 2013Myofibroblasts combine the matrix-producing functions of fibroblasts and the contractile properties of smooth muscle cells. They are the main effectors of fibrosis in...
Myofibroblasts combine the matrix-producing functions of fibroblasts and the contractile properties of smooth muscle cells. They are the main effectors of fibrosis in all tissues and make a major contribution to other aspects of the wound healing response, including regeneration and angiogenesis. They display the de novo expression of α-smooth muscle actin. Myofibroblasts, which are absent from the normal liver, are derived from two major sources: hepatic stellate cells (HSCs) and portal mesenchymal cells in the injured liver. Reliable markers for distinguishing between the two subpopulations at the myofibroblast stage are currently lacking, but there is evidence to suggest that both myofibroblast cell types, each exposed to a particular microenvironment (e.g. hypoxia for HSC-MFs, ductular reaction for portal mesenchymal cell-derived myofibroblasts (PMFs)), expand and exert specialist functions, in scarring and inflammation for PMFs, and in vasoregulation and hepatocellular healing for HSC-MFs. Angiogenesis is a major mechanism by which myofibroblasts contribute to the progression of fibrosis in liver disease. It has been clearly demonstrated that liver fibrosis can regress, and this process involves a deactivation of myofibroblasts, although probably not to a fully quiescent phenotype. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
Topics: Hepatic Stellate Cells; Humans; Liver Cirrhosis; Liver Diseases; Myofibroblasts
PubMed: 23470555
DOI: 10.1016/j.bbadis.2013.02.019 -
The Journal of Experimental Medicine Sep 2021Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1)...
Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.
Topics: Adult; Aged; Animals; Case-Control Studies; Cell Differentiation; Cytoskeleton; Female; Gene Expression Regulation; Homeodomain Proteins; Humans; Male; Mice, Knockout; Middle Aged; Myofibroblasts; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Young Adult; rho-Associated Kinases; Mice
PubMed: 34259830
DOI: 10.1084/jem.20201916 -
Physiology (Bethesda, Md.) Jan 2019Fibrosis is a dynamic process with the potential for reversibility and restoration of near-normal tissue architecture and organ function. Herein, we review mechanisms... (Review)
Review
Fibrosis is a dynamic process with the potential for reversibility and restoration of near-normal tissue architecture and organ function. Herein, we review mechanisms for resolution of organ fibrosis, in particular that involving the lung, with an emphasis on the critical roles of myofibroblast apoptosis and clearance of deposited matrix.
Topics: Animals; Apoptosis; Extracellular Matrix; Fibrosis; Humans; Lung; Myofibroblasts
PubMed: 30540232
DOI: 10.1152/physiol.00033.2018 -
Genomics Sep 2023The matricellular protein, follistatin-like 1 (FSTL1), regulates lung development and saccular formation. Here, we employed single-cell RNA sequencing (scRNA-seq) to...
The matricellular protein, follistatin-like 1 (FSTL1), regulates lung development and saccular formation. Here, we employed single-cell RNA sequencing (scRNA-seq) to construct a transcriptomic atlas of 22,774 individual cells from wild-type (WT) and Fstl1 lung (E18.5) samples and identified 27 cell subtypes. We observed abnormal population sizes and gene expression profiles in diverse cell subtypes in Fstl1 lung samples. We identified Pdgfra and Tgfbi as genetic markers specifically expressed in postnatal myofibroblasts (MyoFBs). Fstl1 deletion decreased the number of MyoFB cells and downregulated their roles in ECM organization and muscle tissue/vasculature development, partly through the TGF-β1/BMP4 signaling pathway. Our data provide a single-cell view of the cellular heterogeneity and the molecular mechanisms underlying abnormal saccular formation and atelectatic lungs in Fstl1 mice.
Topics: Animals; Mice; Follistatin-Related Proteins; Lung; Myofibroblasts; Signal Transduction; Single-Cell Gene Expression Analysis
PubMed: 37406975
DOI: 10.1016/j.ygeno.2023.110677 -
Journal of Thoracic Oncology : Official... May 2014Cancer-associated stromal cells interact with carcinoma cells and thus participate in tumor growth. Our aim was to characterize the ultrastructure and contractile...
BACKGROUND
Cancer-associated stromal cells interact with carcinoma cells and thus participate in tumor growth. Our aim was to characterize the ultrastructure and contractile properties of stromal cells in collagen gel cultured from lung cancer of various histological types and from tumor-free lung.
METHODS
Cells cultured from lung cancer (13 adenocarcinomas, six squamous cell carcinomas, one adenosquamous carcinoma, and one pleomorphic carcinoma) and tumor-free lung were analyzed by transmission electron microscopy and three-dimensional collagen gel contraction assays. The expression of α-smooth muscle actin (α-SMA), a recognized myofibroblast marker, was examined by immunoelectron microscopy from individual cells and by Western blotting from the whole cultured cell population.
RESULTS
According to their ultrastructure, the cell lines were composed of fibroblastic and myofibroblastic cells. In electron microscopy, cells of lung cancer exhibited more myofibroblastic features displaying higher amounts of actin belts (p = 0.057) and α-SMA labeling (p = 0.010) than cells from tumor-free lung. Myofibroblasts cultured from lung cancer of smokers expressed less α-SMA labeling (p = 0.013) than counterparts from nonsmokers. Western blotting revealed that cancer-associated fibroblasts expressed more α-SMA (p = 0.006) than cells from tumor-free lung, whereas cells from tumor-free central lung of smokers showed less α-SMA (p = 0.039) than counterparts from nonsmokers. Cells cultured from cancer contracted more in collagen gel than those from tumor-free lung. The contractile capacity in collagen gel correlated with the frequency of extracellular component of fibronexus by transmission electron microscopy.
CONCLUSIONS
Lung cancer-associated myofibroblasts are different both ultrastructurally and functionally when compared with cells cultured from tumor-free lung. Smoking altered myofibroblastic phenotype, regardless of their origin.
Topics: Actins; Adenocarcinoma; Adherens Junctions; Aged; Aged, 80 and over; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Endoplasmic Reticulum, Rough; Extracellular Matrix; Female; Humans; Lung; Lung Neoplasms; Male; Microscopy, Electron, Transmission; Middle Aged; Myofibroblasts; Smoking; Tumor Cells, Cultured
PubMed: 24662457
DOI: 10.1097/JTO.0000000000000149 -
Scientific Reports Oct 2021Elevated blood serotonin levels have been observed in patients with heart failure and serotonin has a role in pathological cardiac function. The serotonin receptor...
Elevated blood serotonin levels have been observed in patients with heart failure and serotonin has a role in pathological cardiac function. The serotonin receptor system was examined in adult rat isolated cardiac fibroblast and myofibroblast cells. This is one of the first studies that has investigated serotonin receptors and other proteins involved in the serotonin receptor system in rat cardiac fibroblast and myofibroblast cells. Rat primary cardiac fibroblasts were isolated and transformed into myofibroblasts using 5 ng/ml TGF-β1. Transformation of cells to myofibroblasts was confirmed with the presence of α-smooth muscle actin using Western blot. Serotonin metabolism and receptor protein expression was assessed using Western blot techniques and serotonin levels measured using ELISA. The 5-HT, 5-HT and 5-HT receptors were found to be present in both rat cardiac fibroblasts and myofibroblast cells, however no significance in protein expression between the two cell types was found (P > 0.05). In this study a significant increase in the serotonin transporter (SERT), tryptophan hydroxylase 1 and extracellular serotonin levels was observed in rat cardiac myofibroblasts when compared to fibroblasts (P < 0.05). These results suggest that serotonin levels may rise in parallel with cardiac myofibroblast populations and contribute to the pathogenesis of heart failure via serotonin receptors.
Topics: Animals; Cells, Cultured; Heart Failure; Male; Myocardium; Myofibroblasts; Rats; Rats, Wistar; Receptors, Serotonin; Serotonin
PubMed: 34645867
DOI: 10.1038/s41598-021-99632-y