Fibroblast Cell Markers

What are Fibroblasts?

Fibroblasts are cells of mesenchymal origin that are most commonly found in connective tissue. They serve to synthesize components of the extracellular matrix (ECM) and stroma, hence fibroblasts play an important role in cell maintenance and structural homeostasis. Connective tissue structural integrity is sustained by fibroblast secretion of ECM precursors, such as collagen I.

Additionally, fibroblasts are chemotactic and migrate in response to chemokine, cytokine, and growth factor secretion where they function in wound healing and tissue repair and remodeling. While the activation and proliferation of fibroblasts is essential for wound healing, dysregulation of this process is associated with multiple disease pathologies including fibrosis, cancer, and inflammation.

Morphologically, fibroblasts can be quite heterogenous depending on the tissue origin and physiological conditions, but they are typically characterized by a flattened, spindle-like shape, lack of basement membrane, and multiple extended cellular processes.

Bright-field microscopy image of verified fibroblast morphology from cells generated from skin-punch biopsy under 4x (left) and 20x (right) magnification. Image collected and cropped by from the following publication (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043099), licensed under a CC0 1.0 license.

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Fibroblast Cell Markers

Fibroblasts are regarded as one of the more difficult cell types to identify given their heterogeneity. In addition to their location and typical spindle-like shape, fibroblasts have traditionally been identified through positive expression of the mesenchymal markers vimentin and PDGFR alpha. Since the late 1980s a few reported anti-fibroblast antibodies have been identified including TE-7, an antibody against thymic stroma, and 1B10, which is shown to react with human fibroblasts and cell lines, tissue macrophages, and peripheral blood monocytes.

While additional fibroblast cell markers have been identified, none are unique to fibroblasts. Many of these markers are found in other ECM-producing cells, epithelial cells, immune cells, and neurons, but are more predominantly expressed in fibroblasts. Certain cell markers are also differentially expressed, dependent upon subtype, tissue, or organ. For instance, FAP and alpha-Smooth Muscle Actin (SMA) are more highly expressed in activated fibroblasts. Using multiple cell markers including negative, or exclusion, markers will enable more accurate identification and characterization of fibroblasts.

Immunocytochemical analysis of human primary cardiac fibroblasts stained with Rabbit Anti-Vimentin Polyclonal Antibody (Catalog # NBP1-31327] (green) and Smooth Muscle Actin Antibody (red). Image from verified customer review.

Fibroblast Activation Protein alpha/FAP detected in WI-38 human lung fibroblast cell line by flow cytometry. WI 38 fibroblasts stained with either Mouse Anti-Fibroblast Activation Protein alpha/FAP Monoclonal Antibody (Catalog # MAB3715) (orange histogram) or Mouse IgG1 Isotype Control Antibody (Catalog # MAB002) (open histogram), followed by Phycoerythrin-conjugated Anti-IgG Secondary Antibody (Catalog # F0102B).

Cell markers for fibroblast characterization and analysis are used in several research applications including immunohistochemistry (IHC), immunocytochemistry (ICC)/immunofluorescence(IF), flow cytometry, and western blot. Additionally, RNAscope^® is a powerful tool allowing for visualization of fibroblast marker gene expression in situ.

Common Fibroblast Markers:

*Marker is predominantly for activated fibroblasts rather than mature, quiescent fibroblasts.

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Genetic Strategies Validation. Western blot depicting lysates of HeLa human cervical epithelial carcinoma parental cell line and S100A4 knockout (KO) HeLa cell line. The membrane was probed with Mouse Anti-S100A4 Monoclonal Antibody (Catalog # NBP2-36431) followed by HRP-conjugated Anti-IgG Secondary Antibody (Catalog #HAF008). A specific band for S100A4 is detected at ~12 kDa in the parental cell line, but not in the KO cell line.

HeLa cell line expressing Protein Disulfide Isomerase/P4HB detected using Mouse Anti-Protein Disulfide Isomerase/P4HB Monoclonal Antibody (Catalog # MAB4236). Cells were stained with Northern¬Lights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # NL007) (red) and counterstained with DAPI (blue). Specific P4HB staining is localized to the cytoplasm.

Simple Western lane view analysis of Integrin beta 1/CD29 in lysates from L 929 mouse fibroblast cell line, NIH 3T3 mouse embryonic fibroblast cell line, PC 12 rat adrenal pheochromocytoma cell line, and Rat 2 rat embryonic fibroblast cell line. Samples were probed with Goat Anti-Integrin beta 1/CD29 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2405) followed by HRP-conjugated Anti-IgG Secondary Antibody (Catalog # HAF109) and a specific band was detected at ~153-174 kDa. Lysates were run under reducing conditions and using the 12-230 kDa system.

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Fibroblast Cell Origin & Plasticity

The heterogeneity observed in fibroblasts function and lack of unique cellular markers is partially attributed to their multiple developmental origins. Primary fibroblasts arise from the primary mesenchyme during gastrulation. Further along in development, true mesenchyme generated from the mesoderm gives rise to mature, resident fibroblasts. Some studies have also suggested a hematopoietic stem cell origin for fibroblasts.

Besides resident fibroblasts, the mesoderm also contributes to the development of other cell types including mesenchymal stem cells, epithelial cells, endothelial cells, adipocytes, and fibrocytes. The common embryonic origin with other cells of mesenchymal lineage contributes to cellular plasticity amongst these populations. This plasticity between fibroblasts and other cell types is generally observed in the context of injury, inflammation, and cancer or other pathologies. Epithelial cells can convert to fibroblasts through epithelial-to-mesenchymal transition (EMT), which is a hallmark of cancer. Furthermore, during wound healing, adipocytes have been shown to lose their lipid stores, migrate to the wound, and become fibroblasts. Overall, fibroblast plasticity is highest during embryonic and early development and decreases with aging.

Fibroblast Cell Origin and Lineage Plasticity

Fibroblasts may be derived from a number of cell types through a variety of mechanisms including division and proliferation, transdifferentiation such as EMT or EndoMT, differentiation of precursors cells like mesenchymal stem cells or hematopoietic stem cells, and de-differentiation of mature cell types.

Fibroblasts may originate from several cell types, including:

• Resident fibroblasts via proliferation and cell division
• Epithelial cells through EMT
• Endothelial cells through endothelial-to-mesenchymal transition (EndoMT)
• Mature cell types, such as adipocytes and smooth muscle cells, through de-differentiation
• Hematopoietic or mesenchymal stem cells via differentiation

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Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are a subgroup of activated fibroblasts that are found within the tumor microenvironment (TME). CAFs contribute to cancer progression, metastasis, and immune cell reprogramming by secretion of growth factors, cytokines and chemokines, interleukins, matrix metalloproteases (MMPs), and ECM deposition.

Several mechanisms have been shown to activate normal, resident fibroblasts to become CAFs including:

• Contact with cancer cells
• Changes or remodeling in the ECM causing stromal stiffness
• DNA damaging chemotherapy, radiotherapy, or targeted therapy
• Physiological stress and reactive oxygen species (ROS) production
• Growth factor signaling and receptor tyrosine kinase ligand expression such as TGF-β, HGF, PDGF, and FGF
• Exposure to inflammatory signals including IL-1β, IL-6, and TNFα

While CAFs continue to express a number of common fibroblast biomarkers such as alpha-SMA, FSP1 (S100A4), and FAP, they are often upregulated, whereas PDGFR alpha is reduced. CAFs have also been shown to originate from and express markers from cells of other origins. For example, CAFs of endothelial cell origin express Podoplanin, of lymphocyte or dendritic cell origin express CD70, and of neutrophil origin express GPR77.

Furthermore, CAFs can be grouped into different subsets based on their biomarker expression.

*CAFs expressing neutral biomarkers can be subdivided into two groups – myofibroblastic CAFs (myCAFs) (alpha-SMA^highIL-6^low) or inflammatory CAFs (iCAFs) (alpha-SMA^lowIL-6^high). Similar to resident fibroblasts, heterogeneity persists within the CAF subgroup.

Collagen-binding protein HSP47 detection in sections of human normal breast tissue section (left) and cancerous breast tissue section (right) using Mouse Anti-HSP47 Monoclonal Antibody (Catalog # MAB9166). Tissue was stained with Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS002) (brown) and counterstained with hematoxylin (blue). Specific staining was appropriately localized to the cytoplasm of the breast cancer cell tissue, as HSP47 is associated with tumor growth and metastasis.

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Practical Applications of Fibroblasts

Fibroblasts are commonly cultured in the laboratory, both in primary fibroblast cell cultures and transformed cell lines. Given their accessibility, fibroblasts are often used in regenerative medicine. Fibroblasts are easily obtained from donor skin or hair biopsies and can be used as a direct source for cell therapy or easily reprogrammed into induced pluripotent stem cell (iPSCs). Subsequently, iPSCs can be used directly or can be genetically engineered, and differentiated into mature, tissue-specific cells for disease modeling.

Learn More About the Applications of iPSCs

Cell Culture

Commonly, fibroblast cell lines are used as a feeder monolayer to support the survival and growth of other cell types. In general, feeder cells are adherent cells locked in a state of growth arrest that secrete growth factors into the media and provide cell-cell contact. Mouse embryonic fibroblasts (MEFs) and human dermal fibroblasts (HDFs) are two common types of feeder cells that support the culture of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

BG01V Human Embryonic Stem Cells (hESCs) Cultured on Irradiated Mouse Embryonic Fibroblasts (MEFs) Demonstrate a Phenotype that is Consistent with Pluripotency. A. BG01V hESCs colonies cultured on Irradiated MEFs (R&D Systems Catalog # PSC001) were analyzed for pluripotency markers. B. Flow cytometry was used to analyze SSEA-4 expression in cells with PE-conjugated Mouse Anti-SSEA-4 Monoclonal Antibody (Catalog # FAB1435P) (filled histogram) or a PE-conjugated Mouse IgG3 Isotype Control Antibody (Catalog # IC007P) (open histogram). C. & D. BG01V hESC colonies were probed with Anti-Oct-3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) or Anti-Nanog Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1997). Following the primary antibody, cells were stained using NorthernLights™ 557-conjugated Anti-IgG Secondary Antibody (Catalog # NL001). SSEA-4, Oct-3/4, and Nanog expression is indicative of pluripotency.

Regenerative Medicine & Tissue Engineering

The source of fibroblasts in regenerative medicine can be autologous (from the self) or allogeneic (from a donor). While use of autologous cells limits the risk of graft rejection, the process often is more time consuming and requires large-scale cell expansion, whereas harvested allogeneic cells can be banked, cryopreserved, and ready-to-use to treat multiple patients.

In addition to iPSC-based regenerative medicine, dermal fibroblasts can be used in the clinic for wound repair in ulcers, burns, the treatment of fragility skin disorders, and reconstructive surgeries. Besides skin regeneration, fibroblasts can also be utilized for cardiac, liver, and bone and cartilage regeneration.

Gene-corrected autologous or allogeneic fibroblasts can be either directly injected into the patient or grown on a scaffold and engrafted for transplantation. In scaffold-based skin tissue engineering, fibroblasts are mounted onto a synthetic matrix with the addition of growth factors, small molecules, and mechanical stimuli. The scaffold creates a microenvironment conducive to skin organization and generation of an engineered graft.

Autologous fibroblast cell therapy for treatment of skin disorders. A skin biopsy is taken from the patient with a skin disorder (1), fibroblasts are isolated and characterized from the sample (2), and gene corrected (3). The fibroblast population is expanded (4) and can either be directly injected into the patient wound or transplanted via a skin graft from fibroblasts seeded onto a scaffold (5).

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Select References

Chang, Y., Li, H., & Guo, Z. (2014). Mesenchymal stem cell-like properties in fibroblasts. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 34(3), 703–714. https://doi.org/10.1159/000363035

Costa-Almeida, R., Soares, R., & Granja, P. L. (2018). Fibroblasts as maestros orchestrating tissue regeneration. Journal of tissue engineering and regenerative medicine, 12(1), 240–251. https://doi.org/10.1002/term.2405

Fernandes, I. R., Russo, F. B., Pignatari, G. C., Evangelinellis, M. M., Tavolari, S., Muotri, A. R., & Beltrão-Braga, P. C. (2016). Fibroblast sources: Where can we get them?. Cytotechnology, 68(2), 223–228. https://doi.org/10.1007/s10616-014-9771-7

Goodpaster, T., Legesse-Miller, A., Hameed, M. R., Aisner, S. C., Randolph-Habecker, J., & Coller, H. A. (2008). An immunohistochemical method for identifying fibroblasts in formalin-fixed, paraffin-embedded tissue. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 56(4), 347–358. https://doi.org/10.1369/jhc.7A7287.2007

Han, C., Liu, T., & Yin, R. (2020). Biomarkers for cancer-associated fibroblasts. Biomarker research, 8(1), 64. https://doi.org/10.1186/s40364-020-00245-w

Llames, S., García-Pérez, E., Meana, Á., Larcher, F., & del Río, M. (2015). Feeder Layer Cell Actions and Applications. Tissue engineering. Part B, Reviews, 21(4), 345–353. https://doi.org/10.1089/ten.TEB.2014.0547

LeBleu, V. S., & Neilson, E. G. (2020). Origin and functional heterogeneity of fibroblasts. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(3), 3519–3536. https://doi.org/10.1096/fj.201903188R

Mascharak, S., desJardins-Park, H. E., & Longaker, M. T. (2020). Fibroblast Heterogeneity in Wound Healing: Hurdles to Clinical Translation. Trends in molecular medicine, 26(12), 1101–1106. https://doi.org/10.1016/j.molmed.2020.07.008

Nurmik, M., Ullmann, P., Rodriguez, F., Haan, S., & Letellier, E. (2020). In search of definitions: Cancer-associated fibroblasts and their markers. International journal of cancer, 146(4), 895–905. https://doi.org/10.1002/ijc.32193

Sahai, E., Astsaturov, I., Cukierman, E., DeNardo, D. G., Egeblad, M., Evans, R. M., Fearon, D., Greten, F. R., Hingorani, S. R., Hunter, T., Hynes, R. O., Jain, R. K., Janowitz, T., Jorgensen, C., Kimmelman, A. C., Kolonin, M. G., Maki, R. G., Powers, R. S., Puré, E., Ramirez, D. C., … Werb, Z. (2020). A framework for advancing our understanding of cancer-associated fibroblasts. Nature reviews. Cancer, 20(3), 174–186. https://doi.org/10.1038/s41568-019-0238-1

Shams, F., Rahimpour, A., Vahidnezhad, H., Hosseinzadeh, S., Moravvej, H., Kazemi, B., Rajabibazl, M., Abdollahimajd, F., & Uitto, J. (2021). The utility of dermal fibroblasts in treatment of skin disorders: A paradigm of recessive dystrophic epidermolysis bullosa. Dermatologic therapy, 34(4), e15028. https://doi.org/10.1111/dth.15028

Vapniarsky, N., Arzi, B., Hu, J. C., Nolta, J. A., & Athanasiou, K. A. (2015). Concise Review: Human Dermis as an Autologous Source of Stem Cells for Tissue Engineering and Regenerative Medicine. Stem cells translational medicine, 4(10), 1187–1198. https://doi.org/10.5966/sctm.2015-0084

Wong, T., McGrath, J. A., & Navsaria, H. (2007). The role of fibroblasts in tissue engineering and regeneration. The British journal of dermatology, 156(6), 1149–1155. https://doi.org/10.1111/j.1365-2133.2007.07914.x