Fibroblast Cell Markers

Fibroblast Cell Markers | Fibroblast Origin and Plasticity | Cancer-Associated Fibroblasts | Applications of Fibroblasts

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.

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 PDGFRα. 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 α-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.

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 visualization of fibroblast marker gene expression in situ.

Common Fibroblast Markers

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

Fibroblast Cell Origin and 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.

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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) (α-SMA^highIL-6^low) or inflammatory CAFs (iCAFs) (α-SMA^lowIL-6^high). Similar to resident fibroblasts, heterogeneity persists within the CAF subgroup.

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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.

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).