Fibroblast Markers

PDGFRα (CD140a)

PDGFRα is a transmembrane receptor tyrosine kinase that coordinates fibroblast proliferation, survival, and migration. Throughout tissue development and repair, PDGFRα facilitates ECM production by PI3K/Akt and MAPK signaling pathways. Although PDGFRα is expressed in other mesenchymal cells, this protein is a reliable marker for fibroblast identification in different tissues and its strong expression in fibroblast populations enables its isolation through flow cytometry.¹²

Vimentin

Vimentin is a type III intermediate filament protein that forms fibroblast structural networks. It keeps cell shape, fixes organelles, and enables fibroblast migration during wound healing. Although again this protein is found in mesenchymal cells, it still allows the detection of fibroblast morphology and behavior using immunofluorescence. Its reliable expression and function in mechanotransduction make it valuable in marker panels.²

S100A4

S100A4 is an intracellular calcium-binding protein that regulates the cell cycle and differentiation. Single-cell RNA sequencing has shown that S100A4-expressing fibroblasts form a distinct subpopulation in inflammatory conditions like fibrosis and cancer. It was first thought to be fibroblast-specific but is now known to be found in cells involved in inflammation and tissue remodeling. However, S100A4 is useful when combined with additional markers.^2,13

PDGFB

PDGFB is a growth factor, and, in tumors, it promotes fibroblast differentiation into a large population of cancer-associated fibroblasts (CAFs). PDGFB enhances ECM deposition involving a mechanosensitive pathway: it activates myosin light chain phosphorylation, leading to cell contraction and inducing the release of active TGFβ from the ECM. The released TGFβ is involved in further signaling pathways. PDGFB signals by PDGFRα and PDGFRβ receptors facilitating fibroblast expansion—PDGFB loss results in reduced CAF recruitment and ECM deposition, demonstrating its importance in tissue remodeling.¹⁴

Papillary Fibroblasts

Papillary fibroblasts contribute to skin homeostasis and regeneration in the superficial dermis under the dermal-epidermal junction.^1,16 These cells are responsible for the upper dermis's loose connective tissue, and facilitate wound repair and re-epithelialization.^1,2,6,16,17 Their architecture enables them to communicate with both epidermal cells above and reticular fibroblasts in the lower dermal layers.²

These cells house several important markers:

• CD26 (DPP4) identifies populations that produce large amounts of collagen during wound healing.⁶ Studies show CD26-positive fibroblasts are enriched in the papillary dermis and are more proliferative compared to reticular populations.²
• CD34 and CD90 production distinguish these cells from other dermal populations.^2,6
• Single-cell transcriptomics has shown that APCDD1 and PTGDS are also expressed in papillary fibroblast populations.⁴

Reticular Fibroblasts

Reticular fibroblasts produce ECM in the lower dermis and can undergo adipogenic differentiation. Transcriptomics studies have shown that they have distinct molecular signatures appropriate to matrix production and organization.²

Reticular fibroblast markers include:

• CD90, which is consistently more highly expressed in reticular fibroblasts than in papillary fibroblasts.²
• CD36, which indicates cells that may be adipogenic, and is expressed more highly in reticular than papillary fibroblasts.^2,18
• MFAP5 and tetraspanin-8¹⁵

The differential expression of these markers reflects the specialized nature of papillary and reticular fibroblasts in the skin.¹⁵ Understanding these molecular differences has revealed how fibroblast populations (secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory) contribute to skin homeostasis related to the extent of collagen overexpression.¹¹

α-SMA (ACTA2)

Alpha-smooth muscle actin indicates activated myofibroblasts. This protein is not present in quiescent fibroblasts but becomes more expressed during activation.¹¹

FAP (Fibroblast Activation Protein)

FAP is a cell surface protease that is selectively upregulated during tissue remodeling. This protein promotes tumor formation and metastasis by matrix remodeling and influences cancer cell growth. Its expression pattern makes it important for monitoring fibroblast activation in diseased tissue, particularly in cancer.⁷

Flow Cytometry

Flow cytometry can identify and isolate fibroblast populations using specific marker combinations. For broad fibroblast detection, PDGFRα+/CD90+ double-positive staining is widely used.¹⁹ CD26/CD90 markers can separate papillary from reticular fibroblasts² and discern specific subpopulations. Activated fibroblasts are well identified using FAP.²

Immunohistochemistry/Immunofluorescence

IHC and ICC/IF provide valuable spatial data about fibroblast distribution and activation inside tissues. Vimentin reveals fibroblast morphology and distribution, while α-SMA specifically distinguishes activated fibroblast populations.⁵ Parallel to these markers, the ECM protein expression pattern can be monitored to follow cell-matrix interactions. PDGFB production could also be used to follow CAFs.¹⁴

Single-Cell Analysis

When analyzing fibroblasts at single-cell resolution, multiple markers could be used to identify populations, providing high specificity for different subtypes and disease states.^5,11 For example, recent scRNAseq studies have identified CAF subpopulations, including myofibroblast-like CAFs that express high levels of ECM proteins and inflammatory CAFs with a characteristic cytokine profile.¹⁴

References

1. Lynch, M. D. & Watt, F. M. Fibroblast heterogeneity: implications for human disease. The Journal of Clinical Investigation 128, 26-35 (2018).
2. Korosec, A. et al. Lineage Identity and Location within the Dermis Determine the Function of Papillary and Reticular Fibroblasts in Human Skin. Journal of Investigative Dermatology 139, 342-351 (2019).
3. Chitturi, P. & Leask, A. The role of positional information in determining dermal fibroblast diversity. Matrix Biology 128, 31-38 (2024).
4. Łuszczyński, K. et al. Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review. Cells 13 (2024).
5. Lendahl, U., Muhl, L. & Betsholtz, C. Identification, discrimination and heterogeneity of fibroblasts. Nature Communications 13, 3409 (2022).
6. Worthen, C. A. et al. CD26 Identifies a Subpopulation of Fibroblasts that Produce the Majority of Collagen during Wound Healing in Human Skin. Journal of Investigative Dermatology 140, 2515-2524.e2513 (2020).
7. Fitzgerald, A. A. & Weiner, L. M. The role of fibroblast activation protein in health and malignancy. Cancer and Metastasis Reviews 39, 783-803 (2020).
8. Bensa, T., Tekkela, S. & Rognoni, E. Skin fibroblast functional heterogeneity in health and disease. The Journal of Pathology 260, 609-620 (2023).
9. Han, C., Liu, T. & Yin, R. Biomarkers for cancer-associated fibroblasts. Biomarker Research 8, 64 (2020).
10. Venning, F. A. et al. Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer. Journal of Experimental & Clinical Cancer Research 40, 175 (2021).
11. Deng, C.-C. et al. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nature Communications 12, 3709 (2021).
12. Horikawa, S. et al. PDGFRα plays a crucial role in connective tissue remodeling. Scientific Reports 5, 17948 (2015).
13. Tabib, T., Morse, C., Wang, T., Chen, W. & Lafyatis, R. SFRP2/DPP4 and FMO1/LSP1 Define Major Fibroblast Populations in Human Skin. Journal of Investigative Dermatology 138, 802-810 (2018).
14. Zhang, Y. et al. Platelet-Derived PDGFB Promotes Recruitment of Cancer-Associated Fibroblasts, Deposition of Extracellular Matrix and Tgfβ Signaling in the Tumor Microenvironment. Cancers 14 (2022).
15. Solé-Boldo, L. et al. Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming. Communications Biology 3, 188 (2020).
16. Driskell, R. R. et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277-281 (2013).
17. Talbott, H. E., Mascharak, S., Griffin, M., Wan, D. C. & Longaker, M. T. Wound healing, fibroblast heterogeneity, and fibrosis. Cell Stem Cell 29, 1161-1180 (2022).
18. Gao, H. et al. CD36 Is a Marker of Human Adipocyte Progenitors with Pronounced Adipogenic and Triglyceride Accumulation Potential. Stem Cells 35, 1799-1814 (2017).
19. Michelis, K. C. et al. CD90 Identifies Adventitial Mesenchymal Progenitor Cells in Adult Human Medium- and Large-Sized Arteries. Stem Cell Reports 11, 242-257 (2018).