Project description:Fibroblasts are the main dermal cell type and are essential for the architecture and function of human skin. Important differences have been described between fibroblasts localized in distinct dermal layers, and these cells are also known to perform varied functions. However, this phenomenon has not been analyzed comprehensively yet. Here we have used single-cell RNA sequencing to analyze >15,000 cells from a sun-protected area in young and old donors. Our results define four main fibroblast subpopulations that can be spatially localized and functionally distinguished. Importantly, intrinsic aging reduces this fibroblast ‘priming’, generates distinct expression patterns of skin aging-associated genes, and substantially reduces the interactions of dermal fibroblasts with other skin cell types. Our work thus provides comprehensive evidence for a functional specialization of human dermal fibroblasts and suggests that the age-related loss of fibroblast priming contributes to human skin aging.
Project description:Adult human dermal fibroblasts reside in vivo under low oxygen tension. Thus, low oxygen culture conditions represent a physiological state for adult human dermal fibroblasts. We have also previously shown that low oxygen and addition of basic fibroblast growth factor (FGF2) lead to prolonged life-span of adult human dermal fibroblasts. Therefore, we set to determine effects of low oxygen and FGF2 on the gene expression signature of adult human dermal fibroblasts. This global analysis will allow identification of genes affected and pathways regulated by low oxygen and FGF2.
Project description:Background: Skin homeostasis is mediated by dermal fibroblasts and is affected by aging. Although age-related heterogeneity in fibroblasts has been reported, the effects of donor and species on this heterogeneity are unclear. Methods: To analyze age-related transcriptomic changes in human dermal fibroblasts, single-cell RNA sequencing was performed on dermal fibroblasts (ASF-4 cells) collected from the inner forearm of a volunteer over three decades. Results: Four main cell subpopulations changed with donor age and showed proliferative, homeostasis, fibrotic, and senescence functional annotations. The downregulation of the expression of genes encoding key extracellular matrix production and mechanotransduction components decreased with donor age. Interestingly, dermal fibroblasts have two putative differentiation pathways: one that involves the acquisition of senescent properties and the acquisition of fibrotic properties without the suppression of proliferation. Aging induced fibroblast differentiation in a manner involving the acquisition of senescent properties. Conclusion:Reconciling the various aspects of fibroblast heterogeneity may provide insight into the mechanisms underlying human skin aging and associated phenomena, including wrinkles, sagging, delayed wound healing, and suppressed scar formation.
Project description:Fibroblasts are widely used cells for regenerative medicine in clinics, such as gingival or facial skin treatment. In fact, fibroblasts are considered as a mixture of various types of cells with "spindle shape" and there is no available clear marker. Gingival and dermal fibroblasts are similar in their morphology and function; however it is considered that the cultured cells retain their original characteristics depending on the origin, which may contribute to the differential therapeutic effects. For example, gingival wounds are known to heal relatively quickly with less scar formation compared with skin, which may imply their higher capability for regeneration as a therapeutic effect. The reason for this phenomenon may be partly due to characteristic differences between gingival and dermal fibroblasts including the expression of migration stimulating factor and matrix formation but these differences remain largely unknown. Recently, the characteristics of dermal fibroblasts have been reported to be different depending on body sites, such as face, trunk and plamoplantar skin. Although the expression of fibronectin and their splicing variants were known to be different between trunk and oral mucosal fibroblasts, there is still no detailed report on the functional differences between gingival and dermal fibroblasts. In this study, we investigated differential gene expression in normal gingival and dermal fibroblasts using DNA microarray to investigate the difference between the vague fibroblast-type cells from different tissue origin to achieve higher therapeutic effect in cell therapy. Gingival and dermal tissues were collected from healthy patients. After successive stages primary culture and RNA extraction and hybridization on Affymetrix microarrays (Genome Focus Array). Dermal and gingival tissues were obtained from healthy volunteers (8 cases for dermal tissue; 6 females, 2 males , average age 48 and 8 cases for buccal gingival tissue; 6 females, 2 males, average age 43) whose informed consent was obtained according to a protocol approved by the ethics committee of Nagoya University Hospital. After enzymatic digestion, tissues were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C in the presence of 5% CO2 for about 4 weeks as reported previously. Total mRNAs were extracted from cells between passages 4-5 by Trizol reagent (Invitrogen, Carlsbad, CA, USA) and were applied to Human Focus Arrays (Affymetrix, Santa Clara, CA, USA) for microarray analysis according to the manufacturer's protocol (http://www.affymetrix.com/support/technical/manuals.affx). The gene expression data were analyzed by Arrayassist (Stratagene, La Jolla, CA, USA). Briefly, 8,500 probes on the array, normalization and scaling (MAS5), flag-positive gene selection, unpaired t-test, and CV selection (<20) resulted in 5,284 genes to analyze. GO (Gene ontology) analysis was performed using the software default settings to find the gene group related to the same category of biological function by searching common key terms that were reported for each gene.
Project description:The current study was to determine the effects of Fibroblast Growth Factor 2 (FGF2) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in culture medium (with FGF2 or not) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes exposure to FGF2, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts. Methods: Human dermal fibroblasts were FGF2 (10ng/ML) treatment for 48h hours. Then, the RNA was extracted for library construction Results: FGF2-responsive genes were significantly involved in ECM-receptor interaction, PI3K-Akt signaling and Hippo pathway.