Project description:3mm punch biopsies were taken from a healed normotrophic scar and a contralateral matched control site in burn patients with a scar at least 1 year old. Fibroblasts were cultured from explants to passage 2 and DNA was extracted and run on methylation arrays to examine differences in scar and control fibroblast gene expression Normotrophic scar maintains its abnormal scar phenotype for the rest of the patients life, long after the injury has healed. Differences in gene expression may reaveal target genes that can be modulated to improve scar appearance
Project description:In this study, we have analyzed DNA methylation changes upon aging of human dermal fibroblasts by using the HumanMethylation27 BeadChip assessing 27,578 unique CpG sites. Cells were isolated from skin samples donated by young (6-23 years) and elderly (60-73 years) patients undergoing surgical interventions. Strikingly, global DNA-methylation profiles of fibroblasts from the same anatomical site clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture.
Project description:3mm punch biopsies were taken from a healed normotrophic scar in burn patients with a scar at least 1 year old and fibroblasts were cultured from explants. Previous transcriptomic and epigenomic work found MKX and FOXF2 genes were overexpressed and these were knocked down using siRNA. RNA was then extracted and analysed using RNAseq to determine genes and pathways affected by this knockdown
Project description:In this study, we have analyzed DNA methylation changes upon aging of human dermal fibroblasts by using the HumanMethylation27 BeadChip assessing 27,578 unique CpG sites. Cells were isolated from skin samples donated by young (6-23 years) and elderly (60-73 years) patients undergoing surgical interventions. Strikingly, global DNA-methylation profiles of fibroblasts from the same anatomical site clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. Skin samples from younger or elderly donors were treated with dispase (Roche Diagnostics, Mannheim, Germany) for 12 hours to separate the dermis from the epidermis. The dermis was digested with 0,2% collagenase and 1,5% BSA in collagenase buffer (100mM HEPES, 120mM NaCl, 50mM KCl, 1mM CaCl2, 5mM Glucose) for 45 minutes. Dermal remnants were removed by filtering the digest through a 100µm nylon strainer (Falcon, Becton Dickinson [BD], San Jose, USA). The cells were subsequently washed and expanded in standard medium consisting of DMEM (PAA; 1g/L glucose) supplemented with glutamine (PAA), penicillin/sptrepamycin (PAA) and 10% fetal calf serum (Biochrom, Berlin, Germany) in a humidified atmosphere at 5% CO2. Cells were always replated when grown to 80% confluency. For methylation profiles upon aging we have isolated DNA from the samples of passage 3 of younger and elderly donors. For methylation profiles upon long-term culture we have isolated DNA from the samples of early passage (P3) and late passage (P21)
Project description:We describe a so far uncharacterized, embryonic and self-renewing Neural Plate Border Stem Cell (NBSC) population with the capacity to differentiate into central nervous and neural crest lineages. NBSCs can be obtained by neural transcription factor-mediated reprogramming (BRN2, SOX2, KLF4, and ZIC3) of human adult dermal fibroblasts and peripheral blood cells (induced Neural Plate Border Stem Cells, iNBSCs) or by directed differentiation from human induced pluripotent stem cells (NBSCs). Moreover, human (i)NBSCs share molecular and functional features with an endogenous NBSC population isolated from neural folds of E8.5 mouse embryos. Upon differentiation, iNBSCs give rise to either (1) radial glia-type stem cells, dopaminergic and serotonergic neurons, motoneurons, astrocytes, and oligodendrocytes or (2) cells from the neural crest lineage. Here we provide array-based methylation data of iNBSCs reprogrammed from adult dermal fibroblasts (ADF), iPSC-derived NBSCs and adult dermal fibroblasts. The data provided demonstrate robust changes in the methylation landscape after reprogramming of human adult dermal fibroblasts into iNBSCs.
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:3mm punch biopsies were taken from a healed normotrophic scar and a contralateral matched control site in burn patients with a scar at least 1 year old. Fibroblasts were cultured from explants to passage 2 and RNA was extracted and run on expression arrays to examine differences in scar and control fibroblast gene expression Normotrophic scar maintains its abnormal scar phenotype for the rest of the patients life, long after the injury has healed. Differences in gene expression may reaveal target genes that can be modulated to improve scar appearance
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.