Methylation Pattern of Human Dermal Fibroblasts

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)