Nhdfs

Normal human dermal fibroblasts (NHDF) were obtained from Takara Bio (Shiga, Japan). NHDFs were grown in a low-glucose Dulbecco’s modified Eagle medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (Thermo Fisher Scientific).
Replicative senescence was defined as a cell population doubling level greater than 50 and no proliferation for more than 2 weeks. Ionizing radiation-induced senescence was induced in the same manner as previously reported [14 (link)]. Cells were irradiated with 10 Gy of X-rays by AB-160 X-Ray Irradiation System (AcroBio, Tokyo, Japan) and analyzed 10 days later. Control (young; proliferating) cells were mock-irradiated by removal from the incubator, transport to the irradiator, and maintenance outside the irradiator for the same period as the irradiated cells. Intracellular SA-β-gal activity was assessed by staining cells using Senescence β-Galactosidase Staining Kit from Cell Signaling (Danvers, MA, USA).

Primary cultured NHDFs, neonatal hASCs and HUVECs were purchased from Lonza (Walkersville, MD). Each cell type was derived from single donor. NHDFs were cultured in Dulbecco’s modified Eagle medium (DMEM, Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS, SAFC Biosciences; Lenexa, KS) under 5% CO₂ at 37 °C as described in our previous study^{4 (link)}. For cultivation of HUVECs, Endothelial Growth Medium-2 (Lonza; Walkersville, MD) was used as described previously^{3 (link)}. For cultivation of hASCs, Mesenchymal Stem Cell Growth Medium 2 (Takara Bio Inc., Kusatsu, Japan) was used. Transwell inserts with porous polyester bottom (pore size: 0.4 µm) for 24-well culture plate were purchased from CORNING Inc. (New York, NY).

NHDFs were obtained from Lonza (Walkersvill, MD, USA). Leigh syndrome patient-derived skin (7S) fibroblasts were kindly provided by Koinobori Associate Inc., which supports research on mitochondrial diseases under approval from the ethical committees of our institution and Koinobori Associate Inc. The human uterine endometrial gland-derived mesenchymal cell line EPC100 was acquired from the Japanese Collection of Research Bioresources Cell Bank, where it is assigned JCRB1538. NHDFs and EPC100 cells were maintained in high-glucose DMEM (043-30085, Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific Incorporated, Waltham, MA USA). The 7S fibroblasts were maintained in low-glucose DMEM with pyruvate (11885084) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific incorporated). All cell lines were incubated at 37 °C under 5% CO₂ and cultured at ~ 80% confluence.