Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

Figure 2

Induction of premature senescence in MSCs. (a) Induced senescence in MSCs. Senescence was induced by continuous exposure of HMSCs (heart-derived MSCs) and SMSCs (skin-derived MSCs) to a low dose of H₂O₂ (50 µM) and low serum (5% FBS) in the culturing medium. CT: control HMSCs treated with PBS; SN: senescent HMSCs treated with 50 µM H₂O₂ for 14 d (×10). (b) Quantitation of β-Gal cells. Cells were counted under microscopy. The results are expressed as the mean ± standard deviation of β-Gal positive cells per field. as compared with the PBS control. (c) Senescent-related genes. Senescent HMSCs were harvested and RNA were extracted. RT-PCR was carried out to amplify senescence-related genes, including caveolin-1, apolipoprotein J, and OX 1. (d) Apoptosis-related genes. Expression of p53 and p21 genes was measured by RT-PCR. (e) Telomere length in senescent HMSCs. The relative length of telomere was estimated by qPCR as the ratio between the copies of telomere and the copies of β-globin (T/S). as compared with the replicative senescence and high H₂O₂ groups.

(a) β-Gal staining

(b) β-Gal quantitation

(c) Senescence gene

(d) Apoptosis gene

(e) Telomere length