Functional Restoration of Amyotrophic Lateral Sclerosis Patient-Derived Mesenchymal Stromal Cells Through Inhibition of DNA Methyltransferase.

Alteration of DNA methylation is highly associated with aging and neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Remedying these aberrant methylation patterns may serve to improve these diseases. Previously, we reported that human bone marrow mesenchymal stromal cells isolated from ALS patients (ALS-MSCs) have functionally decreased stem cell potency, and excessively express DNA methyltransferases (DNMTs). In this study, we examined the correlation between excessive DNMT expression and functional decline in ALS-MSCs. The DNMT inhibitor RG108 was used for this. RG108-treated ALS-MSCs exhibit increased expression of the anti-senescence genes TERT, VEGF, and ANG, and decreased expression of the senescence-related genes ATM and p21. The activity of SA-ß-galactosidase and the expression of senescence proteins p53 and p16 were reduced in RG108-treated ALS-MSCs. The abilities of cell migration and protection against oxidative damage were improved in the treated ALS-MSCs. In neuronal differentiation experiments, the treated MSCs more effectively differentiated into neuron-like cells. These results suggest that ALS-MSC function can be restored by inhibiting excessively expressed DNMTs, an approach that may ultimately provide better efficacy in stem cell therapy.

Anti-senescence effects of DNA methyltransferase inhibitor RG108 in human bone marrow mesenchymal stromal cells.

Alteration of DNA methylation is highly associated with ageing and ageing-related diseases. Remedy of the altered methylation pattern may provide beneficial efficacy in these diseases. In this study, we used a DNA methyltransferase inhibitor, RG108, to investigate the senescence effects in human bone marrow mesenchymal stromal cells (hBM-MSCs). First, we determined the optimized dose and time of RG108 treatment in hBM-MSCs to be 5 µM for 48 H, respectively. Under these conditions, the anti-senescence genes TERT, bFGF, VEGF, and ANG were increased, whereas the senescence-related genes ATM, p21, and p53 were decreased. The number of ß-galactosidase-positive cells was significantly decreased in RG108-treated MSCs, whereas the rates of MSC migration and cellular protection were increased. We have shown that RG108 significantly induces the expression of TERT by blocking methylation at the TERT promoter region. Thus, these data indicate that an optimized dose of RG108 may improve the cell migration, protection, cellular senescence, which may provide a better efficacy of these cells in stem cell therapy.

Functional restoration of replicative senescent mesenchymal stem cells by the brown alga Undaria pinnatifida.

The brown alga Undaria pinnatifida, which is called Mi-Yoek in Korea, has been traditionally consumed as a health food in East Asian countries. Recent studies have reported that U. pinnatifida has beneficial effects on arteriosclerosis, inflammation, fat metabolism, and tumors. In this study, we examined the anti-senescence effects of ethanol extracts of U. pinnatifida (UP-Ex) in human bone marrow mesenchymal stem cells (hBM-MSCs). UP-Ex protected hBM-MSCs against oxidative injury, as determined by MTT assays. This effect was confirmed by immunoblot analysis of the oxidation-sensitive protein p53 and the apoptotic protein cleaved caspase-3. Excessive intracellular reactive oxygen species (ROS) accumulation induced by oxidative stress was moderated in UP-Ex-treated hBM-MSCs (UP-Ex-MSCs). Similarly, expression of the ROS-scavenging enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase was recovered in UP-Ex-MSCs. Excessive ROS induced by long-term cell expansion (passage 17) was significantly decreased along with restoration of the senescence proteins p53, p21, and p16 in UP-Ex-MSCs. UP-Ex treatment also improved the ability of these replicative, senescent hBM-MSCs (passage 17) to differentiate into osteocytes or adipocytes, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells and may provide better therapeutic efficacy in stem cell therapy. Abbreviations: hBM-MSCs: human bone marrow mesenchymal stem cells; DCF: 2',7'-dichlorodihydrofluorescein; DCFH-DA: 2',7'-dichlorofluorescein diacetate; MTT: 3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazolium bromide; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RIPA: radioimmunoprecipitation assay; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2.