Resveratrol effects on mesenchymal stem cell proliferation and differentiation

T Demirtas1, G Gacar2, N Gacar1, T Utkan1, B Akpinar2, ZS Unal2, Y Yazir3, E Karaoz0. 1Kocaeli University Medical School, Pharmacology 41380, Turkey, 2Kocaeli University, Stem Cells and Gene Therapy 41380, Turkey, 3Kocaeli University Medical School, Histology and Embryology, Turkey

Mesenchymal Stem Cells (MSCs) are a type of cell with the capabilities of self-renewal and multipotency. These cells have the ability to differentiate into many tissue pathways such as osteogenic, chondrogenic, and adipogenic. The most studied bone marrow MSCs decreases significantly with age so we have to find alternative sources of stem cells. For therapeutic purposes, we have to get them pure and in large quantities. In particular, resveratrol can provide a cheap and effective approach. The bone protective effects of resveratrol have been demonstrated in several osteoporosis models. In the present study, we investigated in vitro effects of resveratrol on cell proliferation and osteoblastic differentiation in human mesenchymal stem cell cultures. For this purpose, MSC (1x105) were inoculated in 6-well plates. All cell lines were maintained in Dulbecco\'s modified Eagle\'s medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/l penicillin and 100 U/l streptomycin and were cultured at 37°C in a humid atmosphere consisting of 5% CO2 and 95% air. After 2 hours later, resveratrol was used at various concentrations (0, 1, 10, 20, 40µM) for 1, 4, 7 and 14 days. After each time period, we determined the proliferation capasity and viability using WST-1, and 72 hours later apoptosis level using AnnexinV-PI staining by using flow cytometer. After WST-1 assay and AnnexinV-PI staining, we detected reduction of proliferation in high doses so caspase-3 activity were measured. Significant differences were determined using one-way ANOVA followed by Tukey’s post-hoc tests.

We observed that resveratrol (10 and 20µM) (2.74±0.03 and 3.07±0.04) (p<0.001) stimulated the cell proliferation while high doses (40µM) (1.48±0.09) (p<0.001) were stopped compared the control group (2.54±0.04) in WST-1 proliferation assay. The expression of Alkaline phosphatase (ALP) was detected in the early osteogenic induction on 21st day and the calcified nodules were observed by von Kossa staining. These data suggest that stem cells are enriched by culturing the heterogeneous cell populations with resveratrol.

After incubating with osteogenic differentiation media, we investigated that resveratrol dose-dependently increased the ALP activity of MSCs to a value 2,5 times the control activity at 40 µM( 1.616±0.03) compared to control (0.598±0.01) (p<0.001). Real-time RT-PCR studies indicated a significant increase in BMP-2 mRNA expression levels in hMSCs. We used human dental pulp derived mesenchymal stem cells (hDP-MSC) at passage 3 and we observed an increase in expression of the gene BMP2 and BMP4 with RT-PCR analysis after osteogenic differentiation with resveratrol.

In conclusion, resveratrol directly stimulates both cell proliferation and osteogenic differentiation dose-dependently, also is observed by remarkable changes in the expression of some genes. Increased expression of BMP2 (0.71±0.06) and BMP4 (0.32±0.01) gene shows us that resveratrol has a direct stimulatory effect on bone formation in cultured osteoblastic cell in vitro compared to control for BMP2 (0.18±0.01) and BMP4 (0.02±0.001) (p<0.001). Presumably, resveratrol is a useful tool in the prevention of and therapy for osteoporosis.

Z. Dai et al., Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomedicine; 14 (2007) 806–814.