Resveratrol Exerts Dosage and Duration Dependent Effect on Human Mesenchymal Stem Cell Development

<div><p>Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs.</p> </div

Resveratrol increased the percentage of S-phase cells in a dosage dependent manner.

<p>Flow cytometry was performed to analyze cell cycle distributions on cells pre-exposed to resveratrol or BM for 0 (0D-PT) or 30 (30D-PT) days, followed by 6 (0D-PT) or 4 (30D-PT) more days of treatment respectively after equal density plating, and cells cultured in regular hMSCs media (CM) for 30 days prior to 4 days of resveratrol or BM treatment following equal density plating (30D-CM). Stacked columns represent the relative distribution of cells in S, G2/M or (S+G2/M) phases in each treatment group normalized to the value in the BM treated cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037162#pone.0037162.s003" target="_blank">Table S1</a>). Data presented are the mean values of 2 or 3 independent experiments for each experimental set. All duplicate experiments had similar outcomes.</p

Resveratrol modulates adipogenic differentiation of hMSCs in a dosage dependent manner.

<p><b>A</b>). Oil droplets were stained by Oil-Red-O solution and subsequently quantified under three different treatment schemes: concurrent treatment, pretreatment and equal density. <b>B</b>). Images of Oil-Red-O stained cells under different treatment conditions. Concurrent treatment: cells were exposed to both resveratrol and AIM throughout the differentiation duration; Pretreatment: cells were cultured in BM/resveratrol conditioned media continuously for certain days prior to AIM induction; Equal density: Cells were cultured in BM/resveratrol conditioned media continuously for certain days and re-plated at equal density before AIM induction. Except for the pretreatment groups, all experiments were repeated 3 times independently, with triplicates in each experimental set. A representative data set is presented for each group and data shown are the relative mean values of triplicates normalized to the mean value of the BM control cells in each group. Error bars represent standard deviation. *: p<0.05 vs. BM. Images were taken at 8× except for bottom row in B) (6.3×).</p

Resveratrol pretreatment prolongs cell-doubling time of hMSCs in a dosage dependent manner.

<p>Untreated early passage cells (0D-PT), untreated late passage cells (30D-CM) and cells pretreated with resveratrol or BM for 30 days (30D-PT) were plated at 1–2 cells/well density and exposed to the same resveratrol or BM treatment for 72 hours or longer for cell-doubling time assay. Data presented are the mean values of 2 independent experiments for each experimental set. Error bars represent standard deviation. *: p<0.05 vs. BM.</p

Resveratrol exerts dosage dependent enhancing vs. inhibitory effect on the self-renewal rate of hMSCs.

<p><b>A</b>). Cells were plated at equal density and cultured in different concentrations of resveratrol continuously for 36 days during which cells were counted and split at equal ratio every 6 days. *: p<0.01. <b>B</b>). Cells pretreated with resveratrol or BM for 0 (0D-PT), 9 (9D-PT), 12 (12D-PT) or 30 (30D-PT) days were seeded at 8000 cells/well and continued to culture in corresponding media until resazurin assay. Error bars represent standard deviation (triplicates in each treatment condition). *: p<0.05 vs. BM.</p

Resveratrol regulates the expression of genes implicated in osteogenesis and adipogenesis.

<p><b>A</b>). Representative gel images of gene expression examined by semi-quantitative RT-PCR on cells subjected to concurrent treatment of BM/resveratrol with OIM for 3 days (OIM 3D-CT) or 7 days (OIM 7D-CT), or cells pretreated with BM/resveratrol for 12 days followed by 3 days of OIM (12D PT-3D OIM) or 7 days of AIM (12D PT–7D AIM) exposure. Expression of internal control gene <i>Hsp90</i> from the same batch of <i>cDNA</i> for each gene is shown in the bottom row. <b>B</b>). Expression of each gene in resveratrol treated cells was quantified relative to that in BM treated cells and normalized by the expression of housekeeping gene <i>Hsp90</i>. Data shown are the mean values of three repeats. Error bars represent standard deviation. Concentration unit: µM. *: p<0.05 vs. BM. **: p = 0.055 vs. BM.</p

Resveratrol does not cause acute cytotoxicity in hMSCs at concentrations tested.

<p>Untreated P4 cells (0D-PT) or cells pretreated for 6 (6D-PT), 25 (25D-PT), 35 (35D-PT) or 41 days (41D-PT) were plated at equal density and then subjected to 24 hours of treatment in corresponding BM or resveratrol media before LDH assay. AM: assay media; AM alone: no cells; AM alone w/RSV: no cells and the highest concentration of RSV in the group was used; 1% triton X-100 in AM: positive control (all values >2.5). Data presented were the mean values of each treatment type. Error bars represent standard deviation. *: p<0.05 vs. BM.</p

Resveratrol exerts dosage dependent anti- vs. pro-senescence effect on hMSCs.

<p><b>A</b>). Cells were stained in X-gal (blue color) and neutral red solution (red color). Images were taken at 200× magnification. <b>B</b>). Percentages of senescent cells vs. total cells were determined based on images taken. At least 184 total cells from each treatment condition were counted. Column represents the relative fold changes of percentage of cells undergoing senescence in each treatment group normalized to the value obtained in the BM treated cells. Data was obtained from three independent experiments. Error bars represent standard deviation. *: p≤0.01 vs. BM.</p

Resveratrol regulates the expression of genes implicated in cell cycle, cell senescence and longevity regulation.

<p><b>A</b>). Representative gel images of gene expression examined by semi-quantitative RT-PCR on cells pretreated with BM or resveratrol for 3 or 5 days. Expression of internal control gene <i>Hsp90</i> from the same batch of <i>cDNA</i> for each gene is shown in the bottom row. <b>B</b>). Expression of each gene in resveratrol treated cells was quantified relative to that in BM treated cells and normalized by the expression level of housekeeping gene <i>Hsp90</i>. Data shown are the mean values of three repeats. Error bars represent standard deviation. *: p<0.05 vs. BM.</p

Resveratrol pretreatment affects cell proliferation rate of hMSCs in both time and dosage dependent manner.

<p><b>A</b>). Duplicate wells of cells pretreated with resveratrol or BM for 0 (0D-PT), 12 (12D-PT) or 30 (30D-PT) days, followed by 6 (0D-PT) or 5 (12D-PT & 30D-PT) more days of treatment respectively after similar density plating were fixed and stained at 6 or 12 hrs post EdU addition. Cells labeled with EdU were green (indicated by arrows) whereas unlabeled cells appeared grey from Hoescht 33342 staining (image taken using UV/Blue dual filter at 200× magnification). <b>B</b>). Percentages of green cells were normalized to the value in the BM treated cells. Error bars represent standard deviation. *: p<0.05 vs. BM.</p