Effect of Paclitaxel upon the differentiation ability of hMSCs.

<p>(A) and (B) After 21 days of induced adipogenic differentiation in the presence of either 0, 10, or 10,000 nM Paclitaxel, cells were fixed and stained with the lipophilic dye Nile Red and Hoechst 33342. Image samples were taken of each culture and area of staining was standardized to nuclei counts for that area. A decrease in lipid accumulation of approximately 40% when compared to control was observed in hMSCs treated with either 10 or 10,000 nM Paclitaxel. (C) Microtubule staining via immunocytochemistry reveled an apparent dose-dependence upon cytoskeletal organization in the differentiated hMSCs. At 10 nM characteristic bundling was observed. At 10,000 nM over-accumulation of microtubules was observed. Results are displayed as mean ± standard error. *** P < 0.01. Scale bar (B) 150 μm, (C) 15 μm.</p

Proliferation and viability of hMSCs in the presence of Paclitaxel.

<p>(A) Human MSCs were incubated with 30–250,000 nM Paclitaxel for 72 hrs, then treated with dimethyl thiazolyl diphenyl tetrazolium salt (MTT) and processed. Absorbance was measured at 600 nM and compared with controls to produce relative values. (B) Human MSCs were incubated for 72 hrs with Paclitaxel at various concentrations. After incubation, cells were counted via hemocytometer and compared with controls to determine relative viability. After 72 hrs viability was determined to be above 90% in hMSCs treated with up to 100,000 nM Paclitaxel. (C) Growth curves were generated by counting stained nuclei of hMSCs treated with either 0, 10, or 10,000 nM Paclitaxel for various lengths of time. It was revealed that Paclitaxel at both 10 and 10,000 nM completely inhibited proliferation of hMSCs. Results for each experiment are displayed as mean ± standard deviation.</p

Quantitative real-time PCR of growth arrest specific factor 1 (GAS1) in hMSCs treated with Paclitaxel over time.

<p>Human MSCs were treated with 10 nM Paclitaxel for up to 12 days, with samples being taken at various time points. Relative expression for each gene was calculated via the 2^^(-ΔΔCt) method with Day 0 expression acting as the basis for comparison. Results are displayed as mean ± standard deviation. *** P < 0.01</p

Western blot analysis of P-glycoprotein in hMSCs treated with 10nM Paclitaxel over time.

<p>Results show that the hMSCs do not natively express P-glycoprotein (P-gp), nor is expression induced by exposure to Paclitaxel. Positive control is cell lysate from the human colorectal adenocarcinoma cell line HCT-15, which constitutively expresses high levels of P-gp. β-actin was used as a loading control.</p

Characterization of the fibroblast-like state hMSCs adopt when treated with Paclitaxel.

<p>(A) After 72hrs of exposure to either 10 or 10,000 nM Paclitaxel hMSCs transition from the typical spindle-shaped morphology, seen in the untreated hMSCs, to one that is more broad and flat. Quantitative Real-Time PCR was used to determine expression levels of (B) CD9 (C) CD106 (D) CD146 (E) CD166 (F) integrin alpha 11 (ITGA11) (G) matrix metalloproteinase 1 (MMP-1) and (H) MMP-3 in hMSCs treated with 10 nM Paclitaxel over time. Results are displayed as mean ± standard deviation. ** P < 0.05, *** P < 0.01. Scale bar (A) 150 μm.</p

Cell cycle regulation may provide Paclitaxel resistance in hMSCs.

<p>By inducing cells to enter quiescence before the G2/M transition needed for Paclitaxel action, it is possible that hMSCs utilize cell cycle regulation to protect themselves from the cytotoxic effects of Paclitaxel.</p

Primer list for real-time PCR.

<p>Primer list for real-time PCR.</p