Fig. 3
Generation of therapeutic AD-MSCs that fulfil the clinical requirement. Cells were transfected with LPEI at various pDNA amounts, ± Enhancer. Forty-eight hours later, cells were harvested for flow cytometry analysis to determine the percentage of CD::UPRT::GFP+ cells and b NC-3000 automated cell counting to measure cell viability with PI exclusion method. Graph bars present mean ± SD, n = 3. Significant differences ± Enhancer were calculated using two-tailed Student’s t test. **P < 0.005. AD-MSCs were transfected at 155 ng pDNA/cm2 in the presence of Enhancer. c AD-MSCs and CD::UPRT::GFP_AD-MSCs were labelled with fluorophore-conjugated antibodies and analysed by flow cytometry according to the manufacturer’s instructions. Isotype antibodies served as respective controls for gating. Histograms demonstrate the immunophenotypic profiles. d AD-MSCs and CD::UPRT::GFP_AD-MSCs were cultured in osteogenic or adipogenic differentiation medium for 14 or 21 days, respectively. The presence of calcium deposits stained with Alizarin red S indicates osteogenic differentiation of AD-MSCs. Oil Red O stained for oil droplets visible in the cells and was indicative of adipogenic differentiation. All images were captured at × 20 magnification. Migratory property of MSCs was evaluated using transwell assay. e Target cells (glioma cell lines or fibroblast) were plated in complete media. One day later, media were replaced with serum-free DMEM. CD::UPRT::GFP_AD-MSCs (transfected 1 day before the experiment) and un-transfected AD-MSCs were loaded onto the cell inserts. The inserts were transferred to the target cell cultures, respectively. Twenty-four hours later, cell migration was evaluated under a microscope by taking fluorescent images of cells stained with Hoechst 33342. f A similar study was performed to compare migration of modified MSCs towards parental and TMZ glioma cells. The fold change of AD-MSCs migrated towards cancer cells over fibroblast was calculated. Graph presents the mean of fold change + SD (n = 3)