Fig. 4

Cryopreservation did not adversely affect the migratory potential of MSC overexpressing CD::UPRT::GFP. (A) Unmodified MSCs, freshly modified or thawed MSC overexpressing CD::UPRT::GFP (500,000 cells each group) were stained with anti-human CXCR4 antibody conjugated to a PE fluorophore (Cat. 306,506, Biolegend) at 1:10 dilution. Staining of cells were analyzed by flow cytometry. Percentage of expression was measured using FACS. The population of CXCR4 positive MSCs was gated relative to their isotype controls. (B) Four hundred thousand A549 or RPMI 2650 cells were seeded on the bottom chamber of a trans-well (Corning) plate in DMEM supplemented with 10% FBS. Twenty-four hours later, the media was replaced with serum free DMEM before the adding of 150,000 MSC overexpressing CD::UPRT::GFP (unmodified, freshly modified or cryopreserved modified) in the 8 µM matrigel coated cell inserts. The inserts were transferred into the wells containing the cancer cells. Twenty-four hours later, cells on the flip side of the inserts were stained with Hoechst 33,342 and imaged using a microscope. A total of three frames were imaged and counted. The graph represents mean ± SD of migratory cells per frame (n = 3). MSC overexpressing CD::UPRT::GFP without cancer cells (blank) served as negative control. Significant differences in cell count per frame between unmodified and modified MSCs were calculated using two-tailed Student’s t test. **P < 0.005