Fig. 2.

MSC engineering using mRNA and in vitro functional validation. (a) PSGL-1/SLEX/CD/OPG MSC display functional rolling on an endothelial layer under physiological shear flow. Native MSC, PSGL-1/SLEX MSC and PSGL-1/SLEX/CD/OPG MSC were flowed on a layer of endothelial cells at different physiological flow-rates 24 h post-MSC engineering. HL-60 leukocytic cells were used as a positive control for rolling. Plot: mean + SD, statistical analysis: Two-way ANOVA test with Dunnett's multiple comparison test to compare each column to Native MSC, *** p ≤ .001, **** p ≤ .0001. (b) PSGL-1/SLEX/CD/OPG MSC inhibit osteoclastic differentiation in vitro. Murine osteoclast precursors (RAW264.7 cells) were plated for 6 days in media with no additional treatment (CT), 100 ng/mL recombinant murine RANKL to induce osteoclastogenesis, and day 2 supernatant of MSC (Native and PSGL-1/SLEX/CD/OPG). 100 ng/mL of recombinant human OPG was used as a positive control for osteoclastogenesis inhibition. Pictures show the TRAP stained culture at day 6 for each condition. Plot: mean + SD, statistical analysis: Kruskal-Wallis with Dunn's multiple comparison test, *** p ≤ .001 compared to PBS + RANKL condition. (c) PSGL-1/SLEX/CD/OPG MSC convert 5-FC into 5-FU in vitro in a cell concentration-dependent manner. 24 h post-engineering, MSC were plated at different concentrations in presence of 400 μg/mL 5-FC. LC-MS/MS was done on conditioned media collected at different days to measure the 5-FU converted from 5-FC. Plot shows mean + SD. (d) PSGL-1/SLEX/CD/OPG MSC kill MDA-MB231 cancer cells in vitro. Native MSC and PSGL-1/SLEX/CD/OPG MSC were plated at different ratios (1:2 and 1:10) on top of cancer cells in the presence of increasing doses of 5-FC, and the viability of the co-culture was determined at day 6. 5-FU was used as a positive control. Graph shows mean ± SD.