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. 2021 Mar 26;22(7):3416. doi: 10.3390/ijms22073416

Figure 1.

Figure 1

A conditioning medium primes the immunomodulatory capacity of mesenchymal stromal cells (MSCs). Quantification of immunosuppressive molecules and abundance of extracellular vesicles (EVs) released by telomerase-expressing MSCs conditioned (MSC-Tc) or not (MSC-T) with pro-inflammatory cytokines for 48 h. (a) indoleamine 2,3 dioxygenase (IDO), cyclooxygenase 2 (COX2) and PD-L1 gene expression levels measured by RT-qPCR. Target gene expression was normalized to GAPDH expression and is relative to levels in MSC-T, arbitrarily set to 1. Graphs represent mean ± SD of 3 independent experiments. Paired t-test was used for statistics. (b) Representative Western blots of IDO, COX2 and PD-L1 protein expression; α-tubulin was used as a protein loading control. (c) PD-L1 protein expression measured by flow cytometry using mean fluorescence intensity (MFI) of human telomerase enzyme (MSC-T) (black) and MSC-Tc (grey). Values are represented as relative to MSC-T geometric mean value. Graphs represent mean ± SD of three independent experiments. Paired t-test was used for statistics. (d) Concentration of protein of EVs extracted from 100 mL of culture medium from 1 × 107 cells that were resuspended in 100 μL of PBS. Graphs represent mean ± SD of four independent experiments. Paired t-test was used for statistics. (e) Quantification of acetylcholinesterase activity in EVs. Values are represented as relative to the EVMSC-T value. Graphs represent mean ± SD of three independent experiments. Paired t-test was used for statistics. (f) Amount of EVs per milliliter of supernatant of the same number of MSC-T and MSC-Tc measured by nanoparticle tracking analysis (NTA). Graphs represent mean ± SD of three independent experiments. Unpaired t-test was used for statistics. * p < 0.05, ** p < 0.01. (g) Representative images of EVs analyzed by NTA. (h) Representative Western blot of Hsp70, TSG101 and CD9 proteins in EVs. Absence of calnexin demonstrates a pure EV preparation. Cells were used as calnexin-positive controls. (i) Representative electron microscopy images of isolated EVs collected from different MSC conditions. Scale bar: 200 nm.