Figure 8. The knockdown of HGF and COX-2 partially reverses the effects of MSCs on BM cell differentiation.

(A) Heatmaps of RNA-Seq on MSCs cultured alone without GM-CSF, MSCs cultured under GM-CSF stimulation, and MSCs cocultured with BM cells under GM-CSF. Presented are the gene expression levels in MSCs cultured with GM-CSF (the first column) and in MSCs cocultured with BM cells + GM-CSF (the second column), relative to those in MSCs cultured without GM-CSF. The genes that encode secreted proteins and were upregulated by >2-fold in MSCs cocultured with BM cells under GM-CSF in comparison with MSCs cultured alone under GM-CSF are listed. The whole data are deposited in ArrayExpress (accession E-MTAB-8976). (B) MSCs were transfected with control siRNA (Con KD MSC), HGF siRNA (HGF KD MSC), or COX-2 siRNA (COX-2 KD MSC) and cocultured with BM cells in either direct or Transwell coculture system (trans) in the presence or absence of GM-CSF. The cell-free culture supernatants were evaluated for the secretion of HGF and PGE₂ using ELISA in order to confirm the knockdown of each gene. (C) The flow cytometry results for CD11b^midLy6C^midLy6G^lo cells out of BM cells after 5 days of coculture with MSCs. (D) Real-time RT-PCR for mRNA levels of inducible nitric oxide synthase (Nos2), arginase (Arg1), and Il10 in BM cells. ELISA for TNF-α in the cell-free supernatants of BM cell–MSC coculture. (E) BM cells were cocultured with Con KD MSC, HGF KD MSC, or COX-2 KD MSC for 5 days under GM-CSF stimulation. Then, BM cells were separated and cocultured with CFSE-labeled CD4⁺ cells on anti-CD3 and anti-CD28 antibody–coated plates. After 5 days, CD4⁺ cell proliferation was evaluated by CFSE dilution assay and IFN-γ secreted level was measured by ELISA. (F) ELISA for PGE₂ and HGF in cultures of HGF KD MSC and COX-2 KD MSC, respectively. Three independent sets of experiments in total were performed. Each set contained 2–4 biological samples in each group, and 1 dot depicts data from 1 biological sample. Each biological sample was assayed in 3 technical replicates for RT-PCR and ELISA (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. One-way ANOVA and Tukey’s multiple-comparison test were used in B–E and Mann-Whitney test was used in F.