Irradiated mice given CpG-MSCs have greater myeloid differentiation and proliferation potential after infection compared with mice given MSC and PBS alone, an effect that is partially TLR9 dependent. Representative images of (A) CFU-G, (B) CFU-M, and (C) CFU-GM. Images were taken on a VWR Trinocular Inverted Microscope using an OMAX 18.0MP USB 3.0 digital camera at 4× original magnification acquired on ToupView software and cropped in Fiji ImageJ. Representative colonies were selected to confirm the presence of Ly6G⁺ neutrophils (dashed line) in CFU-Gs, F4/80⁺ macrophages (dashed line) in CFU-Ms compared to unstained controls (blue filled curve), and both Ly6G⁺ neutrophils and F4/80⁺ macrophages in CFU-GMs. (D) 1000 c-kit⁺ cells isolated from the bone marrow of irradiated mice administered PBS, MSC, or CpG-MSC (n = 3 per group) and infected with pulmonary P aeruginosa were seeded into methylcellulose media in triplicate. Cultures were grown for 9 days before blinded manual determination of CFU-GM (purple bars), CFU-M (orange bars), or CFU-G (green bars). Data were assessed by 2-way ANOVA; interaction P = .005. †Significant comparisons of CFU-GMs by Bonferroni’s multiple comparisons test, PBS vs MSC, P = .012; *PBS vs CpG-MSC, P < .0001; and §CpG-MSC vs MSC, P = .011. (E) CFU counts when irradiated and infected mice were given PBS, CpG-MSCs, or CpG TLR9KD-MSCs (n = 3 per group). Data were assessed by 2-way ANOVA; interaction P < .0001. §Significant comparison of CFU-Gs by Bonferroni’s multiple comparisons test, CpG Scr-MSC vs CpG TLR9KD MSC, P = .011. (F) Quantification of the total neutrophil count per plate after 9 days of growth from 1000 c-kit⁺ cells harvested from mice that received PBS (green bars), CpG Scr-MSCs (blue bars), or CpG TLR9KD-MSCs (red bars). Data were assessed by 2-way ANOVA; interaction P = .009. *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSCs, P = .010. (G) TLR9 protein expression by flow cytometry in fluorescence minus 1 (FMO) wild-type control, MSCs transduced with scramble shRNA (MFI 8.6) and MSCs transduced with TLR9 shRNA (MFI 4.4). Based on differences in MFI, there is ∼50% TLR9 protein knockdown in TLR9KD MSCs.