Fig. 5

VEGFA is a potential functional molecule by which MSCs promote MRC regeneration. A The top 10 ligands were screened and ranked via bulk RNA sequencing data from MSCs in vitro and those distributed in aged spleens. B Relative VEGFA mRNA expression was detected through RT‒PCR validation in the MSC^NC, MSC^VEGFA-KD1, MSC^VEGFA-KD2, and MSC^VEGFA-KD3 groups (n = 5). C Flow cytometry analysis of splenic MRC proliferation (CD45^-CD31^-Ter119^-MAdCAM-1⁺KI-67⁺) in the Ctrl, MSC^NC, MSC^VEGFA-KD1, and MSC^VEGFA-KD2 groups; n = 3 mice per group. D Vehicle (Ctrl), nonsense control MSC (MSC^NC) or VEGFA-knockdown MSC (MSC^VEGFA-KD) were administered to aged BALB/C mice (>18 months old), which were analyzed 28 days later (E, F) or then immunized with OVA (G‒J). E Representative immunofluorescence staining of splenic stromal cells (PDGFRβ⁺, fuchsia) in the control, MSC^NC, MSC^VEGFA-KD1, and MSC^VEGFA-KD2 groups. Scale bars: 100 μm. F Statistical analysis of the proportion of splenic stromal cells in the Ctrl, MSC^NC, MSC^VEGFA-KD1, and MSC^VEGFA-KD2 groups; n = 3 mice per group. G Flow cytometry analysis of splenic germinal center B-cell populations (B220⁺IgD^-GL7⁺) 28 days after OVA immunization in the control (OVA Ctrl), MSC^NC + OVA, MSC^VEGFA-KD1 + OVA, and MSC^VEGFA-KD2 + OVA groups. H Representative immunofluorescence staining of follicular B lymphocytes (IgD⁺, fuchsia), germinal centers (GL-7⁺, green), and DAPI (blue) in the OVA Ctrl, MSC^NC + OVA, MSC^VEGFA-KD1 + OVA, and MSC^VEGFA-KD groups; scale bars: 70 μm. I Statistical analysis of the splenic GC area (GL-7⁺) and GL-7⁺ RawIntden population in the OVA Ctrl, MSC^NC + OVA, MSC^VEGFA-KD1 + OVA and MSC^VEGFA-KD2 + OVA groups; n = 3 mice per group. J Titers of serum soluble ovalbumin-specific IgG1 antibodies after 28 days of immunization in the OVA Ctrl, MSC^NC + OVA, MSC^VEGFA-KD1 + OVA and MSC^VEGFA-KD2 + OVA groups; n = 4 mice per group. K Recombinant VEGFA was administered to aged BALB/C mice (>18 months old), and the results were analyzed 3 days later (L‒N). L Flow cytometry analysis of splenic MRC proliferation (CD45^-CD31^-Ter119^-MAdCAM-1⁺KI-67⁺) in the vehicle (control) or recombinant VEGFA delivery (rVEGFA) groups after 3 days; n = 3 mice per group. (M) Representative immunofluorescence images of splenic MRCs (MAdCAM-1⁺, fuchsia), Ki-67 (green), and DAPI (gray) in the control and rVEGFA groups; scale bars: 5 μm. N Statistical analysis of splenic MRC proliferation in the control and rVEGFA groups; n = 5 mice per group. O Recombinant VEGFA was administered to aged BALB/C mice (>18 months old), and the results were analyzed 28 days later (P–R) or after ovalbumin immunization (S–V). P Representative flow cytometry of splenic stromal cells (CD45^-CD31^-Ter119^-PDGFRβ⁺) in the control and rVEGFA groups. O Statistical analysis of the proportion of splenic stromal cells in the control and rVEGFA groups; n = 3 mice per group. R Statistical analysis of splenic stromal cell counts in the control and rVEGFA groups; n = 3 mice per group. S Flow cytometry analysis of splenic germinal center B-cell populations (B220⁺IgD⁻GL7⁺) in the control (OVA Ctrl) and recombinant VEGFA groups 28 days before OVA immunization (rVEGFA +OVA); n = 3 mice per group. T Representative immunofluorescence staining of B lymphocytes (B220⁺, red) and germinal centers (PNA⁺, green) in the OVA Ctrl and rVEGFA +OVA groups; scale bars: 50 μm. U Statistical analysis of the splenic GC area (PNA⁺) in the OVA Ctrl and rVEGFA +OVA groups; n = 4 per group. V Titers of serum soluble ovalbumin-specific IgG1 antibodies after 28 days of OVA immunization in the groups of no immunization (Control), sham control (OVA Ctrl), and recombinant VEGFA administration 28 days before OVA immunization (rVEGFA +OVA), n = 4 mice per group. The data represent the means ± SEMs of three independent experiments. In (C, F, G, I, J), statistical significance was determined via one-way ANOVA with a multiple comparison test. In (L, N, Q, R, S, U, V), statistical significance was determined via a two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns not significant