Fig 2: Stabilization of p53 by blockade of the p53-MDM2 binding inhibits vasculogenic differentiation of dental pulp stem cells.a shRNA-p53 transduced DPSC were treated with 0–2.5 µM MI-773 and 0–10 µM APG-115 for 24 h, western blots were performed for P53, MDM2, and p21, ß-actin was used as loading control. b DPSC were cultured with endothelial differentiation medium in the presence of 50 ng/ml VEGF and 0–0.25 µM MI-773 for 14 days, western blots for p53, MDM2, p21, Bmi-1, VEGFR2, Tie-2, and CD31. The density of protein expression was normalized with ß-actin. c, d 1 × 104 DPSC were seeded in growth factor-reduced matrigel and cultured in endothelial differentiation medium (EGM2) in the presence of 0–0.5 µM MI-773 for 13 days. c photographs of sprout, scale bar:100 µm. d Graph depicting the number of sprouts formed in c. Asterisk indicates p < 0.001, as determined by one-way ANOVA followed by a post hoc test (Tukey’s test).

Fig 3: Characterization of MSC and endothelial markers in dental pulp stem cells.Cell surface markers detected by flow cytometry in DPSC, SHED, and HDMEC under standard culture conditions for each cell type. a Flow plots depicting expression of MSC markers (i.e., CD73, CD90, CD105, CD34, CD44) using IgG-APC as isotype-matched control. b Flow plots depicting expression of endothelial cell-related proteins (VEGFR1 and CD146) using IgG-PE and IgG-FITC were used as isotype-matched controls. c RT-PCR for odontogenic/osteogenic markers (DSPP, DMP-1), endothelial cell-related markers (VEGFR1, VEGFR2, CD146, CD31, VE-cadherin) in primary human odontoblasts, dental pulp tissue, DPSC, SHED, or HDMEC. GAPDH was used as a loading control.

Fig 4: Endogenous T cells that infiltrate glioblastoma express distinct homing marker genes, and complementary chemokine genes are detected in other glioblastoma-associated cell clusters. (A) Representative 7µm glioblastoma tissue section identifying endogenous CD3+ T cells (green) localised adjacent to tumour-associated CD31+ blood endothelial cells (white) and infiltrating GD2+ tumour parenchyma (magenta). DAPI was used as a nuclear stain, scale bar: 50µm, representative of n=6 patient samples. (B) UMAP plot of T cells, NK cells, B cells, and macrophages identified within the lymphocyte cluster from pooled scRNA-seq analysis of n=3 patient glioblastoma biopsies. Cell clusters were identified according to differentially expressed genes (see Supplementary Figure 1 ). (C, D) Frequency of T cells within the lymphocyte cluster positive for (C) chemokine receptor and (D) integrin genes. Each dot represents one patient, bars represent mean ± SEM. nd=not detected. (E) UMAP plot of non-lymphocyte glioblastoma-associated cell clusters from pooled scRNA-seq analysis of n=3 patient glioblastoma biopsies. Endothelial cells (ECs), glioblastoma-associated macrophages and microglia (GAMs), pericytes and tumour cells were clustered and identified according to differentially expressed genes. (F, G) Frequency of ECs, GAMs, pericytes and tumour cells positive for (F) CXCL-, XCL- and CX3CL1, and (G) CCL-chemokine genes. Bars represent mean ± SEM, nd, not detected.

Fig 5: Silencing p53 enhances vasculogenic differentiation of DPSC in vitro and in vivo.a Photomicrographs depicting dental pulp stem cells that differentiated into functional blood vessels in vivo. GFP-transduced DPSC were seeded in scaffolds and transplanted in the subcutaneous space of the dorsum of SCID mice. Six weeks later, scaffolds were retrieved and tissue sections were prepared for immunofluorescence staining. Scattered cells showed positive staining for both, GFP (green), and SMA-alpha (red). In mature blood vessels, GFP was detected in the endothelial cells, SMA-alpha was stained the surrounding smooth muscle cells. Endothelial cells in the inner layer were stained for CD31 (green), while surrounding smooth muscle cells stained positive for SMA-alpha (red). Arrows point to DPSC-derived endothelial cells (green cells). Scale bar: 50 µm. b Western blots for VEGFR2, CD31, PDGFR-a, PDGFR-ß, SMA-a, p53, and Bmi-1 in DPSC, SHED, HUASMC, and HDMEC. c Endothelial cell differentiation: shRNA-transduced DPSC were cultured with 5%FBS–MEM for 14 days, western blots for p53, p21, VEGFR2, Tie-2, CD31, and VE-cadherin. The density of protein expression was normalized with ß-actin. d shRNA-p53-mediated vasculogenic differentiation in vivo: H&E and IHC staining. Blood vessels were revealed by H&E staining and detected with anti-factor VIII antibody, scale bar: 50 µm. e Graph depicting the blood vessel density observed in d stained with Factor VIII. Asterisk indicates p = 0.00035, as determined by t test. f DPSC cells were seeded in matrigel and cultured with EGM2 for 8 days. The Matrigel was fixed, and the sprouts were revealed by IF staining for CD31. Scale bar: 100 µm. g In all, 1 × 104 shRNA-transduced DPSC were seeded in growth factor-reduced matrigel-coated 12-well plate and cultured in endothelial differentiation medium (EGM2) for indicated time points. Sprouts were photographed, scale bar:100 µm. h Graph depicting the numbers of sprout formed in g. Three independent experiments using triplicate wells per condition were performed. Asterisk indicates p < 0.001, as determined by one-way ANOVA followed by a post hoc test (Tukey’s test).