Hypoxia-inducible factor-1α stabilized stem cells from human deciduous teeth in 3D spheroids promote endothelial differentiation and improve hindlimb ischemia

Critical limb ischemia (CLI) remains a major clinical challenge with limited treatment options. This study explores a novel approach to enhance the therapeutic potential of stem cells from human exfoliated deciduous teeth (SHED) by combining hypoxia-inducible factor-1 (HIF-1) stabilization with 3D spheroid culture, with the aim of boosting endothelial differentiation and improving outcomes in a mouse model of CLI. SHED were transfected with small hairpin RNA (shRNA) targeting prolyl hydroxylase domain-containing protein 2 (PHD2) to stabilize HIF-1α, then self-assembled into 3D spheroids on non-adhesive microwells. Spheroid diameters of 200 μm, 400 μm, and 600 μm were tested for endothelial differentiation. After 7 days of 3D culture, spheroids were transferred to 2D culture dishes and maintained for an additional 7 days to obtain monodispersed Induced-SHED. Endothelial differentiation was assessed via qRT-PCR, western blotting, immunofluorescence, Matrigel tube formation, and RNA sequencing. Induced-SHED were encapsulated in fibrin hydrogel and injected into mice with surgically induced hindlimb ischemia (HLI). Therapeutic effects were evaluated by blood perfusion, ischemia scoring, immunohistochemistry, and Masson’s trichrome staining. Statistical analysis used Student’s t-test and one-way ANOVA with Tukey’s post hoc test. PHD2 knockdown led to a sevenfold increase in HIF-1 expression without affecting proliferation. Spheroids of 400 μm in diameter exhibited the highest expression of endothelial markers (CD31, VEGFR2) at both the mRNA and protein levels and were identified as optimal for promoting endothelial differentiation. Induced-SHED formed robust vascular networks on Matrigel, with sprouting lengths significantly greater than controls and exhibited phenotypic and functional properties similar to those of endothelial cells in vitro. In vivo, HLI mice treated with Induced-SHED showed markedly improved blood perfusion, reduced ischemic scores, enhanced angiogenesis (as indicated by increased CD31 + vessels), and less muscle fibrosis compared with controls. RNA sequencing revealed upregulation of insulin-like growth factor 1 (IGF1), which was shown to potentiate cell-extracellular matrix interactions, thereby inducing endothelial differentiation of SHED. The combination of HIF-1α stabilization and 3D spheroid culture significantly enhanced SHED endothelial differentiation and therapeutic potential in treating CLI.