Spheroids of HIF-1α-Stabilized SHED Demonstrate Enhanced Endothelial Differentiation

Abstract

Objectives: To examine the potential of hypoxia inducible factor -1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) and 3D spheroid culture in promoting the differentiation of stem cells from deciduous teeth (SHED) into endothelial cells. Methods: Knockdown of PHD2 in SHED was achieved via target specific shRNA lentiviral particles. Efficiency of PHD2 knockdown and HIF-1α stabilization was examined by western blotting and RT-PCR. Cell proliferation following HIF-1α stabilization was assessed by Cell Counting Kit (CCK)-8 assay. Micro-molds (3D-MicrotissueTM) were used to facilitate the self-assembly of cells into 3D microtissue spheroids. Endothelial growth medium 2 (EGM2) was used to culture HIF-1α-stabilized-SHED in 2D culture or 3D spheroids (2D-Control-SHED, 2D-PHD2KD-SHED, 3D-Control-SHED and 3D-PHD2KD-SHED). Expression of specific endothelial markers, including CD31 and vascular endothelial growth factor receptor-2 (VEGFR2) was determined by real time PCR, western blotting and immunofluorescence. Results: Western blotting and RT-PCR results showed successful knockdown of PHD2 and HIF-1α stabilization with respective protein and m-RNA expression levels. There was no significant difference in cell proliferation following PHD2 knockdown. Expression of CD31 mRNA in 3D-Control-SHED and VEGFR2 in 2D-PHD2KD-SHED was significantly higher than that of the 2D-Control-SHED on day 2 and day 4 (p<0.05), respectively. CD31 and VEGFR2 mRNA expression in the 3D-PHD2KD-SHED was significantly higher than that of the 2D groups while CD31 expression was almost twice as high as that of the 3D-Control-SHED on day 2 (p<0.05). As for protein levels, the highest CD31 and VEGFR2 expression was observed in 3D-PHD2KD-SHED after 7 days of induction, while 2D-PHD2KD-SHED and 3D-Control-SHED showed significantly higher expression levels compared with that of 2D-Control-SHED (p<0.05). Similar expression patterns were observed with immunofluorescence. Conclusions: PHD2 knockdown effectively stabilizes the expression of HIF-1α in SHED. Once assembled into 3D spheroids, HIF-1α-stabilized-SHED could significantly enhance the differentiation of cells into endothelial cells.