HIF-1α-Stabilized SHED Spheroids Enhance Endothelial Differentiation and Perfusion Recovery

Abstract

<p>Objectives: To examine the endothelial differentiation potential of hypoxia inducible factor (HIF)-1α stabilized stem cells from human exfoliated deciduous teeth (SHED) in 3D culture and their capacity in promoting perfusion recovery in mouse hindlimb ischemia model.<br>Methods: Stabilization of HIF-1α in SHED was achieved via knockdown of prolyl hydroxylase domain (PHD)-2 using target specific shRNA lentiviral particles. HIF-1α-stabilized-SHED were seeded onto 3D Petri dishes fabricated using 3D micro-molds (3D-MicrotissueTM) and allowed to self-assemble into 3D microtissue spheroids. The cells were cultured inendothelial growth medium 2 (EGM2) either in 2D plates for 14 days, or in 3D spheroids for 7 days followed by 2D plates for 7 days. Expression of endothelial specific markers, including CD31 and VEGFR2 was determined by western blotting and immunofluorescence. In vitro Matrigel assay was performed to examine the endothelial function of SHED after differentiation. Cells were mixed in fibrin gel and injected into 4 locations of induced ischemic hind limb muscle of immunocompromised mice. Blood perfusion was assessed using Laser Speckle Contrast Imaging at day 7, 14, 21, and 28.<br>Results: HIF-1α-stabilized-SHED secreted significantly high VEGF levels and when cultured in 3D spheroids showed significantly higher expression of CD31 and VEGFR2 in both western blotting and immunofluorescence compared with control groups (p < 0.05). In Matrigel assay, 3D induced HIF-1α-stabilized-SHED formed vascular tubes with significantly higher number of nodes, junctions and total length, which resembled closely with that of HUVECs (p < 0.05). In hindlimb ischemic mice, injection of 3D induced HIF-1α-stabilized SHED resulted in significantly higher perfusion recovery and perfusion ratio with less visible necrosis compared to the control groups (p < 0.05).<br>Conclusions: HIF-1α-stabilized SHED in 3D spheroids can effectively differentiate into functional endothelial cells and promote perfusion recovery in hind limb ischemic mice.<br></p>