Hypoxic Preconditioning Enhances Post-implantation Cell Survival Through Metabolic Adaptations

Objectives: To examine the effects of hypoxia preconditioning by hypoxia-inducible factor-1 alpha (HIF-1α) stabilization on post implantation cell survival of stem cells from human deciduous teeth (SHED).
Methods: SHED were preconditioned to hypoxic environment by knocking down prolyl hydroxylase domain-containing protein 2 (PHD2) using target specific shRNA lentiviral particles. HIF-1α stabilization following PHD2 knockdown was confirmed by examining the expression of PHD2 and HIF-1α mRNA and protein levels by quantitative-PCR and western blot assays, respectively. HIF-1α-stabilized-SHED were encapsulated in Matrigel and injected into the subcutaneous space of the immunocompromised mice. The Matrigel plugs were retrieved after 3 and 7 days of implantation and examined for the cell viability using TUNEL immunohistochemical assay kit. The levels of intracellular ROS, Mitochondrial enzymes, ATP, glycolytic enzymes and glutathione in HIF-1α-stabilized-SHED were examined by qPCR, western blot or relevant assay kits.

Results: HIF-1α expression was significantly upregulated following knockdown of PHD2 in SHED (p<0.05). HIF-1α-stabilized SHED showed significantly enhanced cell survival in Matrigel plugs at days 3 and 7 of implantation compared to that of control-SHED (p<0.05). Furthermore, oxygen consumption rate and ATP levels were significantly reduced in HIF-1α-stabilized SHED (p<0.05) compared to control-SHED, indicating a less O₂ consumption. Mitochondrial enzymes, Cox4-1 and Cox4-2, were downregulated in HIF-1α-stabilized-SHED, resulting in less ROS production, while the total level of glutathione was increased enhancing the ROS scavenging activity (p<0.05). Significantly enhanced transcription of HIF-1α target genes encoding Glut1 and the glycolytic enzymes Hk2, Pfkfb3 and Ldh-a in HIF-1α-stabilized-SHED indicated a metabolic shift from oxidative phosphorylation to glycolysis in order to maintain energy homeostasis and cell survival during oxidative or nutrient stress.
Conclusions: HIF-1α stabilization by PHD2 knockdown enhanced post-implantation cell survival of SHED through adaptations in glutamine-mediated redox homeostasis and glycolytic metabolism. Therefore, targeting cellular metabolism is an appealing strategy for cell-based therapy.