Artificial Hypoxia Inducible Factor-1A (HIF-1A) Stabilisation For Enhanced Cell Survival in Regenerative Medicine

Hypoxia Inducible Factor-1α (HIF-1α) stabilisation activates a number of pro-survival pathways that enables increased cell survival in adverse environments. Hypoxic preconditioning (HP) is, therefore, a proposed strategy to increase cell survival for a number of clinical applications such as cell therapy, tissue engineering and organ transplantation. Changing the oxygen pressure can, however, cause undesirable cell behaviours including increased oxidative stress and cell death, in addition to practical difficulties in controlling oxygen levels in vitro (and in vivo). Here we investigate if stabilising HIF-1α through the use of HIF stabilising mimetics in normoxia, will offer a practical solution to activating prosurvival pathways, whilst limiting the undesirable effects of changing oxygen levels. Various cell survival models were investigated (cryopreservation, hypothermia, low nutrient condition), for their suitability to demonstrate enhanced cell survival following HP (1% O2, 24 h). The hypothermic (4oC) stress model reproducibly demonstrated increased cellular survival, resuscitation and recovery following HP. Using the same model, Hypoxia Mimetic Agents (HMAs): Cobalt (100μM) and DMOG (500μM) showed enhanced cell survival and recovery following hypothermic stress (P ≤ 0.001) in both liver hepatocellular carcinoma cells (HepG2) and adipose-derived mesenchymal stem cells (ADMSC). Interestingly HMAs treated daughter cells, which survived hypothermic stress for the first time, had enhanced cell survival when exposed to further cycles of hypothermic stress, suggesting an epigenetic effect. The HMAs dependent enhanced cell recovery observed in 2D culture was also observed in 3D (collagen type I hydrogel) culture. In 3D culture, the use of HMAs also appeared to enhance spheroid formation (as determined by measuring spheroid size following hypothermic stress). The mechanisms of enhanced cell survival were studied using functional assays and gene expression, where different HMAs showed different effects compared to HP. HMAs treated cells showed increased prosurvival factors expression such as HSP90α, COX2 and BNIP3 to enhance cell survival in adverse conditions. The use of HMAs could have an important role in improving the translation of regenerative medicine therapies