(176f) 3D Aggregation Culture Enhances Therapeutic Outcome of Human Mesenchymal Stem Cells in Ischemic Stroke Treatment

Human mesenchymal stem cells (hMSCs) have been shown to enhance stroke lesion recovery through secretion of trophic factors that mediate inflammation and tissue repair. However, low cell survival and reduced secretory functions post-transplantation of culture-expanded hMSCs are the major barriers limiting the therapeutic efficacy of hMSCs in stroke treatment.¹ To enhance hMSC’s therapeutic efficacy, preconditioning strategies such as hypoxia has shown promising results of lesion and functional recovery for the middle cerebral artery occlusion (MCAO) rat model. In this study, we report the impact of in vitro preconditioning of hMSCs via three-dimensional (3D) aggregation culture of hMSC. 3D aggregation culture enhanced hMSCs secretory profile, resistance to ischemic stress, in vivo survival at stroke lesion site, and stroke lesion recovery. For aggregation process, hMSCs spontaneously formed 3D aggregates on ultra-low adherent (ULA) culture plate. Aggregate-derived hMSCs were acquired via dissociation and labeled with micron-sized paramagnetic iron oxide (MPIO) particles for in vivo magnetic resonance imaging (MRI) analysis, and then injected intra-arterially in a middle cerebral artery occlusion (MCAO) stroke rat model. To assess the influence of 3D preconditioning on hMSC in vivo lifespan and stroke lesion recovery, serial MRI at 21.1 T was performed to acquire images of lesion progression and cell migration. The influence of 3D aggregation culture on the secretion of anti-inflammatory, pro-angiogenic, and anti-apoptotic cytokines was analyzed. Our results show that 3D aggregation culture significantly reduced hMSCs cell size with with enhanced SDF-1α-induced migration and upregulation of CXCR4 gene. Enhanced migratory properties of 3D aggregation culture could be regulated through a caspase-mediated mechanism.^2,3 Aggregate-derived hMSCs also have the increased secretion profile of multiple growth and anti-inflammatory factors. Cell metabolism and redox cycle was reconfigured and activated PI3K/Akt survival pathway contributes to the resistance of in vitro ischemia and reactive oxygen species (ROS) enriched environment in 3D aggregate-derived hMSCs. MRI images of the ischemic stroke lesion showed increased ¹H and ²³Na signal as evidence of the influx of extracellular water and disruption of ionic homeostasis. Lesion volume analysis indicated increased recovery for 3D cultured hMSCs group with statistical significance for ²³Na MRI. After one week of initial transplantation, percentage decrease of MPIO contrast on T₂* weighted images was calculated to evaluate cell clearance. Neurological function recovery post-transplantation was evaluated by behavioral test including open field, elevated maze plus and cylinder test. Together, the results demonstrated that 3D aggregation precondition of hMSCs is an effective strategy that enhances their therapeutic performance in stroke lesion recovery compared to standard adherent culture.