Optimization of intranasal MSC therapy for neonatal hypoxic-ischemic brain injury by osteopontin preconditioning of MSCs

Abstract

Hypoxic ischemic encephalopathy (HIE) is one of the leading causes of infant mortality and development of long-term neurological disabilities. Current treatment options are limited, bringing a need for the development of new innovative treatment methods. Intranasal delivery of mesenchymal stem cells (MSCs) has shown promising results in reducing lesions size and improving motor outcomes in neonatal HIE animal models. However, there is still a room for improvement. MSC preconditioning has been shown as a promising strategy to improve MSC paracrine potency and therapeutic efficacy. This research aims to evaluate the potential of osteopontin (OPN) preconditioning of MSCs prior to intranasal administration as a strategy to increase the MSCs’ treatment efficacy. Additionally, this study aims to elucidate the effect of OPN on the secretome and intracellular pathways of MSCs. This study shows that the secretome of MSCs is changed after preconditioning for 24h with 1000ng/ml OPN. Upregulation of growth supportive and angiogenic genes TGF-b and VEGF was shown using qPCR. Inflammatory genes also seemed to be affected, as indicated by a non-significant upregulation of IL-6 and downregulation of iNOS and COX2 mRNA levels. Finally, there appeared to be an autocrine positive feedback loop activated in MSCs, as OPN mRNA levels were non-significantly upregulated after OPN incubation. Higher sample numbers are needed to confirm these non-significant results. Western blot analysis showed an activation in MSCs of the ERK and AKT pathway after 1 hour of OPN incubation. Additionally, there seemed to be an activation of the NF-κB pathway, most prominently at the early timepoints after incubation. The effect on neurogenesis of OPN preconditioned MSCs (OPN-MSCs) was evaluated in a non-contact MSC/NSC co-culture. OPN-MSCs increased differentiation of neuronal stem cells (NSCs) towards astrocytes and induced NSCs into the formation of more complex neurons. A MSC/microglia non-contact co-culture showed that microglia were activated by LPS and that MSCs were able to reduce activation as measured by TNF-α. OPN-MSC did not further reduce TNF-α secretion compared to naïve MSCs. The overall therapeutic efficacy needs to be elucidated in another in vivo mouse study. The changes in the secretome and the increased neurogenic capacity of the OPN-MSCs indicate that there is a potential for therapeutical benefits compared to naïve MSCs for neonatal HIE.