Hypothermia combined with extracellular vesicles from clonally expanded immortalized mesenchymal stromal cells improves neurodevelopmental impairment in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy following hypoxia-ischemia (HI) is a leading cause of childhood death and morbidity. Hypothermia (HT), the only available but obligatory therapy is limited due to a short therapeutic window and limited efficacy. An adjuvant therapy overcoming limitations of HT is still missing. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promising therapeutic effects in various brain injury models. Challenges associated with MSCs' heterogeneity and senescence can be mitigated by the use of EVs from clonally expanded immortalized MSCs (ciMSCs). In the present study, we hypothesized that intranasal ciMSC-EV delivery overcomes limitations of HT. METHODS: Nine-day-old C57BL/6 mice were exposed to HI by occlusion of the right common carotid artery followed by 1 h hypoxia (10% oxygen). HT was initiated immediately after insult for 4 h. Control animals were kept at physiological body core temperatures. ciMSC-EVs or vehicle were administered intranasally 1, 3 and 5 days post HI/HT. Neuronal cell loss, inflammatory and regenerative responses were assessed via immunohistochemistry, western blot and real-time PCR 7 days after insult. Long-term neurodevelopmental outcome was evaluated by analyses of cognitive function, activity and anxiety-related behavior 5 weeks after HI/HT. RESULTS: In contrast to HT monotherapy, the additional intranasal therapy with ciMSC-EVs prevented HI-induced cognitive deficits, hyperactivity and alterations of anxiety-related behavior at adolescence. This was preceded by reduction of striatal neuronal loss, decreased endothelial, microglia and astrocyte activation; reduced expression of pro-inflammatory and increased expression of anti-inflammatory cytokines. Furthermore, the combination of HT with intranasal ciMSC-EV delivery promoted regenerative and neurodevelopmental processes, including endothelial proliferation, neurotrophic growth factor expression and oligodendrocyte maturation, which were not altered by HT monotherapy. CONCLUSION: Intranasal delivery of ciMSC-EVs represents a novel adjunct therapy, overcoming limitations of acute HT thereby offering new possibilities for improving long-term outcomes in neonates with HI-induced brain injury.

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury.

BACKGROUND: Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action. METHODS: Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples. RESULTS: Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression. CONCLUSION: Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.