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

Abstract

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.

The Long-Term Neuroprotective Effect of the Endocannabinoid 2-AG and Modulation of the SGZ’s Neurogenic Response after Neonatal Hypoxia-Ischemia

“…First, we showed that HI reduced cell proliferation in the SGZ: this effect has been previously described [ 39 , 40 ], whereas others have shown either no differences in the number of proliferating cells between HI and sham-operated animals [ 41 ] or increased [ 42 ] total counts of new cells after HI. The treatment with 2-AG increased the number of Ki67+ proliferating cells in both the whole hippocampus and the SGZ when compared to non-treated HI animals.…”

EV products obtained from iPSC-derived MSCs show batch-to-batch variations in their ability to modulate allogeneic immune responses in vitro

“…To this end, we decided to immortalize MSCs and try to expanded them at the clonal level. Indeed, as described recently, we have successfully established this method and have expanded the first monoclonal cell lines for fare more than 100 population doublings without observing any indication of senescence, thus, clearly overcoming the Hayflick’s limit ( Labusek et al, 2023 ). Notably, being expanded in appropriate media, some of these clonal immortalized MSC (ciMSC) lines repetitively allowed the preparation of EV products with confirmed in vitro immunomodulatory properties, while EV products of other ciMSC lines for now always failed to show such activities.…”

“…Combined to our specific interest in establishing novel treatment strategies for perinatal brain injuries ( Jensen, 2023 ), ciMSC-EVs were administered to a murine model of neonatal hypoxia induced encephalopathy. The ciMSC-EV application significantly suppressed the disease symptoms at various cellular levels implying a multimodal mode of action that can hardly be mimicked by monomolecular compounds ( Labusek et al, 2023 ). Thus, even though we cannot exclude that appropriate production strategies might overcome some of the discussed heterogeneity issues, we consider future MSC-EV therapeutics will derive from monoclonal MSC lines.…”

Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives

“…The authors also demonstrated in vitro using the HL-1 cardiomyocyte line that the protective effect of these exosomes is associated with the activation of the gp130/JAK signaling pathway [ 97 ]. In a recent paper by Labusek et al, exosomes derived from immortalized MSCs are shown to have similar neuroprotective capacity as exosomes from primary MSCs in a mouse model of neonatal hypoxic-ischemic brain injury [ 98 ]. An article by Zhang et al shows a positive effect of topically applied exosomes derived from E1-MYC-immortalized human MSCs in a mouse imiquimod-induced psoriasis model [ 99 ].…”