Mesenchymal Stromal Cell-Derived Extracellular Vesicles Reduce Neuroinflammation, Promote Neural Cell Proliferation and Improve Oligodendrocyte Maturation in Neonatal Hypoxic-Ischemic Brain Injury

Abstract

Background: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury.Methods: Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors.Results: While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination.Conclusion: The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.

Comprehensive Profiling of Secretome Formulations from Fetal- and Perinatal Human Amniotic Fluid Stem Cells

“…Proteomic analysis reported increased expression of proteins related to angiogenesis, like NRP1 [ 86 ] and MMP14 [ 87 ] in hypoxic perinatal hAFS-EVs; such stimulatory profile may explain previous results on the endothelial regenerative properties of III trimester hAFS in a preclinical mouse model of skeletal muscle ischemic injury [ 25 ], despite the evidence of their hAFS-CM being less pro-angiogenic than the corresponding fetal one. Notably, the neural growth factor BDNF was found in both fetal- and perinatal EV cargo, although in low amounts, thus suggesting a putative neurotrophic activity for hAFS-EVs in neuronal survival and neurodevelopmental processes, as also observed for extracellular vesicles secreted by human bone marrow- and umbilical cord blood-MSC [ 88 , 89 ]. Both secretome formulations from fetal- and perinatal hAFS undergoing hypoxic stimulation showed to be enriched with PAI-1, a facilitator of endothelial activation [ 90 ] that has also been involved in the polarization of M2 macrophages in the heart and endowed with cardioprotective and anti-fibrotic potential [ 91 ].…”

Imaging flow cytometry challenges the usefulness of classically used EV labelling dyes and qualifies that of a novel dye, named Exoria™ for the labelling of MSC-EV preparations

“…These protocols allowed us to investigate whether tetraspanins, specifically CD9, CD63 and CD81, whose expression within EV samples has been confirmed by WB, are co-localized on individual EVs or are recovered on distinct EV subsets. With this technology we analyzed EV preparations from mesenchymal stromal cell (MSC) conditioned, human platelet lysate supplemented media, whose therapeutic activities we study in different animal models and confirmed their therapeutic potential in a GvHD patient (Doeppner et al, 2015;Drommelschmidt et al, 2017;Kaminski et al, 2020;Kordelas et al, 2014;Ophelders et al, 2016;Wang et al, 2020). We demonstrated that CD9 and CD81 reside on different EV subpopulations all in the exosomal size range (Görgens et al, 2019).…”

“…Aiming to identify a dye allowing specific labelling of EVs in therapeutically active MSC-EV preparations, we decided to evaluate the accuracy of conventionally used EV labelling dyes, specifically CFSE, Calcein AM, PKH67, BODIPY-TR-Ceramide and a novel lipid dye named Exoria. MSC-EV preparations that have been extensively explored in various animal models had been obtained from supernatants of MSCs raised in 10% human platelet lysate supplemented media by our well established PEG-ultracentrifugation protocol (Borger et al, 2020;Doeppner et al, 2015;Drommelschmidt et al, 2017;Gussenhoven et al, 2019;Kaminski et al, 2020;Kordelas et al, 2014;Ludwig et al, 2018;Ophelders et al, 2016;Wang et al, 2020). Since micelle formation of some of the dyes have been reported and following the MIFlowCyt-EV recommendation (Welsh et al, 2020), we initially added all of the labelling dyes but the EV sample to the NaCl-HEPES buffer, the buffer MSC-EVs are suspended in.…”

Mesenchymal Stem Cell Derived Extracellular Vesicles for Repairing the Neurovascular Unit after Ischemic Stroke

“…In addition, post-stroke dementia may be related to lower cerebral perfusion and impairments of the NVU [ 61 , 62 ]. Research suggests that administration of MSCs and MSC-EVs is able to boost the brain’s regenerative potential [ 63 , 64 , 65 , 66 , 67 ]. The therapeutic effects of MSC-EV administration yield functionally equivalent benefits to MSC administration, including angiogenesis, neuroprotection, neurogenesis, and functional recovery [ 63 , 64 , 65 ].…”

“…In a rat traumatic brain injury (TBI) model, MSC-derived exosomes did not affect lesion volume; however; it did improve functional recovery, increase vascular density, increase the number of new neuroblasts, reduce inflammation, and increase angiogenesis [ 69 ]. Administration of MSC-EVs during the subacute phase of neonatal hypoxic-ischemic (HI) brain injury resulted in increased proliferation of endothelial cells, as well as a reduction in pro-inflammatory astroglia and microglia activations [ 66 ]. These studies show that MSC-EVs exert positive regenerative effects on the ruptured BBB.…”