Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not ‘normoxic’ (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS–MS revealed 52 proteins differentially abundant in sEVs from hypoxic and ‘normoxic’ MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix–receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix–receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

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.

ab CD8 + , gd, and NK lymphocytes are fundamental effector cells against viruses and tumors. These cells can be divided into multiple subsets according to their phenotype. Based on progressive telomere attrition from naive to late effector memory cells, human CD8 + T cell subsets have been positioned along a pathway of differentiation, which is also considered as a process of lymphocyte aging or senescence. A similar categorization has not been clearly established for gd and NK cell populations. Moreover, the distinction between the aging of these populations due to cellular differentiation or due to the chronological age of the donor has not been formally considered. In this study, we performed systematic measurements of telomere length and telomerase activity in human ab CD8 + , gd, and NK lymphocytes based on subset division and across age to address these points and better understand the dichotomy between differentiation and temporal aging. This approach enables us to position phenotypically distinct gd or NK subsets along a putative pathway of differentiation, such as for CD8 + T cells. Moreover, our data show that both cellular differentiation and donor aging have profound but independent effects on telomere length and telomerase activity of lymphocyte subpopulations, implying distinct mechanisms and consequences on the immune system.

Kidney disease is a growing public health problem worldwide, including both acute and chronic forms. Existing therapies for kidney disease target various pathogenic mechanisms; however, these therapies only slow down the progression of the disease rather than offering a cure. One of the potential and emerging approaches for the treatment of kidney disease is mesenchymal stromal/stem cell (MSC) therapy, shown to have beneficial effects in preclinical studies. In addition, extracellular vesicles (EVs) released by MSCs became a potent cell-free therapy option in various preclinical models of kidney disease due to their regenerative, anti-inflammatory, and immunomodulatory properties. However, there are scarce clinical data available regarding the use of MSC-EVs in kidney pathologies. This review article provides an outline of the renoprotective effects of MSC-EVs in different preclinical models of kidney disease. It offers a comprehensive analysis of possible mechanisms of action of MSC-EVs with an emphasis on kidney disease. Finally, on the journey toward the implementation of MSC-EVs into clinical practice, we highlight the need to establish standardized methods for the characterization of an EV-based product and investigate the adequate dosing, safety, and efficacy of MSC-EVs application, as well as the development of suitable potency assays.

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.

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