Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)-derived EVs (MSC-EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC-EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long-term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow-derived MSC lineages. MSC-EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC-EVs, postischemic immunosuppression (i.e., B-cell, natural killer cell, and T-cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC-EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof-of-concept studies in stroke patients. Significance: Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC-induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long-term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post-stroke immune responses. Also, because EVs are well-tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.

Preterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. 3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. MSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. Significance: Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration of living cells. The therapeutic efficacy of systemically administered mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) on hypoxia-ischemia-induced injury was assessed in the preterm ovine brain. Impaired function and structural injury of the fetal brain was improved following global hypoxia-ischemia. A cell-free preparation of MSC-EVs could substitute for the cellular counterpart in the treatment of preterm neonates with hypoxic-ischemic brain injury. This may open new clinical applications for “off-the-shelf” interventions with MSC-EVs.

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However, this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g., LMPPs), implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here, we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP), whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34⁺CD90⁺CD45RA⁻, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.