G·A·B·B·A

Somatic stem cell-based therapies protect the central nervous system (CNS) from chronic inflammation-driven degeneration, such as that occurring in experimental autoimmune encephalomyelitis (EAE) and stroke. It was first assumed that stem cells directly replace lost/damaged cells, but it has now become clear that they are able to protect the damaged nervous system through mechanisms other than cell replacement. In immune-mediated experimental demyelination and stroke we have shown that transplanted neural stem/precursor cells (NPCs) possess a constitutive and inducible ability to mediate efficient bystander myelin repair and axonal rescue. Yet, a comprehensive understanding of the mechanisms by which NPCs exert their therapeutic impact is lacking. To describe the various therapeutic actions of stem cell transplants in vivo, namely their capacity to adapt fate and function(s) to specific microenvironments, we have proposed the concept of therapeutic plasticity. Recent evidence demonstrates that some of the NPC functions, including the immune regulation, require sophisticated mechanisms of intercellular communication. While some communication is delivered by secreted cytokines and/or growth factors, our recent work suggests that a key role might be attributed to a novel mechanism of intercellular communication through the transfer of secreted membrane vesicles (MVs) from donor to recipient cells. Here we have focused on defining whether this form of communication exists for NPCs, and on elucidating its molecular signature and therapeutic relevance. Here we have shown that NPCs derived from cultured neurospheres secrete large numbers of microvesicles comprising a mixed population of exosomes and larger particles, similar to a previously described class of shedding membrane particles. We identified ~900 proteins that are specifically associated with exosomes, of which 11-14% were differentially expressed in response to pro-inflammatory and anti-inflammatory cytokines, with a suppression of proteins involved in RNA translation and processing. Deep RNA sequencing revealed 35 miRs that were highly enriched within exosomes, compared to NPCs, including mmu-miR-9 and mmu-miR-181a that have well characterized pro-neurogenetic and immune-regulatory potential, respectively. We also observed a highly selective depletion of miRNA star (miR*) sequences within exosomes. All miRNAs were repressed in exosomes after exposure to pro-inflammatory cytokines, with the notable exception of mmu-miR-181a and mmu-miR-181b. Similar data were obtained from whole microvesicle preparations. Finally, we show that microvesicle contents can be transferred to recipient fibroblasts, with consequent changes in recipient cell RNA and protein expression, and effects on cell cycle regulation, and the induction of stress response and MHC-class I dependent antigen presentation pathways in the case of exosomes from cells exposed to pro-inflammatory conditions. These results show that there is highly selective protein and miRNA trafficking into secreted vesicles, and that these vesicles can transfer regulatory information to other cells, with important implications for understanding intercellular discourse and the development of cellular therapies.