Abstract
Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
Highlights
Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles with distinct biogenesis, biophysical properties, and functions, which are common to virtually all cells and life forms [1]
We first performed an untargeted multiplex Tandem Mass Tag (TMT)-based proteomic analysis of the whole EV fraction and sucrose gradient–purified exosomes spontaneously released by Neural stem cell (NSC) in vitro and compared them with parental NSC whole-cell lysates (Fig 1A, S1 Data)
Using Gene Ontology Cellular Component (GOCC) annotations, we investigated the subcellular origin of proteins enriched in EVs compared with NSCs (Fig 1B)
Summary
Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles with distinct biogenesis, biophysical properties, and functions, which are common to virtually all cells and life forms [1]. Despite their proven biological potential, the characterisation and classification of this heterogeneous population of membrane vesicles has far been challenging. Exosomes are small vesicles, ranging 30 to 150 nm in diameter, generated from the inward budding of intracellular multivesicular bodies and released after the subsequent fusion with the plasma membrane [3]. As the classification of EVs is continuously evolving, recent consensus has further simplified nomenclature by dichotomising EVs into 2 major categories: ectosomes, for particles released through plasma membrane budding; and exosomes, for particles originated from the endosomal pathway [5]
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