Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal cells were GFP expressing and immunopositive for collagen type I, fibronectin, and CD44. Transplantation of these cells by direct microinjection into the demyelinated spinal cord of the immunosuppressed rat resulted in remyelination. The remyelinated axons showed characteristics of both central and peripheral myelination as observed by electron microscopy; conduction velocity of the axons was improved. GFP-positive cells and myelin profiles were observed in the remyelinated spinal cord region, indicating that the donor-isolated stromal cells were responsible for the formation of the new myelin. The GFP-positive cells were colocalized with myelin basic protein-positive and P0-positive cellular elements. These findings indicate that cells contained within the stromal cell fraction of the mononuclear cell layer of bone marrow can form functional myelin during transplantation into demyelinated spinal cord.

Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSC^exos). In this study, we assessed the possible mechanism of MSC^exos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSC^exos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSC^exos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR “hotspots” were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSC^exos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.

Intravenous (IV) infusion of bone marrow‐derived mesenchymal stem/stromal cells (MSCs) stabilizes the blood‐spinal cord barrier (BSCB) and improves functional recovery in experimental models of spinal cord injury (SCI). Although IV delivered MSCs do not traffic to the injury site, IV delivered small extracellular vesicles (sEVs) derived from MSCs (MSC‐sEVs) do and are taken up by a subset of M2 macrophages. To test whether sEVs released by MSCs are responsible for the therapeutic effects of MSCs, we tracked sEVs produced by IV delivered DiR‐labelled MSCs (DiR‐MSCs) after transplantation into SCI rats. We found that sEVs were released by MSCs in vivo, trafficked to the injury site, associated specifically with M2 macrophages and co‐localized with exosome markers. Furthermore, while a single MSC injection was sufficient to improve locomotor recovery, fractionated dosing of MSC‐sEVs over 3 days (F‐sEVs) was required to achieve similar therapeutic effects. Infusion of F‐sEVs mimicked the effects of single dose MSC infusion on multiple parameters including: increased expression of M2 macrophage markers, upregulation of transforming growth factor‐beta (TGF‐β), TGF‐β receptors and tight junction proteins, and reduction in BSCB permeability. These data suggest that release of sEVs by MSCs over time induces a cascade of cellular responses leading to improved functional recovery.