Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait co-ordination after spinal cord injury.

Autologous cell transplantation is a promising strategy for repair of the injured spinal cord. Here we have studied the repair potential of mesenchymal stromal cells isolated from the human olfactory mucosa after transplantation into a rodent model of incomplete spinal cord injury. Investigation of peripheral type remyelination at the injury site using immunocytochemistry for P0, showed a more extensive distribution in transplanted compared with control animals. In addition to the typical distribution in the dorsal columns (common to all animals), in transplanted animals only, P0 immunolabelling was consistently detected in white matter lateral and ventral to the injury site. Transplanted animals also showed reduced cavitation. Several functional outcome measures including end-point electrophysiological testing of dorsal column conduction and weekly behavioural testing of BBB, weight bearing and pain, showed no difference between transplanted and control animals. However, gait analysis revealed an earlier recovery of co-ordination between forelimb and hindlimb stepping in transplanted animals. This improvement in gait may be associated with the enhanced myelination in ventral and lateral white matter, where fibre tracts important for locomotion reside. Autologous transplantation of mesenchymal stromal cells from the olfactory mucosa may therefore be therapeutically beneficial in the treatment of spinal cord injury. GLIA 2017 GLIA 2017;65:639-656.

A comparative study of glial and non-neural cell properties for transplant-mediated repair of the injured spinal cord.

Cell transplantation is one strategy for encouraging regeneration after spinal cord injury and a range of cell types have been investigated for their repair potential. However, variations in study design complicate determination of which cells are most effective. In this study we have carried out a direct comparison of the regenerative and integrative properties of several cell preparations following transplantation into the lesioned rat spinal cord. Transplants included: (i) purified olfactory ensheathing cells (OECs) and (ii) fibroblast-like cells, from olfactory bulb (OBFB-L), (iii) a 50:50 mixture of (i) and (ii) (OEC/OBFB-L), (iv) dissociated nasal mucosa (OM), (v) purified peripheral nerve Schwann cells (SCs), (vi) peripheral nerve fibroblasts, and (vii) skin fibroblasts (SF). All transplants supported axonal regeneration: OECs and SCs promoted the greatest regeneration while OBFB-like cells were least efficient and mixed cell populations were less effective than purified populations. Tract-tracing experiments demonstrated that none of the cell types promoted regeneration beyond the lesion. Although all cell types prevented cavity formation, the extent of astrocytic hypertrophy [GFAP immunoreactivity (IR) at the transplant/lesion site] differed markedly. OECs and SCs were associated with the least GFAP-IR, fibroblasts and fibroblast-like cells resulted in greater GFAP-IR while hypertrophy surrounding transplants of OM was most extensive. These differences in host-transplant reactivity were confirmed by transplanting cells into normal spinal cord where the cellular interaction is not complicated by injury. Thus, purified glial cells have advantages for transplant-mediated repair, combining maximal support for axonal regeneration with a minimal astrocytic reaction around the transplant site.

Transplant-mediated repair properties of rat olfactory mucosal OM-I and OM-II sphere-forming cells.

Olfactory mucosa is a source of cells for transplant-mediated repair of spinal cord injury (SCI) and is currently being assessed in clinical trials. We previously reported that olfactory mucosa can generate two types of sphere-forming cells with stem cell-like properties. Here we have assessed the repair potential of these cells in a rodent SCI model. Sphere-forming cells transplanted into a dorsal column injury integrated with the host spinal cord, filling the injury cavity, but showed no evidence of differentiation in vivo. Moreover, transplants supported robust axonal regeneration, particularly when suspensions of smaller spheres, rather than large aggregates, were transplanted. However, tract-tracing of dorsal column fibers showed that regenerating axons did not extend beyond the transplant. These observations show that undifferentiated olfactory spheres, though capable of supporting axonal regeneration, do not show any advantage over olfactory ensheathing cells isolated from adult olfactory tissue. In addition, olfactory spheres induced a greater astrocytic hypertrophy at the injury site than previously observed for purified olfactory ensheathing cells.

FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.

After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.

Electrophysiological evidence that olfactory cell transplants improve function after spinal cord injury.

Transplants of cells obtained from the olfactory system are a potential treatment for spinal cord injury and a number of clinical trials are in progress. However, the extent to which transplants improve recovery of function remains unclear and there are contradictory reports on the extent to which they support axonal regeneration. Here, we have used anatomical and electrophysiological techniques to investigate the repair promoted by olfactory cell transplants after a dorsal column lesion. Since the use of olfactory cells of varying type and origin may contribute to the differing outcomes of previous studies, regeneration of dorsal column axons was compared following transplants of pure olfactory ensheathing cells from neonatal animals and mixed olfactory cells from both neonatal and adult rats. Two to three months after lesioning, numerous regenerating fibres could be seen in each type of transplant. However, tracing of ascending dorsal column fibres showed that few regenerated beyond the lesion, even when transplanted with mixed olfactory cells from the adult olfactory bulb which have previously been reported to support regeneration which bridges a lesion. Despite the absence of axonal regeneration across the injury site, olfactory cell transplants led to improved spinal cord function in sensory pathways investigated electrophysiologically. When cord dorsum potentials (CDPs), evoked by electrical stimulation of the L4/L5 dorsal roots, were recorded from the spinal cord above and below a lesion at the lumbar 3/4 level, CDPs recorded from transplanted animals were significantly larger than those recorded from lesioned controls. In addition, sensory evoked potentials recorded over the sensorimotor cortex were larger and detectable over a more extensive area in transplanted animals. These results provide direct evidence that transplants of olfactory cells preserve the function of circuitry in the region of the lesion site and of ascending pathways originating near the injury. These actions, rather than axonal regeneration, may help ameliorate the effects of spinal cord injury.

Olfactory ensheathing cell grafts have minimal influence on regeneration at the dorsal root entry zone following rhizotomy.

The effectiveness of grafts of olfactory ensheathing cells (OECs) as a means of promoting functional reconnection of regenerating primary afferent fibers was investigated following dorsal root injury. Adult rats were subjected to dorsal root section and reanastomosis and at the same operation a suspension of purified OECs was injected at the dorsal root entry zone and/or into the sectioned dorsal root. Regeneration of dorsal root fibers was then assessed after a survival period ranging from 1 to 6 months. In 11 animals, electrophysiology was used to look for evidence of functional reconnection of regenerating dorsal root fibers. However, electrical stimulation of lesioned dorsal roots failed to evoke detectable cord dorsum or field potentials within the spinal cord of any of the animals examined, indicating that reconnection of regenerating fibers with spinal cord neurones had not occurred. In a further 11 rats, immunocytochemical labeling and biotin dextran tracing of afferent fibers in the lesioned roots was used to determine whether regenerating fibers were able to grow into the spinal cord in the presence of an OEC graft. Although a few afferent fibers could be seen to extend for a limited distance into the spinal cord, similar minimal in-growth was seen in control animals that had not been injected with OECs. We therefore conclude that OEC grafts are of little or no advantage in promoting the in-growth of regenerating afferent fibers at the dorsal root entry zone following rhizotomy.