Brain, Vol. 126, No. 3, 598-609,
March 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg055
Meningeal cells enhance limited CNS remyelination by transplanted olfactory ensheathing cells
1 Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge and 2 Department of Neurology, University of Glasgow, Glasgow, UK
Correspondence to: Dr R. J. M. Franklin, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK E-mail: rjf1000{at}cam.ac.uk
Received August 6, 2002. Revised October 11, 2002. Accepted October 16, 2002.
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Olfactory ensheathing cells (OECs) are candidate cells for transplant-mediated repair of persistent demyelination in diseases such as multiple sclerosis. If this approach is to make the transition from laboratory to clinic, an important issue is the most suitable composition of the OEC transplant. Isolation of OECs involves concurrent isolation of other cell types, and specific selection techniques are required to produce purified OECs. In this study we address whether the purity of the OEC transplant affects their ability to remyelinate. Surprisingly, we find that a purified preparation of OECs, selected on the basis of low-affinity nerve growth factor receptor (p75) expression, results in less extensive remyelination than an unpurified preparation following transplantation into areas of persistent demyelination in rodent CNS in the X-irradiation/ethidium bromide (X-EB) model. A distinctive feature of the unpurified preparation both in vitro and following transplantation is the presence of meningeal cells. When meningeal cells are added to purified OECs there is a significant improvement in the extent of remyelination compared with the purified OECs, although if the cells are present in too great an abundance this beneficial effect is lost. These results highlight the important concept that the regenerative properties of OECs are profoundly influenced by the cells with which they are transplanted.
Keywords: demyelination; transplantation; olfactory ensheathing cell; meningeal cell; remyelination
Abbreviations: GFAP = glial fibrillary acidic protein; OEC = olfactory ensheathing cell
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Introduction |
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Although the concept of transplanted-mediated remyelination of the CNS has been demonstrated many times using a variety of experimental models, its progression towards clinical implementation in diseases such as multiple sclerosis has been hampered by the limited availability of suitable cells (Zhang and Duncan, 2000
; Halfpenny et al., 2002). At present, it is not possible to obtain from human sources sufficiently large numbers of progenitor cells committed to becoming oligodendrocytes (Blakemore and Franklin, 2000), and the efficacy of Schwann cells is thought by many to be limited on account of their generally unfavourable interactions with astrocytes (Franklin and Blakemore, 1993; Wilby et al., 1999; Franklin 2002b). The demonstration, first in vitro (Devon and Doucette, 1992) and subsequently in vivo (Franklin et al., 1996; Imaizumi et al., 1998), that olfactory ensheathing cells (OECs) can form myelin sheaths around axons of appropriate diameter has opened up the possibility of using these cells to remyelinate areas of persistent demyelination. This finding, together with the ability of OECs to promote axon regeneration and functional recovery following traumatic injury to the CNS (Li et al., 1997; Imaizumi et al., 2000; Ramon-Cueto et al., 2000; Lu et al., 2001; Pascual et al., 2002), forms the basis of the current interest surrounding the therapeutic potential of these cells (Franklin and Barnett, 2000; Raisman, 2001; Franklin, 2002a).
An unresolved and important issue is whether the efficacy of OEC transplantation is affected by the composition of the transplant. When OECs are prepared from olfactory tissue the resulting culture inevitably comprises many cell types. Is it necessary to generate pure OEC preparations by removing the ‘contaminants’, which may potentially have disadvantageous effects (Brierley et al., 2001), to obtain optimal results? In our own studies we have found that, following transplantation of a clonal cell line into an area of persistent demyelination, the overwhelming majority of transplanted cells adopt a myelinating phenotype. However, when we have transplanted primary preparations of OECs, alternative, non-myelinating cell morphologies are observed in proportions that vary from one preparation to another (Barnett et al., 2000; Smith et al., 2001). These cells have been variously described as being meningeal-like or astrocyte-like and bear some morphological similarity to the ‘A’ cells described by Raisman and colleagues following transplantation of cell preparations containing OECs into sites of electrolytic white matter injury (Li et al., 1998). In order to clarify the identity of the non-myelinating cells seen following transplantation into demyelinating lesions and to investigate the bearing that they may have on the remyelinating potential of OECs, we transplanted different preparations of OECs: an unpurified preparation, an OEC preparation purified on the basis of low-affinity nerve growth factor receptor (also known as p75) expression (Plant et al., 2002), and two purified OEC preparations to which we intentionally added olfactory bulb-derived meningeal cells. We found that purified p75+ OECs have poor remyelinating capacity compared with unpurified preparations of OECs, but that this difference can be overcome by adding a moderate proportion of cotransplanted meningeal cells. These results highlight an important concept: that the regenerative properties of OECs are profoundly influenced by the cells with which they are transplanted and interact, and that the purity of the preparation transplanted is an issue that must be considered in accurately interpreting the regenerative efficacy of OECs.
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Material and methods |
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OEC cultures
Olfactory bulbs were obtained from 7-day-old Fischer rat pups (Harlan, Blackthorn, UK). The outer layer of the rostral tip of the bulb was then dissected and dissociated to a cell suspension using a previously described protocol (Barnett et al., 1993
), and plated onto poly-L-lysine (PLL)-coated flasks (T75, Nunc, Roskilde, Denmark) and initially grown in 10% FBS-DMEM (foetal bovine serum–Dulbecco’s Modified Eagle Medium). This preparation was used as the basis for both unpurified and purified OEC cultures. To generate an unpurified OEC preparation, the flasks of confluent culture of olfactory bulb cells were only subjected to shaking for 3 h (275 r.p.m.), and the supernatant containing floating cells (mainly microglia and oligodendrocyte progenitor cells) was discarded. The remaining cells were grown in 10% FBS-DMEM until harvested for transplantation.
In order to obtain a purer preparation of OECs, the cultures were then treated with cytosine arabinoside (10–5 M; Sigma, Poole, UK) to reduce the number of rapidly growing contaminants, such as endothelial cells, meningeal cells, astrocytes and progenitor cells. Cytosine arabinoside was applied for two 4-day periods from day 2, separated by 2 days in fresh 10% FBS-DMEM lacking cytosine arabinoside. Following the second cytosine arabinoside treatment and a subsequent 3 h shaking period (275 r.p.m.), the floating cells were removed and fresh culture medium was applied for 2–18 h. Remaining cells were further purified by briefly exposing the culture to trypsin (Sigma; 0.01% in Hanks medium) to dislodge the spindle or triangular shaped cells. Detached cells were harvested, washed twice in 10% FBS-DMEM and resuspended in Eagle’s MEM-HEPES [Minimum Essential Medium–4-(2-hydroxyethyl) piperazine-1-ethane sulphonic acid]. The low-affinity nerve growth factor receptor-expressing component of this population was isolated by immunopanning using an antibody against p75 (MA192 hybridoma supernatant; gift from Dr G. Plant). This procedure has been shown previously to produce a contaminant-free population of OECs (Plant et al., 2002). To do this, Petri dishes were prepared by incubating with anti-mouse IgG (immunoglobulin G) antibody overnight (Dako, Ely, UK; 1 : 100, Tris 50 mM, pH 9.5) followed by anti-p75 antibody solution for 2 h at 4°C (1 : 6 solution of the hybridoma supernatant in Eagle’s MEM-HEPES containing 2.5% FBS). The cell suspension was transferred into coated Petri dishes and incubated for 25 min at 4°C. Then the dishes were washed five times with Hanks medium (Ca2+/Mg2+-free) to remove the non-adherent cells. The adherent p75+ cells were detached using a cell scraper (Grenier, Stonehouse, UK). These purified cells were then washed twice in fresh medium and replated onto a fibronectin-coated surface (10 µg/ml; BD Biosciences, Bedford, MA, USA) in T25 culture flasks (Nunc) and then grown to subconfluency in fresh 10% FBS-DMEM supplemented with 2 µM forskolin and 20 ng/ml heregulin (R&D Systems Europe, Abingdon, UK). The cells were maintained under these conditions for at least 2 weeks before transplantation.
Meningeal cell culture
To grow olfactory bulb meningeal cells, the pia-arachnoid was carefully dissected from the olfactory bulbs of 7-day-old Fischer rat pups. The tissue was dissociated and cells were plated onto PLL-coated tissue culture and maintained in 10% FBS-DMEM for 7 days, by which time the cultures had formed a confluent monolayer. The flasks were placed on a rotatory shaker overnight to remove the surface-dwelling cells, a procedure that was repeated 2 days later. In order to ensure that the meningeal cell preparation did not contain OECs, the p75-immunopanning procedure described above was conducted. However, on this occasion the non-adherent cells were retained and replated onto PLL-coated T25 flasks. The cells were grown for a further 5 days, and the remaining O4- and A2B5-expressing contaminant cells were removed by a complement-mediated cytolysis. The monolayer was detached by brief exposure to trypsin (0.025% in Mg2+/Ca2+-free Hanks medium), washed in 10% FBS-DMEM and resuspended in the 1 : 1 mixture of O4 and A2B5 antibody solutions [hybridoma supernatant, IgM (immunoglobulin M) subtype] and was left for 15 min at room temperature. Rabbit complement (Harlan) was then added (1 : 6 final dilution) and the suspension incubated for a further 45 min at 37°C. Following washes in DMEM and in fresh medium, the cells were finally transferred into a PLL-coated T25 flasks and grown to confluency in 10% FBS-DMEM.
Immunocytochemical characterization of cell preparations
To accurately assess the composition of the cultures used for transplantation, an aliquot of each preparation was plated for 24 h in fresh culture medium and characterized by immunocytochemistry. The primary antibodies {anti-p75 [1 : 5, mouse p75-MA192 hybridoma supernatant (IgG1), used as an OEC marker], O4 antibody [1 : 2, mouse, hybridoma supernatant (IgM), an oligodendrocyte lineage marker] and OX42 antibody [1 : 200, mouse (IgG2a) (Serotec, Oxford, UK), a microglia marker)]} were applied to living cells for 30 min at room temperature before the incubation with the secondary antibody. To co-label cells with intracellular markers, following incubation with antibodies for the surface labelling and subsequent fixation with methanol (20 min at –20°C), the cells were immunostained for either glial fibrillary acidic protein (GFAP) (1 : 100; cow polyclonal antibody; Dako) or fibronectin (1 : 400; rabbit polyclonal antibody; Dako). Following incubation with primary antibodies (30 min at room temperature), secondary class-specific FITC (fluorescein isothiocyanate)-conjugated antibodies (1 : 100; Southern Biotechniques, Cambridge Bioscience, Cambridge, UK) and Cy3 conjugates (1 : 300; Jackson Immunoresearch Labs, West Grove, PA, USA) were applied for a further 30 min at room temperature. All washing steps were carried out in 5% FBS-PBS (fetal bovine serum–phosphate-buffered saline). OECs were defined by strong p75 staining and either diffuse GFAP or weak fibronectin immunoreactivity, whereas meningeal cells were identified visualizing the enhanced fibronectin labelling and no p75 labelling. For cell counts, in each sample at least three areas were assessed on multiple coverslips.
X-irradiation/ethidium bromide (X-EB) model of CNS demyelination and cell transplantation
In order to test the remyelinating capacity of the various cell preparations, suspensions of cells were transplanted into focal areas of persistent demyelination in the dorsal funiculus of the spinal cord of adult Fischer rats (
250 g, male). Persistent demyelination was induced using a combination of ethidium bromide injection (to induce demyelination) and local exposure of the spinal cord to X-irradiation (to prevent spontaneous remyelination). This lesion model is referred to as the X-EB lesion and has been used extensively to assess the remyelinating potential of transplanted cells (e.g. Franklin et al., 1996; Imaizumi et al., 1998; Smith et al., 2002). Clear evidence that cells within the lesion are of transplant origin is provided by, inter alia, the presence of non-histocompatible cells in immunosuppressed hosts and their absence when immunosuppression is removed (Smith et al., 2002). In brief, under anaesthesia induced and maintained using halothane, a dorsal laminectomy was performed on the T13 vertebra to expose the dorsal aspect of the spinal cord. With the aid of a three-way manipulator, the tip of a glass micropipette attached to a 10 µl Hamilton syringe was positioned within the dorsal funiculus and 1 µl of a 0.1% solution of ethidium bromide was injected to create a discrete area of demyelination. In order to suppress the remyelination by endogenous cells, the rats were restrained the day following lesion-induction by intramuscular injection of a mixture of fluanisone and fentanyl (Hypnorm; Janssen Pharmaceutica, Beerse, Belgium; 0.30 ml/kg), and a 4 cm long section of the spinal cord including the lesion was exposed to 40 grays of X-irradiation using an orthovoltage radiotherapy machine (Pantak, East Haven, CT, USA). Following a further 48 h period, 1 µl of cell suspension that had been in culture for 3–4 weeks was transplanted into the centre of the lesion in a similar fashion to the procedure used for lesion induction (see Table 2 for transplant composition). After each surgical procedure, a subcutaneous injection of caprofen (Zenecarp, Leyland, UK; 10 mg/kg) was administered to minimize discomfort, together with penicillin antibiosis (Duphapen; Fort Dodge, Southampton, UK; 0.5 ml/kg, intramuscularly). These procedures were performed under a Home Office Licensing protocol.
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Histology and immunohistochemistry
Animals were perfused with 4% glutaraldehyde via the descending aorta under deep pentobarbitone anaesthesia 4 weeks after transplantation. For resin embedding, the spinal cord was cut into a series of 1 mm thick coronal blocks that encompassed the entire lesion and were processed separately so as to maintain the craniocaudal sequence. Serial sections of 1 µm thickness were cut from each block and either stained with alkaline toluidine blue or processed for immunohistochemistry. Selected resin blocks were trimmed for ultrathin sectioning, stained with lead citrate/uranyl acetate and viewed by electron microscopy. Toluidine blue-stained sections from each lesion-containing block were used to score remyelination.
For immunostaining, resin sections mounted on slides treated with Vectabond (Vector Laboratories, Peterborough, UK) were baked for 30 min at 80°C and then incubated in ascending concentrations of alcohol solutions (in 70, 90, 100 and 100% ethanol for 5 min each) using coplin jars. The slides were soaked in a sodium ethoxide–ethanol (1 : 1) solution for 30 min and then washed in ethanol to remove the superfluous resin from sections. After peroxidase blocking with 3% H2O2 in methanol, the sections were incubated firstly in 8% formic acid for 10 min and then in PBS containing 10% NGS (normal goat serum) and 0.1% Triton-X for a further 20 min. Anti-fibronectin antibody (1 : 500, rabbit; Dako; in 2% NGS, 0.1% Triton-X) was applied overnight at 4°C, followed by a biotin-labelled goat anti-rabbit secondary antibody (1 : 400, in PBS) for 1 h at room temperature. Anti-fibronectin antibody (1 : 500, rabbit; Dako; in 2% NGS, 0.1% Triton-X) was applied overnight at 4°C, followed by a biotin-labelled goat anti-rabbit secondary antibody (1 : 400; Sigma, UK; in PBS) for 1 h at room temperature. The staining was detected by applying the ABC complex (Vectastain; Vector Laboratories, Peterborough, UK) for a further 30 min before a colorimetric peroxidase reaction (for 5 min), using a diaminobenzidine substrate kit (Vector Laboratories, Peterborough, UK). All washing steps were carried out in PBS containing 0.1% Triton-X following the application of each reagent. Finally, the sections were air-dried, dehydrated and mounted using dibutyl phthalate.
Remyelination scoring
In resin sections, remyelinated axons can be readily distinguished from normally myelinated axons outside the lesion by their relatively thin myelin sheath. Within the lesion, remyelinated axons can be distinguished from demyelinated axons because the former possess myelin sheaths recognizable as a dark-staining rim around the axon. The myelin of OEC remyelination stains slightly darker than central myelin, and the proximity of the OEC nucleus frequently gives the myelinating OEC axon unit in transverse section a characteristic signet-ring appearance (Franklin et al., 1996). Using these morphological criteria, it was possible to estimate the approximate percentages of demyelinated axons that had been remyelinated by OECs and of those that had remained demyelinated. Comparisons were made independently by the first two authors using coded material, so that they were unaware of the cell preparation each scored section had received. When comparisons were made between transplant preparations, the mean remyelination scores and standard error of the mean for each group of animals were calculated and statistical comparisons were made using a non-parametric test (Kruskal–Wallis test, 5% confidence interval) for comparisons between different transplant groups.
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Results |
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Transplantation of purified OEC preparations results in less extensive remyelination than transplantation of unpurified preparations
In order to establish whether the degree of purity of OEC preparations affected their ability to remyelinate areas of CNS demyelination, preparations of either unpurified OECs (preparation 1; Table 2) or OECs purified by p75 immunopanning (preparation 2; Fig. 1, Table 1), were transplanted into X-EB lesions in the dorsal funiculus of the spinal cord of adult syngeneic rats. The extent of remyelination was compared 3 weeks after transplantation. In lesions from animals in both groups, axons were identified that had been remyelinated in a manner characteristic of transplanted OECs (Franklin et al., 1996
; Imaizumi et al., 1998; Li et al., 1998). Estimation of the extent of remyelination indicated that this was approximately 3-fold greater following transplantation of the unpurified preparation compared with the purified preparation (Fig. 2).
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The results of this comparison suggested that cells other than OECs present within the unpurified preparation might be having a beneficial effect on the remyelinating abilities of the transplanted OECs. Comparing the composition of the two preparations indicated that, whereas the purified preparation consisted almost entirely of p75+, weakly GFAP+ OECs, the unpurified preparation contained a significant proportion of cells that strongly expressed fibronectin, and these were provisionally identified as meningeal cells (Fig. 1, Table 1). The identity of these cells was confirmed by intentionally culturing similar fibronectin-expressing cells from the meningeal layers dissected from the distal end of the olfactory bulb and obtaining cells of similar morphology and fibronectin labelling.
Remyelination by OECs is increased when cotransplanted with small numbers of fibronectin+ meningeal cells
In order to test whether the presence of meningeal cells in the unpurified olfactory bulb preparations contributed favourably to remyelination by transplanted OECs, we next added olfactory bulb-derived meningeal cells to the transplant suspension of purified OECs to a final composition of 30% meningeal cells and 70% OECs (preparation 3). The number of OECs in each transplant was the same as in the previous experiment, when purified OECs alone were transplanted. Histological examination of the lesions 3 weeks after transplantation revealed more widespread remyelination than after transplantation of purified OECs. Frequently, the remyelination extended to the lateral edges of the lesion and had a greater distribution along the lesion’s longitudinal axis. An estimate of the proportion of axons that were remyelinated or that had remained demyelinated revealed a 3-fold increase in the degree of remyelination compared with that achieved following transplantation of purified OECs alone. In order to verify that the increase in the extent of remyelination was not directly attributable to cells present within the meningeal component of the transplant suspension, we transplanted five animals with the meningeal cell preparation only (preparation 5). In no case did we see any remyelination within the lesion (Figs 2 and 3).
We next addressed whether the extent of remyelination could be increased further by increasing the proportion of meningeal cells. When a preparation that contained approximately equal proportions of meningeal cells and OECs (1 : 1) (preparation 4) was transplanted, while maintaining the same number of OECs as in previous transplants, the degree of remyelination was not significantly greater than when the purified OECs were transplanted alone. This indicated that the extent of remyelination achievable by transplanted OECs was not linearly related to the proportion of cotransplanted meningeal cells, and that the beneficial effects of the meningeal presence was only manifested when OECs formed a substantial majority of the cells within the transplant (Figs 2 and 3).
Morphological arrangements adopted by meningeal cells cotransplanted with OECs
In lesions receiving purified OECs (preparation 2) the predominant cell type, in addition to host axons and macrophages, was the remyelinating cell. These tended to be mainly in the centre of the lesion at the point of injection (Fig. 3). The remyelinating cells in the lesions receiving the unpurified preparation (preparation 1) were similar in appearance but more widely distributed. A key difference between the two preparations was that, in the lesions receiving the unpurified preparation, cells were seen extending long processes around remyelinated axons, gathering them together in fascicles bounded in a perineurial-like arrangement (Fig. 4). Such an arrangement has been described as one of two arrangements adopted by meningeal cells following glial cell transplantation, the alternative arrangement consisting of clumps or cords of tightly packed cells (Franklin et al., 1992). Similar fascicles of OEC-myelinated axons have been illustrated in previous OEC transplant studies, although the cells forming the fascicles were not specifically identified (Li et al., 1998; Imaizumi et al., 2000; Smith et al., 2001). If these cells were meningeal cells, one would predict that the frequency of fascicle formation would increase when increasing numbers of meningeal cells were added to the transplant. In the lesions receiving an OEC transplant with 20% meningeal cells, a small number of meningeal cells were indeed observed, and were even more abundant in the lesions transplanted with OECs and meningeal cells in a ratio of 1 : 1. Associated with the presence of meningeal cells was an increase in the abundance of large-diameter collagen within the extracellular space (Fig. 5D and E). However, in both cases the meningeal cells generally formed tightly packed clumps or cords of cells, and only rarely were perineurial-like arrangements of meningeal cells around bundles of OEC-myelinated axons observed. When meningeal cells alone were transplanted into X-EB lesions, they only formed clumps of cells and never adopted the fascicle-forming arrangements around axons seen when transplanted together with OECs (Figs 3 and 4).
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Selected sections were immunostained with fibronectin antibodies to further characterize the arrangements of meningeal cells within the unpurified transplants and when meningeal cells were cotransplanted with OECs. However, while fibronectin remained largely adherent to the surface of meningeal cells in vitro, within the lesions there was strong staining, with fibronectin associated with the surface of myelinating OECs, possibly localized to the basal lamina. This distribution of fibronectin is similar, although more intense, than that seen around myelinating Schwann cells in spinal roots (Fig. 6).
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Discussion |
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Several proof-of-principle studies have been undertaken using a variety of CNS injury models that have established the pro-regenerative effects of transplanting OECs (Franklin and Barnett, 2000
; Raisman, 2001). In order to translate these studies into therapeutic approaches for treating clinical disease, a number of specific issues need to be resolved to render them practicable and to optimize success. Some of these have already begun to be addressed, such as (i) the availability of human OECs (Barnett et al., 2000; Kato et al., 2000) or OECs suitable for xenotransplantation (Imaizumi et al., 2000), (ii) accessible sources of OECs (Lu et al., 2001), (iii) the effectiveness of OEC transplantation in clinically relevant lesion models (Verdu et al., 2001), and (iv) how long OEC transplantation is still efficacious after traumatic injury has occurred (Lu et al., 2002). However, an issue that has received little attention thus far is the composition of the OEC transplant. If OECs are the cells responsible for mediating regeneration, is it beneficial to transplant as pure a population of these cells as possible? In previous studies, OEC transplants of varying degrees of purity have been used, ranging from clonal cell lines of demonstrable purity to preparations in which no purification steps were involved. In the present study we addressed how the purity of an OEC transplant affects the ability of these cells to remyelinate areas of primary demyelination, an issue of particular importance in considering the use of such an approach to repair areas of persistent demyelination in demyelinating diseases, such as multiple sclerosis. We find that the remyelination achieved with an unpurified OEC preparation is significantly greater than that achieved with a purer OEC preparation, which has comparatively poor intrinsic remyelinating capacity. This result would appear to be at variance with the extent of remyelination that we have reported previously following transplantation of a clonal OEC line into the same lesion environment (Franklin et al., 1996). However, this difference almost certainly reflects the greater proliferative propensity of the cell line compared with the primary cells. We also demonstrate that OECs selected on the basis of nerve growth factor receptor expression are unequivocally able to myelinate the large-diameter axons of the X-EB lesion, even though they appear unable to myelinate the very small-diameter axons generated by dorsal root ganglia preparations in vitro (Plant et al., 2002). These results therefore strongly support the concept that the remyelinating capacity of OECs is dependent on axonal diameter rather than that their failure to myelinate in vitro reflects a general property of these cells. We also establish that olfactory bulb meningeal cells cotransplanted with OECs increase the efficiency of remyelination, and that these are likely to be the cells present within unpurified preparations that optimize OEC-mediated remyelination.
By what means might meningeal cells improve remyelination by transplanted OECs? Little is known about what directly effects OEC myelination. However, given the striking similarity between myelination by Schwann cells and by OECs, in terms of ultrastructural morphology (Devon and Doucette, 1992, 1995; Franklin et al., 1996; Li et al., 1998), biochemistry and transcriptional regulation (Smith et al., 2001), studies on factors affecting Schwann cell myelination are likely to be informative. Meningeal cells, like fibroblasts, are mesenchyme-derived cells, and many studies have established the sensitivity of Schwann cells to mesenchymally derived factors (Obremski et al., 1993a, b). A probable beneficial effect of the presence of meningeal cells is their contribution to the extracellular matrix, which plays an important, although not essential, role in Schwann cell myelination (Eldridge et al., 1989; Podratz et al., 2001; Feltri et al., 2002). In support of this notion, a denser and more abundant extracellular matrix was present following transplantation of preparations containing meningeal cells compared with the purified OEC preparation. Moreover, the abundance of fibronectin, a ligand for the ß1 integrin needed by Schwann cells to myelinate (Feltri et al., 2002), also appeared to be increased around remyelinating OECs in the vicinity of meningeal cells. However, promoting the formation of myelin sheaths is unlikely to be the only beneficial effect, since meningeal cell cotransplantation resulted in a greater number of myelinating OECs spread over a wider area, suggesting that their proliferation or migration might also be enhanced. A variety of growth factors are mitogenic for OECs (Yan et al., 2001; Alexander et al., 2002), of which fibroblast growth factor 2 is known to be expressed by meningeal cells (Mercier and Hatton, 2000), and this might have contributed to the increase in remyelination that we have documented.
We also demonstrate that the proportion of OECs and meningeal cells is important in remyelination, because when meningeal cells are transplanted in proportions to OECs of 50%–50% (preparation 4) rather than 30%–70% (preparation 3), their beneficial effect on remyelination is not observed. This is somewhat similar to the observation that a high proportion of contaminating fibroblasts in Schwann cell preparations from adult human peripheral nerve severely impairs remyelination (Brierley et al., 2001). However, we do not observe meningeal cells forming a dense swirling cellular mass with widespread deposition of extracellular matrix, or the consequent axonal injury reported in this earlier study, indicating that the failure of the higher meningeal cell component to promote remyelination cannot be explained solely on the basis of the meningeal cells over-running the lesion.
When meningeal cells were present as a component of unpurified olfactory bulb cultures, they adopted two basic arrangements similar to those described previously for meningeal cells following glial cell transplantation into focal areas of demyelination. The meningeal cells either formed compacted clumps or cords of cells, or, more frequently, had a looser arrangement with long, fine processes that often encircled bundles of OEC-myelinated axons to create a structure resembling a perineurium-bound fascicle in the PNS. These arrangements have been described or illustrated in other studies in which OECs have been transplanted into models of either demyelination or traumatic injury (Li et al., 1998; Imaizumi et al., 2000; Smith et al., 2001). In each case the transplant was not subjected to a substantial degree of OEC purification and was likely to have contained meningeal cells from the surface of the olfactory bulb. The identity of these cells has remained equivocal. In the studies of Raisman and colleagues, in which unpurified OEC preparations were transplanted into electrolytic lesions of spinal cord white matter, these cells are described as ‘A’ cells to distinguish them from the myelin forming ‘S’ cells (Li et al., 1998). One interpretation of this nomenclature is that the S cells represent OECs while the A cells are olfactory bulb meningeal cells (which the authors describe as ‘fibroblast-like’ cells) also present within the transplant suspension (Li et al., 1998; Raisman, 2001). In the PNS, mesenchyme-derived cells are induced to form the perineurium by desert hedgehog (dhh) produced by Schwann cells (Parmantier et al., 1999), and since OECs produce dhh (Smith et al., 2001) it is possible that a similar mechanism accounts for the formation of perineurial structures by meningeal cells. It was somewhat surprising, therefore, to find that when OECs and meningeal cells are cultured separately and then combined in mixed OEC–meningeal cell preparations the meningeal cells generally adopted the compacted arrangements and that the perineurium formation was less frequently observed. The reason why meningeal cells cultured alone should behave differently from those continuously present within an OEC culture is not clear at present.
In this study we demonstrated that the intrinsic remyelinating capacity of OECs is limited but can be augmented by the presence of meningeal cells. It is also suggested that similar beneficial effects may be exerted on OECs in their capacity to support axon regeneration following traumatic injury (Raisman, 2001). Thus, in developing OEC transplantation as a therapeutic approach, there may be little advantage in obtaining pure OEC cultures. Rather, attention should be paid to how their regenerative potential can be optimized by the cells with which they are cotransplanted.
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Acknowledgements |
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This work described in this study was supported by grants from the International Spinal Research Trust and the Myelin Project. We are grateful to Mike Peacock and Anil Kalupahana for their excellent technical assistance.
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References |
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