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JOURNAL OF NEUROTRAUMAVolume 23, Number 3/4, 2006© Mary Ann Liebert, Inc.Pp. 468–478

Olfactory Ensheathing Cells: Characteristics, GeneticEngineering, and Therapeutic Potential

MARC J. RUITENBERG,1,2 JANA VUKOVIC,1,2 JULIJANA SARICH,2SAMANTHA J. BUSFIELD,2 and GILES W. PLANT1,2

ABSTRACT

Injured neurons in the mammalian central nervous system (CNS) do not normally regenerate theiraxons after injury. Neurotrauma to the CNS usually results in axonal damage and subsequent lossof communication between neuronal networks, causing long-term functional deficits. For CNS re-generation, repair strategies need to be developed that promote regrowth of lesioned axon projec-tions and restoration of neuronal connectivity. After spinal cord injury (SCI), cystic cavitations areoften found, particularly in the later stages, due to the loss of neural tissue at the original impactsite. Ultimately, for the promotion of axonal regrowth in these situations, some form of transplan-tation will be required to provide lesioned axons with a supportive substrate along which they canextend. Here, we review the use of olfactory ensheathing cells: their location and role in the olfac-tory system, their use as cellular transplants in SCI paradigms, alone or in combination with genetherapy, and the unique properties of these cells that may give them a potential advantage overother cellular transplants.

Key words: gene therapy; neurotrophic factors; olfactory ensheathing glia; regeneration; spinal cord

1Red’s Spinal Cord Research Laboratory, School of Anatomy and Human Biology, University of Western Australia, Crawley,Perth, Western Australia, Australia.

2Western Australian Institute for Medical Research and UWA Centre for Medical Research, University of Western Australia,Crawley, Perth, Western Australia, Australia.

INTRODUCTION

AQUARTER OF A CENTURY has passed since Richardsonet al. (1980) published their pioneering experiments

on peripheral nerve grafting in the injured central ner-vous system (CNS). A study that revolutionized scien-tific thinking about the (in)ability of adult CNS neuronsto regenerate following axotomy. Up to then, neurons inthe adult CNS were thought to lack the intrinsic capac-ity to regenerate their axons after injury. Severed centralaxons fail to regenerate across the injury site and to re-

establish synaptic contacts with target neurons, a processrequired to restore communication between the higherbrain centers and affected areas of the body. Conse-quently, depending on the severity and vertebral level,spinal trauma normally leads to long-term deficits in vol-untary motor and sensory performance as well as im-pairments in the autonomic nervous system functioning.Most human injuries to the spinal cord are characterizedby the presence of one or more cystic cavitations at theoriginal impact site (Bunge et al., 1993). For the promo-tion of axonal regrowth in these situations, some form of

transplantation will be required to provide lesioned ax-ons with a scaffold along which they can extend.

Since the original publication by Richardson et al.(1980), an ever growing number of papers have been pub-lished using some form of (neural) transplantation for thepromotion of axonal regrowth and spinal cord repair.Apart from Schwann cell–containing grafts, a variety ofother cellular bridging materials have been trialled, forexample, pieces of embryonic spinal cord, engineered fi-broblasts, neural stem cells, bone marrow–derived cells,and olfactory ensheathing cells (Bunge and Pearse, 2003;Myckatyn et al., 2004; Reier, 2004). Here, we will specif-ically focus on the use of olfactory ensheathing cells(OECs), that is, their location and role in the olfactorysystem, use as cellular transplants in spinal cord injury(SCI) paradigms, alone or in combination with gene ther-apy, and the unique characteristics they possess, whichmay give them potential advantages over Schwann cellsand other cellular conduits.

PRIMARY OLFACTORY SYSTEM AS ASOURCE OF OLFACTORY

ENSHEATHING CELLS

The adult primary olfactory system is a unique part ofthe mammalian nervous system that has preserved the ca-pacity to continuously regenerate during adulthood(Schwob, 2002). Olfactory receptor neurons, located inthe olfactory mucosa of the nose, are liable to wear as aconsequence of respiration and have an average lifespanof 6–8 weeks (Carr and Farbman, 1993). Throughout life,dying olfactory receptor neurons are replaced by new-born cells that differentiate from a stem cell layer at thebase of the epithelium (Weiler and Farbman, 1997).These new, differentiating odorant receptor progenitorsascend their axons via discrete bundles (fila olfactoria)into the olfactory bulb, which is part of the CNS. Theability of growing primary olfactory axons to navigatethrough a CNS environment is thought to relate to theglial composition and cytoarchitecture of the olfactorybulb (Doucette, 1990, 1991), in particular the presenceof OECs (Goodman et al., 1993; Ramon-Cueto et al.,1993; Kafitz and Greer, 1998; Ramon-Cueto and Avila,1998).

OEC progenitors are thought to originate from the ol-factory placode and can be seen during early embryo-genesis migrating along with olfactory axons towards thedeveloping olfactory bulb (Doucette, 1989; Chuah andAu, 1991; Valverde et al., 1993; Treloar et al., 1996;Chuah and West, 2002). These observations suggest aunique developmental origin of OECs compared to othernervous system macroglia. In the adult, OECs are found

in both the peripheral and central compartments of theprimary olfactory system, that is, the olfactory epitheliumand bulb (Fig. 1). In the nasal cavity, OECs reside in thelamina propria, where they closely associate with affer-ent axon bundles of olfactory receptor neurons (Doucette,1991; Field et al., 2003). In the cranium, OECs are foundin the nerve layer of the first cranial nerve that covers thesurface of the olfactory bulb. Similar to Schwann cells,OECs upregulate low-affinity nerve growth factor re-ceptor p75 following experimental injury (Gong et al.,1994; Turner and Perez-Polo, 1994) and, deafferentedOECs are thought to provide a cellular matrix for bothnew and regenerating primary olfactory axons (Williamset al., 2004). Thus, these unique cells, which share com-mon characteristics with both CNS and peripheral ner-vous system (PNS) glia, enfold growing axons in the bulband are thought to play a key role in supporting regen-eration of olfactory axons. These features have turnedOECs into attractive transplantation candidates to fosteraxonal growth in other areas of the CNS.

OLFACTORY ENSHEATHING CELL TRANSPLANTS

Several spinal cord lesion models, involving eitherdorsal root, specific tract, or partial spinal cord lesions(Ramon-Cueto and Nieto-Sampedro, 1994; Li et al.,1997, 1998; Navarro et al., 1999; Nash et al., 2002;Gomez et al., 2003; Li et al., 2003; Ruitenberg et al.,2003, 2005; Verdu et al., 2003; Andrews and Stelzner,2004; Chuah et al., 2004; Li et al., 2004; Polentes et al.,2004; Ramer et al., 2004a,b; Riddell et al., 2004), pho-tochemical damage (Garcia-Alias et al., 2004), completespinal cord transection (Ramon-Cueto et al., 1998, 2000;Lu et al., 2001, 2002; Cao et al., 2004; Fouad et al., 2005)or contusive injury (Takami et al., 2002; Plant et al.,2003), have been used to test the potential of these en-sheathing cells to promote axonal regeneration outsidethe olfactory bulb. The first evidence that OECs wereable to support regenerative growth of lesioned axons inthe damaged spinal cord was obtained in an experimen-tal model for dorsal root injury. Following rhizotomy andsubsequent microsurgical anastomosis of the dorsal rootstump to the spinal cord, engraftment of OECs at the in-terface of the PNS and CNS allowed sensory axons ofthe dorsal root, which are normally refractory to regen-erate, to cross the non-permissive barrier of the dorsalroot entry zone and to re-enter the spinal (Ramon-Cuetoand Nieto-Sampedro, 1994). These findings were con-firmed in a later study by Navarro et al. (1999) and, morerecently, in a separate report by Li et al. (2004). Threeother independent reports, however, have not been able

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to reproduce these results (Gomez et al., 2003; Ramer etal., 2004a; Riddell et al., 2004). It is unclear whether thesediscrepancies represent true differences in outcome orwhether they are related to experimental differences intransplant preparation, grafting procedures and source oftransplanted OECs (i.e., lamina propria vs. olfactorybulb). Several studies have also demonstrated that trans-plantation of OECs at sites of SCI induced modest re-growth of a variety of propriospinal and descending axonprojections, including fibers of raphespinal, coeru-lospinal, and/or corticospinal (CST) origin (Li et al.,1997, 1998; Ramon-Cueto et al., 1998, 2000; Cao et al.,2004) as well as a certain degree of functional recovery(Li et al., 1997, 1998, 2003; Ramon-Cueto et al., 2000;Lu et al., 2001, 2002; Keyvan-Fouladi et al., 2003; Plantet al., 2003; Ruitenberg et al., 2003; Cao et al., 2004).Some controversy, which remains to be elucidated, hasarisen on the regenerative response of the CST, since oth-ers have found that regrowth of lesioned CST axons wasrestricted to a modest sprouting response at the proximalinjury and OEC transplantation site (Takami et al., 2002;Plant et al., 2003; Ruitenberg et al., 2005). Comparativestudies will need to be undertaken to assess whether thesediscrepancies represent true differences in outcomes orwhether they are related to inter-experimental variables(e.g., the injury model of choice and transplantation pro-cedures). Bridging of the completely transected spinalcord via Schwann cell–seeded guidance channels suc-cessfully joined the two spinal stumps together but didnot allow regenerating axons to exit the graft (Xu et al.,1997; Plant et al., 2001a). Interestingly, transplantationof OECs at both ends of the Schwann cell bridge enabledaxons to grow across the grafted area and to regeneratefor long distances (up to 1.5–2.5 cm) into host spinal cord(Ramon-Cueto et al., 1998). These remarkable observa-tions suggest that the presence of OECs positively influ-enced the growth-impeding properties of the neural scar.Originally, it was hypothesized that OECs chaperone re-generating axons through the spinal cord, perhaps isolat-ing them from the hostile CNS environment, whichwould explain the remarkable potential of these cells tosupport regenerative growth. Based on Hoechst labellingprior to transplantation, OECs were reported to migrateover large distances in host spinal cord (Ramon-Cueto etal., 1998; Boruch et al., 2001). However, possible leaki-ness of Hoechst label from transplanted cells and subse-quent redistribution to host tissue has been observed(Iwashita et al., 2000; Ruitenberg et al., 2002), indicat-ing that this dye is not a reliable marker to track trans-planted cells in host tissue. Two studies have used OECtransplants that were pre-labelled with superparamag-netic iron oxide, allowing visualization of labelled cellsin vivo by MRI imaging (Dunning et al., 2004; Lee et al.,

2004). Although this technique allows live imaging oftransplanted cells, a potentially confounding factor instudying the migratory behaviour of transplanted cellswith this technique is the uptake of superparamagneticiron oxide from dead cells by macrophages. Ex vivo vi-ral transduction of OECs with green fluorescent protein(GFP) has unequivocally demonstrated that transplantedcells do migrate towards the lesion epicenter but not overlarge distances rostral or caudal from the injury site(Ruitenberg et al., 2002; Li et al., 2003, 2004). These re-sults have been confirmed in xenografting experimentsusing OECs from GFP mice (Ramer et al., 2004a,b).Taken together, these findings suggest that the regener-ation-supporting capacity of OECs is more likely relatedto a different interaction with scar-associated cells. It ap-pears that OECs have the capacity to intermingle withcells that are related to the neural scar such as reactiveastrocytes (Lakatos et al., 2000; Verdu, 2001) andmeningeal fibroblasts (Ramon-Cueto et al., 1998). OECtransplants have been shown to not dramatically upreg-ulate inhibitory proteoglycan expression at the graft–hostinterface (Lakatos et al., 2003), a phenomenon that hasbeen described for Schwann cell transplants (Plant et al.,2001a). This could create a more permissive microenvi-ronment in the scar, allowing axonal elongation throughand beyond the site of injury. Interestingly, in a recentpublication by Fouad et al. (2005), a combinatory ap-proach of OEC or Schwann cell transplantation withchondroitinase ABC treatment, in order to reduce the in-hibitory proteoglycan component in the glial scar, furtherimproved axonal regeneration through these grafts aswell as functional recovery.

A few reports have described Schwann cell–like myeli-nation of spinal axons by transplanted OECs (Frances-chini and Barnett, 1996; Franklin et al., 1996; Li et al.,1998; Barnett et al., 2000; Kato et al., 2000), but thesefindings were inconclusive as unpurified OECs cultureswere used for transplantation (Li et al., 1998) and no re-liable labels were used to identify transplanted cells. Invitro, pure cultures of adult OECs failed to producemyelin under conditions that triggered Schwann cells todo so (Plant et al., 2002). The latter observation may sug-gest the presence of another cell type that supports myeli-nation following certain transplant preparation protocols,or the existence of a separate signalling cascade that isneeded by these cells to produce compact myelin. Re-cently, two independent, conflicting reports were pub-lished studying the in vivo myelination potential of em-bryonic or adult OECs. These cells, expressing the LacZreporter gene or GFP, respectively, were transplanted intothe spinal cord in an attempt to obtain conclusive evi-dence whether these cells were capable of myelination(Boyd et al., 2004; Sasaki et al., 2004). In the first study

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by Boyd et al. (2004), OEC processes were found in closeassociation with axon bundles but failed to myelinate sev-ered axons. These findings contrast earlier observationsin vitro where unlabelled embryonic OECs were believedto myelinate dorsal root ganglia (DRG) neurites (Devonand Doucette, 1992). The observed remyelination of CNSaxons was due to unlabelled host Schwann cells, whichmay have been recruited in increased numbers to the OECtransplantation site (Takami et al., 2002; Ramer et al.,2004b). Intriguingly, transplantation of adult GFP-la-

belled OECs reportedly led to some remyelination ofspinal axons by OECs (Sasaki et al., 2004), although theauthors did not confirm their findings in a standardizedin vitro myelination model as used by Devon andDoucette (1992) and Plant et al. (2002).

Taken together, both the olfactory bulb (Plant et al.,2001b) and lamina propria can be used as a source ofOECs for transplantation purposes (Au and Roskams,2003; Bianco et al., 2004). Transplantation of OECs re-mains one of the most promising strategies available toaugment axonal regeneration and functional recovery af-ter SCI. There are, however, conflicting reports on thepotential of these cells to promote regeneration of spe-cific pathways (e.g., corticospinal axons), as well as ontheir ability to myelinate axons in the damaged CNS.Careful evaluation of the literature does not provide afundamental unifying difference between conflicting re-ports (i.e., isolation and transplantation procedures, aswell as source, purity, and age of transplanted cells). Fur-ther analysis is therefore required in comparative studiesaddressing these issues using OEC transplants from em-bryonic, postnatal and adult rats as well as standardiza-tion of OEC isolation and culturing protocols (Plant etal., 2001b; Barnett and Roskams, 2002).

COMBINING GENE THERAPY AND OEC TRANSPLANTATION

Recent studies have demonstrated that OECs expressseveral molecules that are known to promote axonalgrowth (Ramon-Cueto and Avila, 1998), including NGF,BDNF, GDNF, and NT-4/5 (Boruch et al., 2001; Wood-hall et al., 2001). The expression of neurotrophins by

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FIG. 1. The primary olfactory system as a source for olfac-tory ensheathing cells (OECs). (A) Sagittal preparation throughthe head of an adult rat. Lamina propria-derived OECs can beobtained following biopsies from the olfactory epithelium (OE)and underlying structures in the nasal cavity (dashed line), orfrom the olfactory nerve layer of the olfactory bulb (OB; out-lined area). (B) Pure cultures of OECs, expressing low-affinitynerve growth factor receptor p75 (green) and glial fibrillaryacidic protein (GFAP; red), can be isolated from the OB nervelayer following immunopanning for p75, as described in Plantet al. (2002). (C) Immunopurification of p75-expressing cells(red) from adult epithelial preparations significantly enrichedOEC numbers but no pure cultures could be obtained. Signifi-cant numbers of contaminating cells—i.e., Thy-1 expressing fi-broblasts (green) and another yet unidentified population ofcells (nuclear Hoechst dye; blue)—were still present in thesecultures (Ruitenberg and Plant; unpublished data).

OECs may contribute to and, in part, explain the under-lying mechanism of the growth-promoting properties ofOECs. When compared to Schwann cells, however, lowerlevels of neurotrophins seem to be produced by OECs.For instance, endogenous BDNF expression by OECs(0.15 ng/day/105 cells) (Woodhall et al., 2001) appearsapproximately threefold lower when compared toSchwann cells (5 ng/day/106 cells) (Menei et al., 1998).This suggests that other yet unidentified differences inprotein expression, which remain to be explored, con-tribute to the remarkable regeneration-supporting prop-erties of OECs compared to other glial cell types. Addi-tionally, this opens the possibility for genetic engineeringof OECs in order to elevate neurotrophin expression,which could significantly improve regenerative growththrough these grafts.

Over the last decade, a number of genetically engi-neered cells have been transplanted into the injured CNSto study their neuroprotective and neurite regrowth pro-moting effects (Hendriks et al., 2004). To date, only threestudies have been published using OEC transplants thatwere engineered to secrete neurotrophic factors (Ruiten-berg et al., 2003, 2005; Cao et al., 2004). In an initialcollaborative study by our laboratory and the Neurore-generation Laboratory (Netherlands Institute for BrainResearch, Amsterdam, The Netherlands), we examinedif adenoviral (AdV) vector–mediated expression of theneurotrophins BDNF and NT-3 by transplanted OECscould induce improved regenerative growth of lesionedRST axons through these grafts (Ruitenberg et al., 2003).Ex vivo transduction of primary OECs with AdV vectorsresults in robust but transient expression of the transgenefor a period of approximately 30 days following trans-plantation in the lesioned spinal cord (Ruitenberg et al.,2002). In all experimental animals, the left lateral fu-niculus of the cervical spinal cord was lesioned. Spinalcord injured animals then received an OEC transplant thatwas ex vivo transduced with an AdV vector encodingBDNF and/or NT-3, or a marker protein, respectively.Additional control groups received either no transplantor a graft of non-transduced OECs. Monitoring of thehind limb function during horizontal rope walking re-vealed enhanced recovery in rats that received a trans-plant of OECs transduced with a neurotrophin-encodingAdV vector. Analysis of RST regeneration showed thatAdV vector–mediated production of BDNF, but not NT-3, from transplanted OECs significantly improved sprout-ing of RST axons without promoting robust long-distanceregeneration.

Consequently, this could not have accounted for thefunctional improvements as no anatomical restoration ofthe rubrospinal pathway was obtained. Volumetric analy-sis of lesion size, measured in all experimental animals,

revealed that lesions were smaller in all OEC-trans-planted rats but only significantly reduced in size in animals that received OEC grafts secreting the neu-rotrophins BDNF and NT-3. Interestingly, lesion vol-umes were directly correlated to the functional perfor-mance of experimental animals. These findings suggestthat neurotrophin-mediated tissue preservation can havea beneficial role in the functional outcome after SCI,which stresses the importance of limiting secondary dam-age to the spinal cord in the initial stages after injury. Ina more recent publication, we also studied the regenera-tive response of the dorsal CST projection following in-jury and NT-3 production from AdV vector–transducedOEC transplants (Ruitenberg et al., 2005). Similar to ear-lier findings by Blits et al. (2000), transient productionof NT-3 from engineered cells resulted in long-distancemaintenance/regeneration of CST axons in these animals,at least up to 1 cm caudal to the injury and transplanta-tion site as revealed by anterograde tracing of the lesionedCST projection. No such effect was observed in any ofthe control groups which received an injection of trans-plantation medium only, or a transplant of OECs cellsthat were either non-infected or transduced with an AdVvector encoding a marker gene. All OEC grafts reducedsecondary tissue loss, which resulted in a significant spar-ing of spinal cord tissue at the lesion site commensuratewith other studies (Takami et al., 2002; Plant et al., 2003;Ruitenberg et al., 2003; Ramer et al., 2004b). No addi-tional preservation of spinal tissue by AdV vector–de-rived NT-3 was detected in this particular experimentalmodel. Different regenerative responses of CST and RSTmotor pathways were observed following AdV vec-tor–mediated expression of BDNF and NT-3, indicatingthat individual pathways may require different, specificconditions for optimal regrowth, which correlates withreceptor expression studies in relevant brain areas (Lieblet al., 2001). Nevertheless, transplantation of these engi-neered cells did provide proof of principle that geneticengineering increased growth of lesioned CNS axonsthrough these transplants. Persistent transduction ofOECs has been achieved following lentiviral (Ruitenberget al., 2002) and retroviral (Boyd et al., 2004; Cao et al.,2004) transduction. To date, the publication by Cao et al.(2004) is the only study that has investigated the effectsof chronic neurotrophic factor expression on spinal axonregeneration. In vitro, transduced OECs had an approxi-mate 300-fold increase in the levels of GDNF expression.Transduced cells appeared normal, and no change in theexpression of antigenic markers was observed, suggest-ing that GDNF production did not seem to alter cell phe-notype or functioning. Following transplantation of theseengineered OECs into the spinal cord, high levels ofGDNF expression were detected for up to 8 weeks after

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injury. Neurofilament staining revealed robust ingrowthof axons into areas containing GDNF-secreting OECswhen compared to control cell transplants. Furthermore,retrograde tracing suggested augmented regrowth of CSTand RST axons. Interestingly, analysis of anterogradeCST tracing showed vigorous axon sprouting at the le-sion and transplantation area in animals that receivedGDNF secreting OEC transplant, but no significant dif-ferences in CST axon numbers were found further dis-tally. These observations could indicate that chronic ex-pression of GDNF from OEC transplants entrappedregenerating axons due to high neurotrophic factor con-tent at the site of engraftment. A similar phenomenon wasrecently described in the spinal cord after direct adeno-associated viral (AAV) vector–mediated gene transfer ofGDNF to ventral motoneurons (Blits et al., 2004), andstresses the importance of vector development with reg-ulatory transgene expression (Blesch et al., 2001).

CONCLUSION

Combination of OEC transplantation with neurotrophingene therapy is emerging as a promising strategy to pro-mote regeneration of lesioned spinal cord axons in rats.With the development of non-toxic gene therapy systemssuch as lentiviral (LV) vectors, robust and long-term trans-gene expression in transplanted cells following ex vivogene transfer is now a realistic option (Ruitenberg et al.,2002; Cao et al., 2004). Further improvements are re-quired, however, to develop more sophisticated strategiesthat promote strong directional regrowth of targeted path-ways in the spinal cord, resulting in the reconnection ofneural networks. Advanced tissue engineering procedureswill hopefully lead to the development of cellular con-duits that are more effective in the sparing of spinal cordtissue and the promotion of regeneration. Although thereis much optimism about the clinical potential for OECtransplantation in the treatment of CNS trauma, surpris-ingly little is known about the basic biology of these cellsand the mechanism of action by which they promote ax-onal growth.

Questions that need answering include the following:(1) Are there specific markers that can be used to iden-tify OECs (and subtypes) (Franceschini and Barnett,1996)? (2) What genes do OECs express that are ben-eficial to regeneration (Woodhall et al., 2003)? (3) Dothey also express inhibitory molecules or guidancecues? (4) Related to the last point, what differences be-tween OECs and Schwann cells allow integration andintermingling of OECs with host astrocytes whereSchwann cells seem to aggregate and induce increasedneural scar formation (Lakatos et al., 2003; Barnett et

al., 2004)? With the development of genome-widescreening techniques (i.e., microarray technology), it isnow scientifically feasible to perform large-scale analy-sis of differences in gene expression between differentcell types. In an initial attempt to start addressing thesequestions, we have compared OEC and Schwann celltranscriptomes using mRNA, isolated from purified, pri-mary cell cultures that were obtained from adult rats asdescribed in Plant et al. (2002) and hybridized it againstthe rat genome high-density oligonucleotide array U34(Affymetrix) (Fig 2A). From a total number of just over8800 genes present, expression of approximately 4800genes appeared absent or could not be reliably be de-tected in both cell types. Another 3236 genes werefound to be expressed by both cell types. From the to-tal pool, 745 genes seemed differentially expressed be-tween the cells—that is, 450 genes expressed exclu-sively in OECs, whereas a total of 295 gene transcriptsseemed solely present in Schwann cells (Fig. 2B). Toidentify potential key molecules in OECs that could ac-count for their unique capability to intermingle withscar-associated cells and to stimulate regenerative ax-onal growth, we have confined our studies to those mol-ecules that were exclusively expressed by OECs, in par-ticular, cell surface and secreted proteins. We have sofar confirmed differential expression of a subset of thesegenes in independent RNA samples from separate OECcultures using RT-PCR (Fig. 2C). Similarities in OECcharacteristics in vivo and in vitro have yet to be as-certained. Our ongoing research now focuses on the ex-pression of these molecules in vivo and the role thatthese molecules may play during development and re-generation of the primary olfactory system. It is hopedthat these experiments will increase our understandingof OEC biology and provide further insight into novelmolecules that are produced by these cells which couldplay a crucial role during CNS regeneration.

ACKNOWLEDGMENTS

We would like to thank Seok Voon Lee and AjanthyArulpragasam for their assistance in this work. We arealso very grateful for assistance with viral vectors fromthe ongoing collaboration with Dr. Joost Verhaagen andWilliam Hendricks. We are grateful to Dr. P. Wood (Uni-versity of Miami) for providing the p75 antibody. Workin the laboratory of Giles W. Plant was supported by theNational Health and Medical Research Council (NHMRCresearch grant no. 9935975 and RD Wright fellowshipno. 303265), the Western Australian Institute for Med-ical Research, the Neurotrauma Research Program ofWestern Australia, Spinal Cure Australia, Australian Ro-

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FIG. 2. Preliminary data on comparative gene expression profiling between olfactory ensheathing glia cells (OEG) and Schwanncells (SC). (A) Scatter plot of gene expression data following hybridization of OEC and Schwann cell transcriptome against Af-fimetrix U34 oligonucleotide arrays. (B) Venn diagram showing the overlap in gene expression between OECs and Schwanncells. A total of 450 genes seemed selectively expressed in OECs, whereas 295 gene transcripts were only detected in RNA prepa-rations from Schwann cells. (C) Confirmation of differential expression of a subset of genes, including several growth factors,between OECs and Schwann cells using reverse transcriptase–polymerase chain reaction (RT-PCR).

tary Health Research Fund, Spinal Cord Society of Aus-tralia, the Ramaciotti Foundation, and the generous sup-port of Eileen Bond (RED).

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Address reprint requests to:Dr. Giles W. Plant

Red’s Spinal Cord Research LaboratorySchool of Anatomy and Human Biology

Mail Bag Delivery Point M309University of Western Australia

35 Stirling HighwayCrawley, Perth, WA 6009, Australia

E-mail: gplant@anhb.uwa.edu.au

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