Intranasal Borna Disease Virus (BoDV-1) Infection: Insights into Initial Steps and Potential Contagiosity.

Mammalian Bornavirus (BoDV-1) typically causes a fatal neurologic disorder in horses and sheep, and was recently shown to cause fatal encephalitis in humans with and without transplant reception. It has been suggested that BoDV-1 enters the central nervous system (CNS) via the olfactory pathway. However, (I) susceptible cell types that replicate the virus for successful spread, and (II) the role of olfactory ensheathing cells (OECs), remained unclear. To address this, we studied the intranasal infection of adult rats with BoDV-1 in vivo and in vitro, using olfactory mucosal (OM) cell cultures and the cultures of purified OECs. Strikingly, in vitro and in vivo, viral antigen and mRNA were present from four days post infection (dpi) onwards in the olfactory receptor neurons (ORNs), but also in all other cell types of the OM, and constantly in the OECs. In contrast, in vivo, BoDV-1 genomic RNA was only detectable in adult and juvenile ORNs, nerve fibers, and in OECs from 7 dpi on. In vitro, the rate of infection of OECs was significantly higher than that of the OM cells, pointing to a crucial role of OECs for infection via the olfactory pathway. Thus, this study provides important insights into the transmission of neurotropic viral infections with a zoonotic potential.

Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation.

Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide evidence for regeneration promoting cells recruitable for therapeutic purposes. There is evidence that neurotrophin receptor p75 (p75NTR)-expressing cells emerge in the lesioned CNS. However, the phenotype and identity of these cells, and signals triggering their in situ generation under normal conditions and certain pathological situations has remained enigmatic. In the present study, we used a spontaneous, idiopathic and inflammatory CNS condition in dogs with prominent lympho-histiocytic infiltration as a model to study the phenotype of Schwann cells and their relation to Schwann cell remyelination within the CNS. Furthermore, the phenotype of p75NTR-expressing cells within the injured CNS was compared to their counter-part in control sciatic nerve and after peripheral nerve injury. In addition, organotypic slice cultures were used to further elucidate the origin of p75NTR-positive cells. In cerebral and cerebellar white and grey matter lesions as well as in the brain stem, p75NTR-positive cells co-expressed the transcription factor Sox2, but not GAP-43, GFAP, Egr2/Krox20, periaxin and PDGFR-α. Interestingly, and contrary to the findings in control sciatic nerves, p75NTR-expressing cells only co-localized with Sox2 in degenerative neuropathy, thus suggesting that such cells might represent dedifferentiated Schwann cells both in the injured CNS and PNS. Moreover, effective Schwann cell remyelination represented by periaxin- and P0-positive mature myelinating Schwann cells, was strikingly associated with the presence of p75NTR/Sox2-expressing Schwann cells. Intriguingly, the emergence of dedifferentiated Schwann cells was not affected by astrocytes, and a macrophage-dominated inflammatory response provided an adequate environment for Schwann cells plasticity within the injured CNS. Furthermore, axonal damage was reduced in brain stem areas with p75NTR/Sox2-positive cells. This study provides novel insights into the involvement of Schwann cells in CNS remyelination under natural occurring CNS inflammation. Targeting p75NTR/Sox2-expressing Schwann cells to enhance their differentiation into competent remyelinating cells appears to be a promising therapeutic approach for inflammatory/demyelinating CNS diseases.

Transcriptional profiling predicts overwhelming homology of Schwann cells, olfactory ensheathing cells, and Schwann cell-like glia.

Schwann cells (SCs), olfactory ensheathing cells (OECs), and central nervous system Schwann cell-like glia (SG) represent a group of nerve growth factor receptor p75 (NGFR)-positive cells, originating from different tissues. Because of their pro-regenerative capacities, these cells are subjects in experimental transplantation-based therapies of spinal cord trauma. The objective of this study was to compare the transcriptomes of uninfected and canine distemper virus-infected OECs, SCs, SG and fibroblasts (FBs) derived from four beagle dogs and cultured under identical conditions in vitro, employing canine genome 2.0 arrays (Affymetrix). Here, we observed a complete lack of transcriptional differerences between OECs and SG, a high similarity of OECs/SG to SCs, and a marked difference of SCs and OECs/SG towards FBs. Differentially expressed genes possibly involved in the maintenance of cell type-specific identity included an up-regulation of HOXD8 and HOXC4 in SCs, and an up-regulation of CNTNAP2 and EFEMP1 in OECs/SG. We identified cell type-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection. Thereby AQP1 and SCRG1 were predicted to be the most powerful biomarkers distinguishing SCs from OECs/SG. Immunofluorescence confirmed a higher expression of SCRG1 in OECs and SG, and conversely a higher expression of AQP1 in SCs in vitro. Furthermore, canine and murine olfactory nerves showed SCRG1-positive, AQP1-negative OECs and/or axons, whereas sciatic nerves displayed multifocal non-myelinated, AQP1-positive, SCRG1-negative cells. Conclusively, OECs/SG are suggested to be a uniform cell type differing only in the tissue of origin and highly related to SCs.

Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory ß-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory ß-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

Schwann cell-free adult canine olfactory ensheathing cell preparations from olfactory bulb and mucosa display differential migratory and neurite growth-promoting properties in vitro.

BACKGROUND: Transplantation of olfactory ensheathing cells (OEC) and Schwann cells (SC) is a promising therapeutic strategy to promote axonal growth and remyelination after spinal cord injury. Previous studies mainly focused on the rat model though results from primate and porcine models differed from those in the rat model. Interestingly, canine OECs show primate-like in vitro characteristics, such as absence of early senescence and abundance of stable p75NTR expression indicating that this species represents a valuable translational species for further studies. So far, few investigations have tested different glial cell types within the same study under identical conditions. This makes it very difficult to evaluate contradictory or confirmatory findings reported in various studies. Moreover, potential contamination of OEC preparations with Schwann cells was difficult to exclude. Thus, it remains rather controversial whether the different glial types display distinct cellular properties. RESULTS: Here, we established cultures of Schwann cell-free OECs from olfactory bulb (OB-OECs) and mucosa (OM-OECs) and compared them in assays to Schwann cells. These glial cultures were obtained from a canine large animal model and used for monitoring migration, phagocytosis and the effects on in vitro neurite growth. OB-OECs and Schwann cells migrated faster than OM-OECs in a scratch wound assay. Glial cell migration was not modulated by cGMP and cAMP signaling, but activating protein kinase C enhanced motility. All three glial cell types displayed phagocytic activity in a microbead assay. In co-cultures with of human model (NT2) neurons neurite growth was maximal on OB-OECs. CONCLUSIONS: These data provide evidence that OB- and OM-OECs display distinct migratory behavior and interaction with neurites. OB-OECs migrate faster and enhance neurite growth of human model neurons better than Schwann cells, suggesting distinct and inherent properties of these closely-related cell types. Future studies will have to address whether, and how, these cellular properties correlate with the in vivo behavior after transplantation.

Clonal in vitro analysis of neurotrophin receptor p75-immunofluorescent cells reveals phenotypic plasticity of primary rat olfactory ensheathing cells.

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75(NTR))-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell™) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75(NTR) expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75(NTR) at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.

Cell type- and isotype-specific expression and regulation of ß-tubulins in primary olfactory ensheathing cells and Schwann cells in vitro.

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on ß-tubulin isotype expression in primary adult canine glia as a translational large animal model. ß-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed ßII-V isotype mRNA. Interestingly, ßIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to ßV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of ßIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of ßIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (ßIII) and isotype-specific regulation (ßIII, ßV) of ß-tubulin isotypes in OECs and SCs. While differential expression of ßIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong ßIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.

Toward defining the regenerative potential of olfactory mucosa: establishment of Schwann cell-free adult canine olfactory ensheathing cell preparations suitable for transplantation.

Olfactory mucosa (OM)-derived olfactory ensheathing cells (OECs) are attractive candidates for autologous cell transplantation-based therapy of nervous system injury. However, defining the regenerative capacity of OM-derived OECs is impeded by the fact that cell cultures used for transplantation may contain significant amounts of contaminating trigeminal nerve Schwann cells that escape identification by sharing in vitro expression of OEC markers. The aim of the present study, therefore, was to quantify contaminating Schwann cells in OEC preparations and to develop a protocol for their specific depletion. Based on the observation that freshly dissociated, but not cultured, OECs and Schwann cells display differential expression of HNK-1 and p75(NTR), magnet-activated cell sorting (MACS) was used to deplete myelinating (HNK-1-positive) and nonmyelinating (p75(NTR)-positive) Schwann cells from primary cell suspensions containing HNK-1-/p75(NTR)-negative OECs. Upregulation of p75(NTR) expression in OECs during culturing allowed their subsequent MACS-based separation from fibroblasts. Immunofluorescence analysis of freshly dissociated OM prior to MACS depletion revealed that 21% of the total and 56% of all CNPase-positive cells, representing both OECs and Schwann cells, expressed the Schwann cell antigens HNK-1 or p75(NTR), indicating that freshly dissociated OM prior to culturing contained as many Schwann cells as OECs, while olfactory bulb (OB) primary cell suspensions revealed lower levels of Schwann cell contamination. Interestingly, neurite growth of neonatal rat dorsal root ganglion (DRG) neurons cocultured with OM-OECs, OB-OECs, and fibular nerve (FN) Schwann cells used as control was significantly higher in the presence of OECs than of Schwann cells. The first report on identification and specific depletion of Schwann cells from OEC preparations provides a solid basis for future efforts to fully define the regenerative potential of nasal mucosa OECs.

Comparison of Schwann cells and olfactory ensheathing cells for peripheral nerve gap bridging.

INTRODUCTION: Previously, we introduced the biogenic conduit (BC) as a novel autologous nerve conduit for bridging peripheral nerve defects and tested its regenerative capacity in a short- and long-term setting. The aim of the present study was to clarify whether intraluminal application of regeneration-promoting glial cells, including Schwann cells (SC) and olfactory ensheathing cells (OEC), displayed differential effects after sciatic nerve gap bridging. MATERIAL AND METHODS: BCs were generated as previously described. The conduits filled with fibrin/SC (n = 8) and fibrin/OEC (n = 8) were compared to autologous nerve transplants (NT; n = 8) in the 15-mm sciatic nerve gap lesion model of the rat. The sciatic functional index was evaluated every 4 weeks. After 16 weeks, histological evaluation followed regarding nerve area, axon number, myelination index and N ratio. RESULTS: Common to all groups was a continual improvement in motor function during the observation period. Recovery was significantly better after SC transplantation compared to OEC (p < 0.01). Both cell transplantation groups showed significantly worse function than the NT group (p < 0.01). Whereas nerve area and axon number were correlated to function, being significantly lowest in the OEC group (p < 0.001), both cell groups showed lowered myelination (p < 0.001) and lower N ratio compared to the NT group. DISCUSSION: SC-filled BCs led to improved regeneration compared to OEC-filled BCs in a 15-mm-long nerve gap model of the rat.

The long-term functional recovery of repair of sciatic nerve transection with biogenic conduits.

INTRODUCTION: The aim of this study was to evaluate long-term regenerative capacity over a 15-mm nerve gap of an autologous nerve conduit, the biogenic conduit (BC), 16 weeks after sciatic nerve transection in the rat. METHODS: A 19-mm long polyvinyl chloride (PVC) tube was implanted parallely to the sciatic nerve. After implantation, a connective tissue cover developed around the PVC-tube, the so-called BC. After removal of the PVC-tube the BCs filled with fibrin (n = 8) were compared to autologous nerve grafts (n = 8). Sciatic functional index (SFI) was evaluated every 4 weeks, histological evaluation was performed at 16 weeks postimplantation. Regenerating axons were visualized by retrograde labelling. RESULTS: SFI revealed no significant differences. Nerve area and axon number in the BC group were significantly lower than in the autologous nerve group (P < 0.05; P < 0.01). Analysis of myelin formation showed no significant difference in both groups. Analysis of N-ratio revealed lower values in the BC group (P < 0.001). CONCLUSION: This study reveals the suitability of BC for nerve gap bridging over a period of 16 weeks with functional recovery to comparable extent as the autologous nerve graft despite impaired histomorphometric parameters.

Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and nondemyelinated central nervous system.

Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFRα, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.

Increased p75 neurotrophin receptor expression in the canine distemper virus model of multiple sclerosis identifies aldynoglial Schwann cells that emerge in response to axonal damage.

Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide regeneration-promoting cells recruitable for therapeutic purposes. There is accumulating evidence that aldynoglial cells with Schwann cell-like growth-promoting properties emerge in the lesioned CNS. However, the characterization of these cells and the signals triggering their in situ generation have remained enigmatic. In the present study, we used the p75 neurotrophin receptor (p75(NTR) ) as a marker for Schwann cells to study gliogenesis in the well-defined canine distemper virus (CDV)-induced demyelination model. White matter lesions of CDV-infected dogs contained bi- to multipolar, p75(NTR) -expressing cells that neither expressed MBP, GFAP, BS-1, or P0 identifying oligodendroglia, astrocytes, microglia, and myelinating Schwann cells nor CDV antigen. Interestingly, p75(NTR) -expression became apparent prior to the onset of demyelination in parallel to the expression of ß-amyloid precursor protein (ß-APP), nonphosphorylated neurofilament (n-NF), BS-1, and CD3, and peaked in subacute lesions with inflammation. To study the role of infiltrating immune cells during differentiation of Schwann cell-like glia, organotypic slice cultures from the normal olfactory bulb were established. Despite the absence of infiltrating lymphocytes and macrophages, a massive appearance of p75(NTR) -positive Schwann-like cells and BS-1-positive microglia was noticed at 10 days in vitro. It is concluded that axonal damage as an early signal triggers the differentiation of tissue-resident precursor cells into p75(NTR) -expressing aldynoglial Schwann cells that retain an immature pre-myelin state. Further studies have to address the role of microglia during this process and the regenerative potential of aldynoglial cells in CDV infection and other demyelinating diseases.

Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury.

Better understanding of the pathogenesis of spinal cord injury (SCI) is needed for the development of new therapeutic strategies. Spinal cord injury has been investigated in various rodent models, but extrapolation to humans requires the use of a large animal model that more closely mimics human SCI. Dogs frequently develop spontaneous SCI with features that bear a striking resemblance to the human counterpart. We investigated the temporal course of the immune response during naturally occurring canine SCI and in organotypic canine spinal cord slice cultures that are devoid of peripheral immune cells. By immunohistochemistry, the inflammatory response in subacute canine SCI was largely restricted to resident immune cells as demonstrated by activation of major histocompatibility complex class II-expressing microglia/macrophages. By quantitative polymerase chain reaction, there was parallel upregulation of proinflammatory cytokine gene expression (i.e. of interleukin 6 [IL-6] and IL-8 with a trend toward upregulation of tumor necrosis factor) in acute canine SCI. Expression of neuroprotective cytokines (e.g. IL-10) remained unchanged, and transforming growth factor ß upregulation was delayed. In organotypic spinal cord slices, there was similar activation of major histocompatibility complex class II-positive microglia and prolonged upregulation of inflammatory cytokines, indicating that resident rather than infiltrating cells play major roles in the postinjury immune response. Thus, canine SCI represents a bridge between rodent models and human SCI that may be relevant for clinical and preclinical treatment studies.

Defining the morphological phenotype: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a novel marker for in situ detection of canine but not rat olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are the non-myelinating glial cells of the olfactory nerves and bulb. The fragmentary characterization of OECs in situ during normal development may be due to their small size requiring intricate ultrastructural analysis and to the fact that available markers for in situ detection are either expressed only by OEC subpopulations or lost during development. In the present study, we searched for markers with stable expression in OECs and investigated the spatiotemporal distribution of CNPase, an early oligodendrocyte/Schwann cell marker, in comparison with the prototype marker p75(NTR). Anti-CNPase antibodies labeled canine but not rat OECs in situ, while Schwann cells and oligodendrocytes were positive in both species. CNPase immunoreactivity in the dog was confined to all OECs throughout the postnatal development and associated with the entire cell body, including its finest processes, while p75(NTR) was mainly detected in perineural cells and only in some neonatal OECs. Adult olfactory bulb slices displayed CNPase expression after 4 and 10 days, while p75(NTR) was detectable only after 10 days in vitro. Finally, treatment of purified adult canine OECs with fibroblast growth factor-2 significantly reduced CNPase expression at the protein and mRNA level. Taken together, we conclude that CNPase but not p75(NTR) is a stable marker suitable for in situ visualization of OECs that will facilitate their light-microscopic characterization and challenge our general view of OEC marker expression in situ. The fact that canine but not rat OECs expressed CNPase supports the idea that glia from large animals differs substantially from rodents.

Cell surface expression of 27C7 by neonatal rat olfactory ensheathing cells in situ and in vitro is independent of axonal contact.

Olfactory ensheathing cells (OECs) are Schwann cell-like glial cells of the olfactory system that promote neural regeneration after transplantation into the injured central nervous system. Compared to the closely related Schwann cells, however, the biological characterization of OECs has remained fragmentary. This is due to the fact that the expression of OEC-specific markers is subject to complex regulation and that intricate ultrastructural analysis is essential to determine their localization. The p75 neurotrophin receptor (p75(NTR)) as the prototype OEC marker, for example, is only expressed by a minor population of neonatal rat OECs in situ. The major population carries O4-positive axonal fragments on their surface after dissociation and up-regulates p75(NTR) during culturing (Wewetzer et al. in Glia 49:577-587, 2005). In the present study, we investigated whether the cell surface determinant 27C7, defined by a monoclonal antibody to Schwann cells, is also expressed by neonatal rat OECs in situ and in vitro. Primary cell suspensions of the olfactory bulb displayed 27C7 expression of both p75(NTR)-negative and p75(NTR)-positive OECs, while immature oligodendrocytes and astrocytes were devoid of any 27C7 labeling. This together with the finding that the intrafascicular OECs of the olfactory nerves in the mucosa expressed 27C7 but not p75(NTR), suggests that 27C7 was expressed by the entire OEC population in situ. Maintenance of OECs in the absence of olfactory neurons in organotypic slice culture up-regulated p75(NTR) but did not alter 27C7 expression. It is concluded that 27C7 unlike p75(NTR) is constitutively expressed by OECs and may, therefore, be a useful marker for characterization of neonatal OECs in situ and in vitro.

Species-specific control of cellular proliferation and the impact of large animal models for the use of olfactory ensheathing cells and Schwann cells in spinal cord repair.

Autologous transplantation of olfactory ensheathing cells (OECs) and Schwann cells (SCs) is considered a promising option to promote axonal regrowth and remyelination after spinal cord injury in humans. However, if the experimental data from the rodent model can be directly extrapolated to humans, as widely believed, remains to be established. While limitations of the rodent system have recently been discussed with regard to the distinct organization of the motor systems, the question whether OECs and SCs may display species-specific properties has not been fully addressed. Prompted by recent studies on canine and porcine glia, we performed a detailed analysis of the in vitro and in vivo properties of OECs and SCs and show that rodent but not human, monkey, porcine, and canine glia require mitogens for in vitro expansion, display a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen stimulation. These data indicate fundamental inter-species differences of the control of cellular proliferation. Whether OECs and SCs from large animals and humans share growth-promoting in vivo properties with their rodent counterpart is not yet clear. Autologous implantation studies in humans did not reveal adverse effects of cell transplantation so far. However, in vivo studies of large animal or human glia and rodent recipients mainly focused on the remyelinating potential of the transplanted cells. Thus, further experimental in vivo studies in large animals are essential to fully define the axonal growth-promoting potential of OECs and SCs. Based on the homology of the in vitro growth control between porcine, canine and human glia, it is concluded that these species may serve as valuable translational models for scaling up human procedures. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Transplantation of olfactory ensheathing cells as adjunct cell therapy for peripheral nerve injury.

Traumatic events, such as work place trauma or motor vehicle accident violence, result in a significant number of severe peripheral nerve lesions, including nerve crush and nerve disruption defects. Transplantation of myelin-forming cells, such as Schwann cells (SCs) or olfactory ensheathing cells (OECs), may be beneficial to the regenerative process because the applied cells could mediate neurotrophic and neuroprotective effects by secretion of chemokines. Moreover, myelin-forming cells are capable of bridging the repair site by establishing an environment permissive to axonal regeneration. The cell types that are subject to intense investigation include SCs and OECs either derived from the olfactory bulb or the olfactory mucosa, stromal cells from bone marrow (mesenchymal stem cells, MSCs), and adipose tissue-derived cells. OECs reside in the peripheral and central nervous system and have been suggested to display unique regenerative properties. However, so far OECs were mainly used in experimental studies to foster central regeneration and it was not until recently that their regeneration-promoting activity for the peripheral nervous system was recognized. In the present review, we summarize recent experimental evidence regarding the regenerative effects of OECs applied to the peripheral nervous system that may be relevant to design novel autologous cell transplantation therapies.

Theiler's murine encephalomyelitis virus preferentially infects immature stages of the murine oligodendrocyte precursor cell line BO-1 and blocks oligodendrocytic differentiation in vitro.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination is an important animal model for multiple sclerosis. The presence of oligodendrocyte precursor cells (OPCs) within demyelinated lesions together with the limited extent of remyelination has raised the question of how OPCs are affected by TMEV. It is well established that oligodendrocytes, astrocytes and microglia are targets during the chronic phase of the disease. However, whether TMEV infection interferes with the capacity of OPCs to generate oligodendrocytes has remained unclear. In the present study, a bipotential murine OPC cell line termed BO-1 was used to determine the antigenic phenotype susceptible to TMEV and the impact of TMEV infection upon cell differentiation. We show here that retinoic acid increased oligodendrocytic differentiation and decreased proliferation and TMEV infection rates. TMEV under serum-free conditions infected about 75% and 60% of early OPCs (NG2(+) and A2B5(+)) and immature oligodendrocytes (CNPase(+)), respectively, but only approximately 18% of mature oligodendrocytes (MBP(+)). Infection with TMEV prior to application of retinoic acid significantly reduced the percentage of MBP(+) BO-1 cells. These data demonstrate that TMEV preferentially infects early stages of the oligodendrocytic lineage and blocks oligodendrocyte maturation. The first demonstration of TMEV-mediated effects on OPC differentiation may shed new light on the pathogenesis of TMEV-induced demyelination and offers an explanation for the limited remyelination observed in vivo.

Impaired spinal cord remyelination by long-term cultured adult porcine olfactory ensheathing cells correlates with altered in vitro phenotypic properties.

BACKGROUND: Extensive studies in rodents have identified olfactory ensheathing cells (OECs) as promising candidates for cell-based therapies of spinal cord and peripheral nerve injury. Previously, we demonstrated that short-term cultured adult porcine OECs can remyelinate the rodent and non-human primate spinal cord. Here, we studied the impact of the culturing interval on the remyelinating capacity of adult porcine OECs. METHODS: Cells were maintained for 1, 2, and 4 to 6 weeks in vitro prior to transplantation into the demyelinated rat spinal cord. Parallel to this, the in vitro phenotypic properties of the OEC preparations used for transplantation were analyzed with regard to morphology, low affinity nerve growth factor receptor (p75(NTR)) expression and proliferation. RESULTS: We report that prolonged culturing of adult porcine OECs resulted in impaired remyelination of the adult rat spinal cord. Animals receiving transplants of OECs maintained in vitro for 2 weeks displayed significantly less remyelinated axons than those animals that received OEC transplants cultured for 1 week. There was virtually no remyelination after transplantation of OECs cultured for 4 to 6 weeks. The adult porcine OECs displayed a progressive lost of p75(NTR)-expression as determined by immunostaining and flow cytometry with time in culture. CONCLUSIONS: Taken together, the results indicate that porcine OECs undergo systematic changes with time in culture that result in reduced p75(NTR)-expression, decreased proliferation, and reduced remyelinating capability with time in vitro indicating that relatively short term cultures with limited expansion would be required for transplantation studies.