A. Jane Roskams

Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord injury

Bridging of a lesion site and minimizing local damage to create an environment permissive for regeneration are both primary components of a successful strategy to repair spinal cord injury (SCI). Olfactory ensheathing cells (OECs) are prime candidates for autologous transplantation to bridge this gap, but little is known currently about their mechanism of action. In addition, OECs from the accessible lamina propria (LP) of the olfactory mucosa are a more viable source in humans but have yet to be tested for their ability to promote regeneration in established SCI models. Here, mouse LP‐OECs expressing green fluorescent protein (GFP) transplanted directly into both rat and mouse dorsolateral spinal cord lesion sites demonstrate limited migration but interact with host astrocytes to develop a new transitional zone at the lesion border. LP‐OECs also promote extensive migration of host Schwann cells into the central nervous system repair zone and stimulate angiogenesis to provide a biological scaffold for repair. This novel environment created by transplanted and host glia within the spinal cord inhibits cavity and scar formation and promotes extensive sprouting of multiple sensory and motor axons into and through the lesion site. Sixty days after rat SCI, serotonin‐ and tyrosine hydroxylase‐positive axons sprouted across the lesion into the distal cord, although axotomized rubrospinal axons did not. Thus, even in a xenotransplant paradigm, LP‐OECs work collaboratively with host glial cells to create an environment to ameliorate local damage and simultaneously promote a regenerative response in multiple axonal populations. J. Comp. Neurol. 473:1–15, 2004.

Olfactory ensheathing cells of the lamina propria in vivo and in vitro

Olfactory ensheathing cells (OECs) continuously support the regeneration of olfactory receptor neurons (ORNs). In addition, OECs promote regeneration of neurons within the CNS in a number of transplantation paradigms, but details of exactly how they support regeneration remain elusive. The majority of studies using OECs to promote regeneration have thus far focused on understanding the cell biology of OECs purified from the olfactory bulb (OB). Here we show that a population of OECs similar to those obtained from the OB is present in the lamina propria (LP) beneath the olfactory epithelium (OE). These OECs are the first glial cells encountered by the axons of developing ORNs as they exit the OE and display distinct and variable expression of p75, S100β, GFAP, and O4, characteristic markers of bulb OECs. Once purified in vitro, they display Schwann cell‐like and astrocyte‐like properties and expand rapidly. In addition to resembling OB‐OECs, LP‐OECs also express a unique combination of developmentally important proteins—CD 44, β1 integrin, P200, Notch 3, NG2, VEGF, and PACAP and CREB binding protein (CBP/p300)—not previously reported in OB‐OECs. These data suggest that LP‐OECs, like OB‐OECs, are a developmentally distinct class of glia that are capable of both immature and mature function, depending on environmental stimuli, within the adult nervous system. GLIA 41:224–236, 2003.

Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype.

Stem cells of adult regenerative organs share a common goal but few established conserved mechanisms. Within the neural stem cell niche of the mouse olfactory epithelium, we identified a combination of extracellular matrix (ECM) receptors that regulate adhesion and mitosis in non-neural stem cells [intercellular adhesion molecule-1 (ICAM-1), β1, β4, and α-1, -3, and -6 integrins] and on horizontal basal cells (HBCs), candidate olfactory neuro-epithelial progenitors. Using ECM receptors as our guide, we recreated a defined microenvironment in vitro that mimics olfactory basal lamina and, when supplemented with epidermal growth factor, transforming growth factor α, and leukemia inhibitory factor, allows us to preferentially expand multiple clonal adherent colony phenotypes from individual ICAM-1+ and ICAM-1+/β1 integrin+-selected HBCs. The most highly mitotic colony-forming HBCs demonstrate multipotency, spontaneously generating more ICAM-positive presumptive HBCs, a combination of olfactory neuroglial progenitors, and neurons of olfactory and potentially nonolfactory phenotypes. HBCs thus possess a conserved adhesion receptor expression profile similar to non-neural stem cells, preferential self-replication in an in vitro environment mimicking their in vivo niche, and contain subpopulations of cells that can produce multiple differentiated neuronal and glial progeny from within and beyond the olfactory system in vitro.

Lamina Propria and Olfactory Bulb Ensheathing Cells Exhibit Differential Integration and Migration and Promote Differential Axon Sprouting in the Lesioned Spinal Cord

Olfactory bulb-derived (central) ensheathing cell (OB OEC) transplants have shown significant promise in rat models of spinal cord injury, prompting the use of lamina propria-derived (peripheral) olfactory ensheathing cells (LP OECs) in both experimental and clinical trials. Although derived from a common embryonic precursor, both sources of OECs reside in different nervous system compartments postnatally, and their ability to promote regeneration and efficacy after transplantation may differ depending on both their source and mode of transplantation. Here, we have purified green fluorescent protein-expressing LP and OB OECs, assayed their biological differences in vitro, and transplanted them acutely either directly into or rostral and caudal to a dorsolateral funiculus crush. LP and OB OECs exhibit multiple morphological and antigenic similarities in vitro, and, after transplantation, they both attenuate lesion and cavity formation and promote angiogenesis, endogenous Schwann cell infiltration, and axonal sprouting. However, an increased mitotic rate and migratory ability of LP OECs in vitro was reflected in vivo by their superior ability to migrate within the spinal cord, reduce cavity formation and lesion size, and differentially stimulate outgrowth of axonal subpopulations compared with OB OECs. An undesired behavior (autotomy) was also significantly enhanced by LP OEC, over OB OEC, transplantation. These results suggest that LP and OB OECs exhibit intrinsic biological differences that, after transplantation into the lesioned CNS, result in differences in postlesion spinal cord neuropathology and anatomical and behavioral regeneration outcomes that also vary depending on direct versus rostrocaudal transplantation.

Histone deacetylases 1 and 2 are expressed at distinct stages of neuro-glial development

The deacetylation of histone proteins, catalyzed by histone deacetylases (HDACs), is a common epigenetic modification of chromatin, associated with gene silencing. Although HDAC inhibitors are used clinically to treat nervous system disorders, such as epilepsy, very little is known about the expression pattern of the HDACs in the central nervous system. Identifying the cell types and developmental stages that express HDAC1 and HDAC2 within the brain is important for determining the therapeutic mode of action of HDAC inhibitors, and evaluating potential side effects. Here, we examined the expression of HDAC1 and HDAC2 in the murine brain at multiple developmental ages. HDAC1 is expressed in neural stem cells/progenitors and glia. In contrast, HDAC2 is initiated in neural progenitors and is up‐regulated in post‐mitotic neuroblasts and neurons, but not in fully differentiated glia. These results identify key developmental stages of HDAC expression and suggest transitions of neural development that may utilize HDAC1 and/or HDAC2. Developmental Dynamics 237:2256–2267, 2008.

Olfactory ensheathing cell transplantation following spinal cord injury : Hype or hope?

Olfactory ensheathing cells (OECs) are unique glia found only in the olfactory system that retain exceptional plasticity, and support olfactory neurogenesis and the re-targeting across the PNS:CNS boundary in the olfactory system. Because they are also relatively accessible in an adult rodent or human, OECs have become a prime candidate for cell-mediated repair following a variety of CNS lesions. A number of different labs across the world have applied OECs prepared in many different ways in several different acute and chronic models of rodent SCI, some of which have suggested surprising degrees of functional recovery. OECs can stimulate tissue sparing and neuroprotection, enhance outgrowth of both intact and lesioned axons (to different degrees), activate angiogenesis, change the response status of endogenous glia after lesion and remyelinate axons after a range of demyelinating insults. Their ability to stimulate regeneration in specific tracts is, however, limited. Despite this, the ongoing clinical use of cell preparations containing OECs has proceeded as a therapeutic approach for human spinal cord injury (SCI). Here, we review the current status of OEC research in SCI, and focus on potential mechanisms for OECs in the SCI repair response that may help to explain the biological reasons underlying the wide variation of results obtained in this promising, yet contentious, field.

Peripherally-derived olfactory ensheathing cells do not promote primary afferent regeneration following dorsal root injury

Olfactory ensheathing cells (OECs) may support axonal regrowth, and thus might be a viable treatment for spinal cord injury (SCI); however, peripherally– derived OECs remain untested in most animal models of SCI. We have transplanted OECs from the lamina propria (LP) of mice expressing green fluorescent protein (GFP) in all cell types into immunosuppressed rats with cervical or lumbar dorsal root injuries. LP‐OECs were deposited into either the dorsal root ganglion (DRG), intact or injured dorsal roots, or the dorsal columns via the dorsal root entry zone (DREZ). LP‐OECs injected into the DRG or dorsal root migrated centripetally, and migration was more extensive in the injured root than in the intact root. These peripherally deposited OECs migrated within the PNS but did not cross the DREZ; similarly, large‐ or small‐caliber primary afferents were not seen to regenerate across the DREZ. LP‐OEC deposition into the dorsal columns via the DREZ resulted in a laminin‐rich injection track: due to the pipette trajectory, this track pierced the glia limitans at the DREZ. OECs migrated centrifugally through this track, but did not traverse the DREZ; axons entered the spinal cord via this track, but were not seen to reenter CNS tissue. We found a preferential association between CGRP‐positive small‐ to medium‐diameter afferents and OEC deposits in injured dorsal roots as well as within the spinal cord. In the cord, OEC deposition resulted in increased angiogenesis and altered astrocyte alignment. These data are the first to demonstrate interactions between sensory axons and peripherally– derived OECs following dorsal root injury.

A Novel Embryonic Nestin-Expressing Radial Glia-Like Progenitor Gives Rise to Zonally Restricted Olfactory and Vomeronasal Neurons

Persistent neurogenesis is maintained throughout development and adulthood in the mouse olfactory epithelium (OE). Despite this, the identity and origin of different embryonic OE progenitors, their spatiotemporal induction and contribution to patterning during development, has yet to be delineated. Here, we show that the embryonic OE contains a novel nestin-expressing radial glia-like progenitor (RGLP) that is not found in adult OE, which is antigenically distinct from embryonic CNS radial glia. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate “CNS-specific” nestin regulatory elements, and produce spatially restricted olfactory receptor neurons (ORNs) in zone 1 of the OE, and vomeronasal receptor neurons restricted to the VR1 zone. This dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, in which green fluorescent protein (GFP) is found in a subpopulation of GFP+Mash1+ neuronal progenitors, despite the fact that endogenous Nestin expression is found in RGLPs throughout the OE. Embryonic OE progenitors produce three biologically distinct colony subtypes in vitro, a subpopulation of which include nestin-expressing RGLPs during in vitro colony formation. When generated from Nestin-cre/ZEG mice, neurogenic colonies also produce GFP+Mash1+ progenitors and ORNs. We thus identify a novel neurogenic precursor, the RGLP of the OE and vomeronasal organ (VNO), and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during development of the OE and VNO.

Axonal Dynactin p150Glued Transports Caspase-8 to Drive Retrograde Olfactory Receptor Neuron Apoptosis

Olfactory receptor neurons (ORNs) undergo caspase-mediated retrograde apoptosis after target removal (bulbectomy), in which axonal caspase-9 and caspase-3 activation leads to terminal apoptosis in ORN soma of the olfactory epithelium. Here, we show that caspase-8 can act as an initiator of ORN apoptosis after bulbectomy and also after synaptic instability is induced by NMDA-mediated excitotoxic death of ORN target neurons in the olfactory bulb. Caspase-8 and caspase-3 are sequentially activated within ORN presynaptic terminals, and caspase-8 complexes with dynactin p150Glued, (a retrograde motor protein) and is transported retrogradely, preceding axonal caspase-3 activation and apoptosis of ORN cell bodies. Focal in vivo inhibition of initiator caspase activation or microtubule-dependent transport (with Taxol) at the lesioned axon terminus results in a significant reduction in retrograde axonal caspase-8 and caspase-3 activation and inhibition of retrograde ORN death. Caspase-8 activation and retrograde transport after NMDA lesion is similarly reduced in mice null for p75, the low-affinity nerve growth factor receptor. The retrograde apoptosis of ORNs thus involves a novel mechanism that used p75 in the local activation of caspase-8. Once caspase-8 is maximally activated in the presynaptic terminal, it is transported retrogradely by the motor complex dynactin/dynein, a process that can be inhibited focally to inhibit ORN apoptosis after acute axonal lesion. These data have revealed a novel mechanism of retrograde apoptosis, in which caspase-8 complexes directly with axonal dynactin p150Glued to reveal a differential vulnerability of subpopulations of ORNs to undergo apoptosis after axonal damage and the loss of olfactory bulb target neurons.

Undesired effects of a combinatorial treatment for spinal cord injury--transplantation of olfactory ensheathing cells and BDNF infusion to the red nucleus.

Transplantations of olfactory ensheathing cells (OECs) have been reported to promote axonal regeneration and functional recovery after spinal cord injury, but have demonstrated limited growth promotion of rat rubrospinal axons after a cervical dorsolateral funiculus crush. Rubrospinal neurons undergo massive atrophy after cervical axotomy and show only transient expression of regeneration‐associated genes. Cell body treatment with brain‐derived neurotrophic factor (BDNF) prevents this atrophy, stimulates regeneration‐associated gene expression and promotes regeneration of rubrospinal axons into peripheral nerve transplants. Here, we hypothesized that the failure of rubrospinal axons to regenerate through a bridge of OEC transplants was due to this weak intrinsic cell body response. Hence, we combined BDNF treatment of rubrospinal neurons with transplantation of highly enriched OECs derived from the nasal mucosa and assessed axonal regeneration as well as behavioral changes after a cervical dorsolateral funiculus crush. Each treatment alone as well as their combination prevented the dieback of the rubrospinal axons, but none of them promoted rubrospinal regeneration beyond the lesion/transplantation site. Motor performance in a food‐pellet reaching test and forelimb usage during vertical exploration (cylinder test) were more impaired after combining transplantation of OECs with BDNF treatment. This impaired motor performance correlated with lowered sensory thresholds in animals receiving the combinatorial therapy – which were not seen with each treatment alone. Only this combinatorial treatment group showed enhanced sprouting of calcitonin gene‐related peptide‐positive axons rostral to the lesion site. Hence, some combinatorial treatments, such as OECs with BDNF, may have undesired effects in the injured spinal cord.