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Crucial roles for olfactory
ensheathing cells and olfactory
mucosal cells in the repair of damaged
neural tracts
Author
A.K. Ekberg, Jenny, St John, James
Published
2014
Journal Title
The Anatomical Record
DOI
https://doi.org/10.1002/ar.22803
Copyright Statement
Copyright 2014 Wiley Periodicals, Inc.. This is the pre-peer reviewed version of the following article:
Crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of damaged neural
tracts, The Anatomical Record, Vol. 297, 2014, pp. 121-1283, which has been published in final form at
http://dx.doi.org/10.1002/ar.22803.
Downloaded from
http://hdl.handle.net/10072/63829
Crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of
damaged neural tracts.
Jenny A.K. Ekberga,b and James A. St Johna*
a
Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan 4111,
Queensland, Australia
b
School of Biomedical Sciences, Queensland University of Technology, Brisbane 4000,
Queensland, Australia
Corresponding author:
James St John (family name = St John)
Eskitis Institute for Cell and Molecular Therapies,
Griffith University, Nathan 4111,
Queensland, Australia
Phone: +61 7 3735 3660
Fax: +61 7 3735 4255
Email: [email protected]
Running title: Neural regeneration using olfactory cells
Grant sponsor: Australian Research Council, DP0986294 to J.E.
1
Abstract
Olfactory ensheathing cells, the glial cells of the olfactory nervous system, exhibit unique
growth-promoting and migratory properties that make them interesting candidates for cell
therapies targeting neuronal injuries such as spinal cord injury. Transplantation of olfactory
cells is feasible and safe in humans; however, functional outcomes are highly variable with
some studies showing dramatic improvements and some no improvements at all. We propose
that the reason for this is that the identity and purity of the cells is different in each individual
study. We have shown that olfactory ensheathing cells are not a uniform cell population and
that individual subpopulations of OECs are present in different regions of the olfactory
nervous system, with strikingly different behaviours. Furthermore, the presence of fibroblasts
and other cell types in the transplant can dramatically alter the behaviour of the transplanted
glial cells. Thus, a thorough characterisation of the differences between olfactory
ensheathing cell subpopulations and how the behaviour of these cells is affected by the
presence of other cell types is highly warranted.
2
Repairing spinal cord injury by transplanting OECs.
Neural injuries are almost always serious with life-changing consequences. Injuries to the
central nervous system (CNS) are particularly devastating as central neural tracts cannot
regenerate themselves after injury. Therefore, many serious spinal cord injuries lead to
permanent disability. The main problem arising after injury to the CNS is the fact that newly
sprouting axons cannot find their target due to the presence of a glial scar, inflammatory
mediators, inhibitory factors and reactive oxygen species (Hulsebosch, 2002; Jones et al.,
2003). To date, there are no therapies that can address this problem, but one methodology that
has shown encouraging results for spinal cord injury is to transplant glial cells, the supporting
cells of the nervous system which are crucial for the survival of all types of neurons, and for
extension of axons.
Transplantation of the glial cells of the olfactory nervous system, olfactory ensheathing cells
(OECs) has shown promise in both animals and humans with spinal cord injury. In humans,
transplantation of OECs that had been purified from the nasal cavity of patients with spinal
cord injury (autologous transplantation) showed that the procedure was safe in a Phase I
clinical trial (Feron et al., 2005; Mackay-Sim et al., 2008). In animals, transplanted OECs
have been shown to survive and actively migrate into the injury site (Boruch et al., 2001;
Deng et al., 2006), reduce scar and cavity formation (Ramer et al., 2004; Li et al., 2011), to
improve locomotion and restore moveability of the hindlimbs (Gorrie et al., 2010; Takeoka et
al., 2011) and restore breathing and climbing ability (Li et al., 2003) even after complete
transections of the spinal cord (Takeoka et al., 2011; Ziegler et al., 2011). While migration of
transplanted OECs is often limited, some studies showed that OECs have migrated much
further than the immediate injury site which may be due to their attraction to reactive
astrocytes (Chuah et al., 2011). Other studies have shown that OEC transplantation leads to
3
no functional improvement (Centenaro et al., 2013; Deumens et al., 2013) and that migration
in the injured spinal cord can be quite limited. In a canine model of spinal cord injury, OEC
transplantation resulted in some improvements of fore-hind limb coordination, but no
changes in functionality of the long tracts in the spinal cord, which was attributed to limited
OEC migration (Granger et al., 2012).
Approaches that combine transplanted glia with growth factors have also been trialled.
Transplantation of OECs in combination with glial cell-derived neurotrophic factor (GDNF)
into the injured optic nerve has been shown to promote regrowth of axons, beyond the results
seen with OECs alone, however, the mechanisms behind the GDNF-mediated effect remain
unknown (Liu et al., 2010). It is also possible to genetically modify OECs to express
significant levels of GDNF, which in turn further promotes nerve repair (Cao et al., 2004).
Transplanted OECs can also stimulate Schwann cells endogenous to the injury site to in turn
promote neuronal survival and growth and potentially also remyelination (Ramer et al., 2004;
Zhang et al., 2011). Taken together, these findings show that the roles of OECs need to be
considered within a multicellular environment.
One variable factor likely to account for some of the differences in functional outcomes is the
source of OECs. We have shown that the behaviour of OECs is strikingly different between
individual populations of OECs isolated from distinct regions of the olfactory nervous system
(Windus et al., 2007; Windus et al., 2010; Windus et al., 2011). Another reason for
discrepancy is the purity of the cells. While some studies have found that inclusion of
olfactory fibroblasts in transplants may be beneficial (Li et al., 2008; Teng et al., 2008; Li et
al., 2012), others have suggested that purified OECs yield better functional outcomes (Toft et
al., 2013). Thus, the most important question in the field that needs to be addressed is which
4
combination of cells from the olfactory mucosa yields the optimal functional outcomes in
spinal cord injury repair? To answer this question, the characteristics of different OEC
subpopulations and the effects of olfactory fibroblasts and other cell types on OEC behaviour
and resultant axonal extension must be addressed.
What are olfactory ensheathing cells?
Primary olfactory receptor neurons arise from stem cells residing in the epithelium of the
nasal cavity. Their axons extend through the basal side of the epithelium and converge into
fascicles (axon bundles), constituting the highly branched olfactory nerve which ends in the
olfactory bulb within the brain. Olfactory neurons only live for 1-3 months after which they
undergo apoptosis and are replaced by new neurons originating from precursor cells in the
nasal epithelium (Mackay-Sim and Kittel, 1991). Thus, the primary olfactory nervous system
constantly regenerates itself throughout life. OECs arise from neural crest to populate the
olfactory placode (Barraud et al., 2010). They then differentiate and migrate together with the
extending axons of primary olfactory neurons that are forming the developing olfactory
nerve. Thus, unlike other glia, OECs are unique in that they migrate from the peripheral
nervous system into the central nervous system. Here, OECs always extend ahead of the
pioneer olfactory axons, leading to the assumption that OECs guide axons along a defined
path (Valverde et al., 1992; Tennent and Chuah, 1996; Chehrehasa et al., 2010; Windus et al.,
2011). In contrast, the dominant glial type in the rest of the peripheral nervous system,
Schwann cells, are known to follow paths already consisting of axons during development
(Jessen and Mirsky, 2005).
In the olfactory nerve, OECs surround large bundles of axons with the cells bodies of the
OECs located mainly on the exterior of the fascicles with the lamellipodial penetrating
5
between the axons (Fig. 1, Movie 1). In contrast, Schwann cells in the neighbouring
trigeminal nerve wrap up individual or small groups of axons (Fig. 1E). The olfactory nerve
passes from the peripheral nervous system through the cribriform plate to enter the central
nervous system. Within the nerve fibre layer, the outer layer of the olfactory bulb, the
primary olfactory axons defasciculate, sort out into specific subtypes and then project to their
correct topographical targets (Fig. 1B). The OECs are intermixed amongst the axons with
distinctly different arrangement and morphology (Movie 2) compared to OECs in the
peripheral nerve (Movie 1). The OECs within the nerve fibre layer are thought to mediate
sorting and guidance of the axons to their targets within the olfactory bulb. The nerve fibre
layer consists of an outer and an inner layer, both populated by OECs. In the outer nerve fibre
layer, OECs are thought to facilitate defasciculation of the mixed bundles of axons which
then extend into the inner nerve fibre layer, where the OECs are instead involved in sorting
and refasciculation of axons based on which type of odorant receptor they express. Therefore,
OECs in the peripheral nerve are most likely to promote cell-cell adhesion and extension of
axons in fascicles, whereas OECs in the olfactory bulb contribute to more complex and
variable cellular behaviours (Fig. 1D) (Au et al., 2002; Ekberg et al., 2012).
Why is transplantation of OECs a promising approach for repairing damaged nerves?
The main challenge in repairing injuries to the central nervous system is for newly sprouting
axons
to
find
their
target,
despite
the
presence
of
physical
barriers
and
inflammatory/inhibitory mediators. Novel therapies for repairing damaged neural tract
therefore need to address this particular problem. While the use of artificial substrates can
promote axon growth (Chehrehasa et al., 2006) it is likely that a combination of glia and
growth factors is needed.
6
To date, the majority of trials using transplanted glial cells have been using either Schwann
cells or OECs, or in some instances, a combination of both. After transplantation, Schwann
cells myelinate individual axons (Li et al., 2007), but do not migrate far into the injury site
and do not integrate well with other cells in the CNS (Andrews and Stelzner, 2007; Franssen
et al., 2009). In contrast, OECs fasciculate axon bundles rather than individual axons (Fig. 2)
and integrate within both peripheral and central neural tracts. This property can be attributed
to the fact that they are normally present at the PNS-CNS interface in the olfactory nervous
system. Indeed one of the properties that makes OECs such an attractive candidate for
transplant therapies in comparison to other glia, such as Schwann cells, is their ability to
freely associate with other cell types, in particular with astrocytes (Lakatos et al., 2003). The
ability of OECs to myelinate axons is widely debated in the field, with some studies showing
that transplanted OECs can myelinate axons under certain conditions (Babiarz et al., 2010),
but others studies suggesting that they cannot (Li et al., 2007). OECs are now considered
crucial to the unique ability of the olfactory system to continuously regenerate itself both
under normal physiological conditions and after injury (Graziadei and Graziadei, 1979;
Farbman and Squinto, 1985; Mackay-Sim and Kittel, 1991). Therefore the contribution of
OECs to olfactory neural regeneration is thought to be worthy of translation into repair of
other regions of the nervous system.
OECs produce numerous factors that may aid nerve repair such as nerve growth factor
(NGF), vascular endothelial growth factor (VEGF), GDNF, fibroblast growth factor (FGF),
insulin-like growth factor (IGF) as well as cell adhesion and extracellular matrix molecules
(Chuah and Teague, 1999; Woodhall et al., 2001; Woodhall et al., 2003; Vincent et al., 2005;
Mackay-Sim and St John, 2011). OECs also produce carbohydrate-binding molecules,
galectins (St John and Key, 1999) that are likely to promote fasciculation or be involved in
7
guidance of axons that express cell surface carbohydrates (St John and Key, 2001; St John et
al., 2006; Lineburg et al., 2011). All of these molecules can aid the growth and regeneration
of neurons.
OECs also express molecules that can be beneficial to repairing an injury site and reducing
astrocytic scar tissue, and they express matrix metalloproteinases MMP-2 and MMP-9
(Gueye et al., 2011) which enable OECs to migrate and penetrate the extracellular matrix.
Within their endogenous environment OECs are known to phagocytose axonal debris that
arises from the turnover of olfactory receptor neurons (Su et al., 2013; Ekberg and St John,
unpublished data) . Further, we have recently shown that they can phagocytose bacteria (E.
coli and B. thailandensis in vitro) (Panni et al., 2013). Thus, OECs that are transplanted into
an injury site are likely to contribute to the immune response to remove cellular debris and
foreign matter.
Together, these various characteristics of OEC biology make them excellent candidates for
neural repair therapies, however, the use of OECs needs to be considered within a complex
multicellular environment.
OECs: a heterogeneous population of cells
One of the problems in making comparisons of published experiments is that different
sources of OECs have been used in different studies. OECs exist in the peripheral nervous
system (the nasal epithelium) and in the central nervous system (the olfactory bulb in the
brain) (Fig. 1) and cells that have been purified from either of these regions have been
transplanted into an injury site in the spinal cord. We have demonstrated that peripheral
8
OECs have distinctly different behaviours and effects on axon growth compared to central
OECs (Windus et al., 2010) but it is yet to be determined which subpopulation of OECs has
the most potential for neural regeneration therapies. In addition, the purity of the cells varies
depending on the protocols used and the methods used for identification of OECs. Unless
there is 100% purity of OECs, other cell types are included in the transplanted cell mix but it
is unknown what the contributions of the different cells are, whether they are crucial to the
role of OECs and if they enhance or impede regeneration. These variations in the source and
purity of the cell preparations are likely to lead to variations in therapeutic outcomes.
Apart from their different proposed roles for olfactory axon guidance in vivo, it is clear that
OEC populations also differ in their molecular composition. In the peripheral nerve and the
outer nerve fibre layer of the olfactory bulb, OECs express high levels of S100ß and the p75
neurotrophin receptor (p75ntr) (Au et al., 2002; Windus et al., 2010). In contrast, OECs of the
inner nerve fibre layer do not express p75ntr and only low levels of S100ß (Au et al., 2002;
Windus et al., 2010). Instead, OECs in the inner nerve fibre layer express neuropeptide Y
(NPY), which is not expressed at all by OECs in the outer nerve fibre layer and the olfactory
nerve (Ubink and Hokfelt, 2000; Windus et al., 2010).
OECs show a remarkable plasticity when cultured in vitro (Pixley, 1992); the morphology
and molecular composition is highly dependent on culture conditions (Vincent et al., 2003;
Huang et al., 2008). OECs can change morphology very rapidly; when studied with timelapse imaging, OECs can transition from being bipolar to flattened within an hour after
changing the composition of the culture medium (Fig. 1F-G) (Huang et al., 2008). Here, the
bipolar, spindle-shaped OECs migrate three-fold faster than the OECs with a flattened
morphology (Huang et al., 2008) so morphology may reflect a different functional state. We
9
have developed transgenic mice in which OECs express a bright fluorescent red protein
(S100ß-DsRed mice), from which we have purified individual OEC populations and studied
their behaviour (Windus et al., 2007; Windus et al., 2010). We compared peripheral OECs
from the olfactory nerve, and central OECs from the olfactory bulb. We found that OECs
from the olfactory nerve adhered to and migrated in close contact with each other, whereas
bulb OECs were only loosely associated and showed complex mixed behaviours involving
both adhesion and repulsion (Windus et al., 2010). These findings reflect the proposed roles
of OECs in vivo: in the olfactory nerve, OECs are thought to mediate fasciculation of axons,
and in the bulbs, OECs are suggested to mediate a variety of functions including
defasiculation, sorting and refasciculation of olfactory axons. We also showed that OECs
from the olfactory bulb can be further divided into individual subpopulations with distinct
characteristics depending on their location within the bulb (dorsal/ventral, rostral/caudal)
(Windus et al., 2010). Thus, OECs are not a uniform population of cells; at least three
subpopulations are now clearly defined with strikingly different behaviours and proposed
roles in the olfactory nervous system. What is yet to be addressed in detail is whether the
subpopulations of OECs have distinct characteristics following transplantation and if so
which subpopulation is best for neural repair therapies?
Purification of OECs for transplantation
The variations in the outcomes in terms of anatomy and functionality in the different studies
of neural repair can be attributed to both the anatomical source of the OECs and to the cell
purity. Each individual laboratory optimises their own protocols for dissection, purification
and culturing of OECs. For example, “peripheral OECs” from the olfactory epithelium and
lamina propria have been used in many studies (Ramer et al., 2004) as they are easily
dissected from the nasal cavity and therefore are most relevant to straightforward autologous
10
transplantation in humans (Mackay-Sim et al., 2008). Alternatively, “central OECs” are often
acquired from the entire nerve fibre layer of the olfactory bulb (Li et al., 1997; Ramon-Cueto,
2000; Lopez-Vales et al., 2006; Li et al., 2007), however, other groups have used a more
defined population of central OECs from a distinct region of the olfactory bulb, that is, rostral
(Lankford et al., 2008) or ventral (Guest et al., 2008). In some studies, the term “central
OECs” has been used without further explanation of their anatomical origin (Teng et al.,
2008).
When OECs are isolated from any site within the olfactory nervous system, in particular the
olfactory mucosa, they are “contaminated” with many other cell types (Fig. 2). The olfactory
epithelium is populated by olfactory neurons, OECs, neural stem cells, other protenitors and
sustentacular cells. Within the lamina propria immediately beneath the epithelium, olfactory
receptor axons are ensheathed by OECs which are themselves surrounded by fibroblasts (Fig.
2). In close proximity to the olfactory nerve bundles are the trigeminal nerve bundles in
which Schwann cells wrap up the trigeminal axons (Fig. 1E). In rodents, the accessory
olfactory nerve is also present (Fig. 2) and the role/benefit of accessory OECs is not known,
but these could also contaminate “main OEC” cultures. The purification of OECs often relies
on the use of immunostaining for the marker p75ntr, but OECs within both the main olfactory
nervous system and the accessory olfactory systems express p75ntr, as do Schwann cells of
the trigeminal nerve which also innervates the nasal cavity. Further complicating the
identification of cells is the fact that cultured fibroblasts have very similar morphology to
OECs and have been shown to express p75 in vitro (Garcia-Escudero et al., 2012) under
some conditions. Thus cultures of cells from the olfactory mucosa which are reported to
contain bipolar cells that express p75ntr may potentially include OECs from the main and
accessory olfactory system, Schwann cells and fibroblasts.
11
One example that highlights the variability in functional outcomes after transplantation of
OECs from the olfactory mucosa is a recent study investigating the method in a canine
model. Here, transplantation had quite positive therapeutic outcomes with some dogs
regaining considerable function and others showing no improvement (Granger et al., 2012).
The variability in the results is likely due in part to the natural variations in the injuries that
the dogs sustained, but may also be due to the cellular mix that was used. The purity of the
cells was reported to be around 50% p75 positive, but with considerable variation between
preparations (+/- 30%). If fibroblasts in culture can also express p75 under certain conditions
(Garcia-Escudero et al., 2012) then without futher markers the identity of the cells becomes
less clear.
It is also possible that combining OECs with other cell types may be beneficial in terms of
functional outcomes. In the olfactory nerve fascicles, OECs and fibroblasts together form
tunnel-like structures through which bundles of axons extend (Li et al., 2005) and fibroblasts
have been shown to promote the proliferation of OECs in the olfactory bulb (Yui et al.,
2011). After transplantation into the injured corticospinal tract, OECs and fibroblasts in
conjunction form a “bridge” that creates a path for regenerating axons (Li et al., 1998).
Similarly, OECs and fibroblasts transplanted together can enhance regeneration of
nigrostriatal dopaminergic axons (Teng et al., 2008). Indeed, it has been proposed that the
combination of fibroblasts together with OECs is needed for maximal therapeutic potential
(Raisman and Li, 2007). In support of this idea, it has been shown that fibroblasts can
dramatically enhance the migration and organisation of Schwann cells at an injury site
(Parrinello et al., 2010), which may confer superior capacity for neural regeneration.
12
If Schwann cells or fibroblasts are included in the semi-purified “OEC” mix of cells,
differences in the proliferation rates could result in the cultures being overrun by one or other
cell type. Without precise tracking of the relative proportions of the cells while in culture and
after transplantation it would difficult to replicate therapeutic outcomes as variations in the
contributions of the different cell types could alter the regenerative capacity of the cell
transplant therapy. At present, there are not other markers or purification protocols that
clearly separate the different glia or fibroblasts and thus there is limited capacity to improve
the purification techniques. Thus, we need to consider both the source of OECs in terms of
subpopulations, and their behaviours in a multicellular environment containing other cell
types, in particular fibroblasts. Recently the regenerative ability of OECs, Schwann cells and
fibroblasts has been examined in the spinal cord of rat and the results showed that the purified
glia performed better than fibroblasts (Toft et al., 2013). In particular, the use of dissociated
mucosa resulted in higher levels of hypertrophy compared to the purified glia preparations
which indicates that the use of undetermined mixed cell preparations may not be ideal (Toft
et al., 2013).
Human spinal cord injury and autologous olfactory ensheathing cell transplantation
The promising studies using animal models have led to the use of OECs in human spinal
transplant therapies. In 2005, a clinical trial showed that transplantation of OECs from the
nasal cavity into the spinal cord of the same individual (autologous transplantation) is
feasible and safe in humans (Feron et al., 2005). The study was a Phase I/IIa clinical trial; this
type of trial is generally conducted to determine whether a procedure is safe (Phase I) and to
initiate evaluation of efficacy/functional outcomes in a small group of patients (IIa). OECs
were isolated from the nasal epithelium of spinal cord injury patients, cultured and
transplanted into the injury site. The quantity of OECs transplanted ranged from 12-20
13
million, and the patients were males aged 18-55 who had all sustained complete thoracic
paraplegia 18-32 months prior to transplantation. Patients were monitored for 3 years using a
range of standard methods for evaluating anatomical and functional outcomes such as
magnetic resonance imaging (MRI), AIS scores (which determines completion of a spinal
cord injury, ranging from A to E; a score of A shows that the entire spinal cord is severed
with little hope of regaining motor control below the site of injury), FIM/SCIM scores (which
determines the degree of disability/independence) and others. The results demonstrated that
the procedure is safe; at 3 years, there were no alterations in MRIs, and no adverse effects in
any of the patients. While the patient number was too low (3 individuals/group) for any real
meaningful evaluation of functional improvements, one patient exhibited increased sensation
to light touch and pin prick tests, suggesting an increased sensation in the zone of partial
preservation (Mackay-Sim et al., 2008).
Using olfactory mucosa (mixed cell source) for neural regeneration in humans
Since this study, autologous transplantation of nasal mucosa (containing OECs, but also other
cell types and potentially including neural stem cells) has been performed in several clinics;
however, the procedure does not yet follow formal clinical trial protocols. Together, these
reports represent 32 cases of autologous transplantation of olfactory mucosa in spinal cord
injury patients. The outcomes suggest that the transplantation was well tolerated, although
15% of patients showed meylomalacia (softening of the spinal cord primarily caused by
bleeding) or syringomyelia (development of a fluid-filled cavity) that could have been caused
by the procedure. AIS scores showed improvement in 13 patients (40%) and decreased in
only one patient. So far, these studies show that the procedure is relatively safe (Lima et al.,
2010) and may improve therapeutic outcomes, although no real conclusions can be drawn
until a proper Phase II clinical trial is conducted.
14
The way forward
The main challenge is the fact that the purity and identity of the cells transplanted has not yet
been fully addressed in any study. As discussed above “nasal mucosa” contains not only
OECs, but also Schwann cells, fibroblasts, olfactory stem cells and other cell types.
Furthermore, as we have shown, OECs consist of many different subpopulations with
strikingly different behaviours (Windus et al., 2010). Thus, the differences in functional
outcomes are most likely attributable to the fact that each transplant contained an individual
composition of cells. In order to improve reproducibility, it is therefore critical to determine
how the presence of other cell types, in particular fibroblasts and olfactory stem cells, affect
the behaviour of OECs, and to determine how individual subpopulations of OECs modulate
axonal extension.
15
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Figure legends
Figure 1. Olfactory ensheathing cells are the glia of the olfactory system. (A) A sagittal
view of an OMP-ZsGreen mouse head showing the olfactory system (Ekberg et al., 2011).
Axons of olfactory receptor neurons express ZsGreen fluorescent protein. Axons project from
the nasal cavity in the peripheral nervous system into the olfactory bulb within the central
nervous system. (B-C) A coronal view of the olfactory system in a OMP-ZsGreen mouse
crossed with a S100ß-DsRed mouse. Axons express ZsGreen (shown in B) and OECs express
DsRed (shown in C); chrondrocytes also express DsRed. OECs are present in the peripheral
olfactory nerve nerve bundles and in the nerve fibre layer surrounding the exterior of the
olfactory bulb. Boxed area in C is shown in D. (D) OECs (red) help fasciculate olfactory
receptor axons (green) in the lamina propria (LP) which underlies the olfactory epithelium
(OE). When axons (green) enter the nerve fibre layer (NFL) of the olfactory bulb, OECs (red)
help axons defasciculate, sort out and project to their targets. (E) Within the lamina propria,
olfactory nerve bundles are adjacent to trigeminal nerve bundles. (F-G) Cultured OECs can
alter morphology rapidly and sometimes exhibit the bipolar morphology but then switch to
the flattened morphology. Scale bar is 1000 µm in A, 500 µm in B-C, 100 µm in D, 8 µm in
E, 5 µm in F-G.
Figure 2. The olfactory mucosa. The olfactory mucosa consists of numerous cells types.
Within the outer olfactory epithelial layer are the stem cells (horizontal basal cell and globose
basal cell) that give rise to immature olfactory receptor neurons which mature and migrate
apically and are supported by sustentacular cells. The axons of the receptor neurons penetrate
through the basement membrane into the lamina propria where they are ensheathed by OECs
which together with fibroblasts form the olfactory nerve bundles. Also located within the
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lamina propria are bundles of the accessory olfactory nerve which are surrounded by
accessory OECs, as well as the trigeminal nerve bundles which are surrounded by Schwann
cells.
Movie 1. Morphology of peripheral OECs. Three-dimensional reconstruction of Ds-Red
OECs surrounding an olfactory nerve bundle within the lamina propria. The cell bodies of the
OECs are largely restricted to the exterior of the bundles with the lamellipodia penetrating
into the bundles.
Movie 2. Morphology of central OECs. Three-dimensional reconstruction of Ds-Red OECs
within the nerve fibre layer of the olfactory bulb. The cell bodies of the OECs have no
distinct arrangement and the lamellipodial protrude in all directions.
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