OPEN
Citation: Transl Psychiatry (2015) 5, e527; doi:10.1038/tp.2014.141
http://www.nature.com/tp
Web End =www.nature.com/tp
REVIEW
Translational potential of olfactory mucosa for the study of neuropsychiatric illness
K Borgmann-Winter1,2, SL Willard1, D Sinclair1, N Mirza3, B Turetsky1, S Berretta4,5 and C-G Hahn1
The olfactory mucosa (OM) is a unique source of regenerative neural tissue that is readily obtainable from living human subjects and thus affords opportunities for the study of psychiatric illnesses. OM tissues can be used, either as ex vivo OM tissue or in vitro OM-derived neural cells, to explore parameters that have been difcult to assess in the brain of living individuals with psychiatric illness. As OM tissues are distinct from brain tissues, an understanding of the neurobiology of the OM is needed to relate ndings in these tissues to those of the brain as well as to design and interpret ex vivo or in vitro OM studies. To that end, we discuss the molecular, cellular and functional characteristics of cell types within the olfactory mucosa, describe the organization of the OM and highlight its role in the olfactory neurocircuitry. In addition, we discuss various approaches to in vitro culture of OM-derived cells and their characterization, focusing on the extent to which they reect the in vivo neurobiology of the OM. Finally, we review studies of ex vivo OM tissues and in vitro OM-derived cells from individuals with psychiatric, neurodegenerative and neurodevelopmental disorders. In particular, we discuss the concordance of this work with postmortem brain studies and highlight possible future approaches, which may offer distinct strengths in comparison to in vitro paradigms based on genomic
reprogramming.
Translational Psychiatry (2015) 5, e527; doi:10.1038/tp.2014.141; published online 17 March 2015
INTRODUCTIONA critical component of neuropsychiatric research is the delineation of neurobiological abnormalities in patients brains. Although decades of postmortem studies have yielded vital insights, the lack of access to living patients brain tissues has long been a major hurdle in the eld. Recently, several in vitro paradigms have emerged, such as induced pluripotent stem cell (iPSC)1 and induced neuronal2 cell technologies, which offer unique and unprecedented opportunities to reprogram patients cells into developing neurons and glial cells.
This review focuses on another paradigm with a similar purpose and with distinct strengths; the olfactory mucosa (OM) tissue approach. The OM harbors neurons and glial cells residing in the nasal cavity and is readily accessible via biopsy. Neural tissues without genomic reprogramming can be captured via olfactory biopsy. OM tissues offer ex vivo and in vitro neuronal cells that may more closely reect in vivo neural characteristics of the donors. OM cells also have regenerative potential, which permits them to propagate in vitro, yielding neuronal or glial cells in sufcient quantity for various assays.
Another unique advantage of this paradigm arises from the fact that the OM is the peripheral component of the olfactory circuitry, connecting olfactory sensory neurons (OSNs) to the olfactory bulb, the neurons of which then connect to the olfactory cortex. As such, neurobiological alterations in patients limbic regions may affect or be reected in the olfactory circuitry, including neural cells in the OM. Indeed, Alzheimers disease,3,4 schizophrenia59 and mood and anxiety disorders (review10) are associated with
olfactory dysfunction, and OM derived from these patients exhibit cellular and molecular alterations.1120
Recently, a growing number of groups have examined OM biopsies in these disorders11,12,17,2123 and others in which
olfactory dysfunction is yet to be characterized as a key phenotype.14,2426 An important question is to what degree and/ or in what ways these OM-derived cells reect the pathophysio-logic mechanisms in patients brains. Given that the OM is a specialized neural tissue containing a regenerative neuroepithelium whose identity is distinct from brain cells, alterations in the OM cells can best be appreciated in the context of the neurobiological characteristics specic to the OM tissue. The goal of this paper, therefore, is to consider how the OM paradigm can contribute to pathophysiologic understanding of neuropsychiatric illnesses by reviewing the neurobiological context in which these OM cells arise in vivo and in vitro.
CYTOARCHITECTURE AND MOLECULAR CHARACTERISTICS OF THE OM
A distinctive characteristic of the OM is that it contains a regenerative neuroepithelium. In this neural tissue, cells continually slough off and are replenished by newborn cells including neurons and other cell types. Multipotent cells of the OM must continuously produce replacement OSNs and other cell types in the adult mammalian OM.27,28 This continual process of renewal from multipotent cells to differentiated neurons offers a unique
1Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA; 2Childrens Hospital of Philadelphia, Philadelphia, PA, USA; 3Department of Otolarnyngology, Head and Neck Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA; 4Translational Neuroscience Laboratory, McLean Hospital, Belmont, MA, USA and
5Department of Psychiatry, Harvard Medical School, Harvard University, Boston, MA, USA. Correspondence: Dr C-G Hahn, Neuropsychiatric Signaling Program, Center for Neurobiology and Behavior, University of Pennsylvania, 125S 31st Street, Philadelphia, PA 19104, USA.
E-mail: mailto:[email protected]
Web End [email protected] Received 11 July 2014; revised 22 October 2014; accepted 17 November 2014
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Figure 1. Cytoarchitecture and molecular characteristics of the olfactory mucosa (OM). The multilaminar organization of the rodent OM is depicted on the left, and core molecular markers that dene and distinguish mammalian OM cell types in vivo are listed on the right. Most of the studies identifying these markers have been reviewed elsewhere.3236 *Markers that distinguish lamina propria mesenchymal stem cells (LP-MSCs) have not been studied to the extent of markers for other OM cell types, but see Tome et al.,37 Lindsay et al.38 and Delorme et al.39 for MSC-specic cluster of differentiation (CD) expression in these cells. GBC, globose basal cell; HBC, horizontal basal cell; INP, intermediate neural progenitor; OEC, olfactory ensheathing cell; OSN, olfactory sensory neuron; SUS, sustentacular cell.
opportunity to capture the processes of neurodevelopment in human studies.
Although the OM is located external to the brain, OSN axons extend through the lamina propria (LP) and then the cribiform plate to the brain where they synapse onto neurons of the olfactory bulb. These olfactory bulb neurons then project to primary olfactory regions, including the piriform and entorhinal cortices and the amygdala.29 Cells within the projection regions then synapse onto cells in other neuropsychiatric disease-related regions, including the hippocampus, prefrontal cortex and hypothalamus, thus enabling olfactory information to inuence emotional, cognitive and visceral functions.10,29
The current knowledge of the cytoarchitecture and molecular proles of the OM stems largely from rodent studies. The human olfactory neuroepithelium (ON) is thought to recapitulate histo-logic and molecular characteristics of the rodent counterpart, though the precise degree of similarity remains under investigation. The ON exists as a pseudostratied epithelium in adult mammals, comprising three primary layers; a basal layer, an intermediate region and an apical layer (as depicted in Figure 1). The apical layer is exposed to the nasal cavity and contains glial-like supporting cells called sustentacular cells. The intermediate region contains layers of immature and mature OSNs and exhibits a distinct laminar organization in the rodent ON (Figure 1), with the immature OSN layer residing closer to the basal lamina. In the human ON, however, immature and mature OSNs are more dispersed throughout, suggesting a less pronounced laminar organization of OSNs than observed in the rodent ON.30,31
Deep to the intermediate region lies the basal layer that contains globose and horizontal basal cells (GBCs and HBCs, respectively), and is separated from the underlying LP by the basal lamina. As shown in the mouse ON, GBCs and HBCs are the stem cells that initiate the OSN lineage by proliferating into transit-amplifying progenitors that give rise to immediate neuronal precursors (INPs), which differentiate into immature and ultimately mature OSNs.32 Several rodent studies have delineated the expression of transcription factors in ON basal cells to dene the lineage of ON cells.33,34 These dening factors and associated cell
types are listed in Figure 1. Depending upon the niche, both HBCs and GBCs comprise stem cell populations in the rodent ON that can be both neuro-competent and multipotent.28,40
In the mouse ON, the basal layer exhibits a distinct organization. HBCs form a single layer next to the basal lamina, exhibit a attened morphology and express cytokeratin-5 and -14, whereas
GBCs exhibit a round morphology, do not express cytokeratins and reside in a single layer supercial to the HBCs (Figure 1).41,42
Similarities between rodent and human ON basal cells have been observed,30,31,43 yet the laminar organization and morphologies of
the different basal cell types (attened versus rounded) appear to be less clearly dened in human ON.30,31 In addition, molecular
expression patterns of these basal cells may differ between species, as robust expression of the low afnity nerve growth factor p75NGFR has been observed in rst- and second-layer basal cells in human ON, but not in adult rodent ON.13,30,44,45 These
basal cells, whether analogous to rodent HBCs, GBCs or both, are thought to comprise the presumptive neural precursor population in human ON.
Juxtaposed to the basal cell layer, separated by the basal lamina, is the LP. Rodent studies have shown this to be a neural crest-derived ectomesenchymal tissue, which contains multi-potent mesenchymal stem cells (LP-MSCs), olfactory ensheathing cells (OECs), axons originating from the OSNs and the acynus of Bowman glands.37,46,47 Studies of LP-MSCs in rodents and humans
have shown that these cells are able to generate multiple cell phenotypes, including, but not limited to, OECs and neural lineages, both during development and in response to regenerative cues.35,37,38,46,47,48 The glial potential of OECs has been
extensively studied in the context of spinal cord transplantation and has been reviewed elsewhere.49,50 OECs represent a highly specialized, likely heterogeneous glial cell population that share morphological and neurochemical features with Schwann cells and astrocytes, although they differ from both. Their neurochemical composition highlights their functional heterogeneity, as they express sets of proteins shared with astrocytes (for example, glial brillary acidic protein (GFAP) and s100), oligodendrocytes and oligodendrocyte precursors (O4, NG2), CNS radial glia (basic lipid binding protein, BLBP), as well as developmentally important proteins CD 44, 1 integrin, P200 and Notch 3.36
USE OF EX VIVO OLFACTORY TISSUE IN THE STUDY OF NEUROPSYCHIATRIC AND NEURODEVELOPMENTAL DISEASES
As a regenerative neuroepithelium containing a range of morphologically and molecularly distinct neuronal and glial cells, the ex vivo ON represents a useful tool for examining cell type-specic biological changes in neuropsychiatric illness. Compared with all other in vitro neuronal models or blood cell studies, this cell type-specic resolution is a unique feature of this paradigm.
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As such, the ON has been utilized as a platform for histologic assessment, investigation of intracellular signaling and gene expression proling.
Using a histologic approach to study neuronal differentiation, Arnold et al.13 conducted immunohistochemical assessment of the ON using markers for specic stages of neuronal differentiation in ex vivo tissue from schizophrenic patients and controls. In this study, basal cells, immature and mature neurons were marked with antibodies for p75NGFR, GAP-43 and OMP, and densities of immunoreactivity for these markers were used as indices for specic stages of differentiation. Compared with controls, patients exhibited decreased density of p75-labeled basal cells, a higher density of GAP-43-labeled immature OSNs and an increased ratio of immature to OMP-labeled mature OSNs. Together these ndings led to the postulate that neuronal lineage may be disrupted in the ON and by extension in the CNS of patients with schizophrenia.13
In a similar paradigm, Pantazopoulos et al.20 examined chondroitin sulfate proteoglycans, prominent components of the extracellular matrix, in ex vivo ON from schizophrenic patients compared with controls. These extracellular matrix proteins are critical for cellular differentiation and migration, and are postulated as a possible mechanism for the altered neuronal lineage as observed in by Arnold et al. In that study, the decreased chondroitin sulfate proteoglycan density reported in mature OSNs in schizophrenia is consistent with previous ndings of reduced proteoglycans in multiple brain regions of postmortem brains of schizophrenic patients.20,51 Thus, some of the neurobiological
characteristics observed in the ON can be extrapolated to those of the brain.
Histologic changes in the ON that are specic to particular neuropsychiatric illnesses may hold promise as potential biomarkers. In the postmortem ON of patients with Alzheimer disease, Arnold et al. found higher frequency and abundance of amyloid- and paired helical lament-tau pathologies in ON derived from Alzheimer patients compared with controls,11 essentially mirroring previous observations in postmortem brains of patients (reviewed in Hardy and Selkoe52). If these and other ndings described above are extended to ON biopsy tissues of living patients, and are correlated with clinical severity or diagnostic subtypes, they could serve as cellular biomarkers of psychiatric illnesses.
The ON can be utilized for gene expression proling of neural cells with cell type-specic resolution. McCurdy et al.14 used
microarray analyses in ex vivo tissues to demonstrate altered expression of genes relating to cell cycle and neurogenesis in schizophrenic patients, ndings that may be consistent with neurodevelopmental dysregulation. More recently, Mor et al.21
used laser capture microdissection in ex vivo tissues to isolate mature OSNs, and observed increased miRNA expression of MiR-382 in schizophrenic patients versus controls. Notably, similar dysregulation in MiR-382 has been observed in the postmortem dorsolateral prefrontal cortex,53 again supporting the notion that certain aspects of brain pathology may be represented in the ON.
Ex vivo ON tissues can also be examined for their electro-physiological properties. OSNs can be dissociated from biopsy tissues, readily identied by their morphology and examined using calcium imaging, voltage sensitive dye imaging and other electrophysiological measures. Indeed, Hahn et al.19 assessed odorant induced calcium signaling in OSNs derived from medication-free bipolar disorder patients and their matched controls, and demonstrated decreased intracellular calcium signaling in bipolar disorder patients. Given that dysregulated calcium signaling has been consistently reported in bipolar disorder patients in peripheral blood and brain,5456 ON derived
from bipolar patients may be useful for furthering understanding of the molecular pathologies of the disorder.
Ex vivo ON tissues also provide a unique paradigm for the study of neurodevelopmental disorders, given the lifelong regeneration
of ON and the presence of OSNs at all the stages of development. One such developmental disorder where ex vivo ON has been studied is Rett syndrome, which results in profound intellectual disability and has been associated with decits in neuronal maturation. Ronnett et al.26 reported fewer mature OSNs, increased immature OSNs and abnormal morphology of mature OSNs in Rett syndrome patients of various ages compared with age-matched controls. These observations support the notion that ON is a viable tool for studying neurodevelopmental aspects of diseases characterized by abnormal neuronal development.
IN VITRO USE OF CULTURED OM-DERIVED CELLS IN THE STUDY OF NEUROPSYCHIATRIC ILLNESSES
A second approach to the use of the OM in psychiatric research, which complements the study of ex vivo OM tissues, is the in vitro culture of OM-derived cells. Due to the regenerative nature of the OM, OM-derived cells can be readily propagated and are increasingly yielding insights into the cellular and molecular underpinnings of neuropsychiatric illness.
Properties of adult human OM-derived cells in vitroWhen developing in vitro models for neuropsychiatric illnesses, it is critical to consider the specic characteristics of cell types arising in culture, which are determined by the tissue compartment from which they originate and conditions in which they are grown. In diverse variations of these factors, multiple approaches to the culture of OM-derived cells have been developed including the organotypic explants, dissociated cultures and neurospheres (see Box 1 for specics of culture paradigms). Common to all these paradigms is that they seek to utilize regenerative characteristics of the ON and its underlying LP as neural tissues, within which progenitors differentiate into neurons, sustentacular cells and OECs.5759
The experimental utility of cultured OM cells as a model for neuropsychiatric illness is, in part, related to the extent to which cultured cells recapitulate the characteristics of in vivo OM cells or of donors neurobiological makeup. To dene neuronal precursors, immature neurons or OSNs, a rapidly increasing database on the expression of molecular markers in cell types of each differentiation stage can be used as a guide. As described in Figure 1, human and rodent ON cells at different stages of neuronal differentiation in vivo are characterized by expression of molecular markers such as Cytokeratin-5, Sox2, Pax6, Ascl1, Neurog1, NeuroD1, Gap43 and OMP. Neuronal precursors can be dened as proliferative cells whose progeny ultimately become neuronal cells. Immature neurons and OSNs do not share this proliferative capacity, and OSNs are further dened functionally by their capacity to respond to odorants.70,71
Numerous markers have been used to characterize OM-derived cells in vitro (Figure 2). While some markers, such as NeuroD1 and NCAM, are selectively expressed in neuronal cells at varying stages of differentiation in vivo, the selectivity or appropriateness of other markers is less clear. For example, -tubulin III and vimentin are expressed not only in olfactory neurons in vivo, but also in other cell types,72,73 thus limiting their utility as sole markers for neurons in vitro despite their widespread expression. While nestin has been recognized as a neural stem cell/progenitor marker in the mammalian brain, its expression in adult human ON in vivo has been demonstrated exclusively in sustentacular cells as opposed to basal cells of neural lineage.45 LP-MSCs, however, do express nestin in vitro, suggesting that the use of nestin immunoreactivity as a stem cell marker applies specically to LP-derived neural stem cells.3739
Adult human OM-derived cells, originating from non-dissociated biopsy tissue and cultured in a monolayer in serum-containing media, are capable of expressing mRNA transcripts or
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Box 1 In vitro culture paradigms of adult human olfactory mucosa (OM)-derived cells
Human OM is harvested from the nasal septum or turbinates typically by biopsy, or also by exfoliation. Cells can be subsequently propagated from the olfactory neuroepithelium (ON),57 its underlying lamina propria (LP)60 or from tissue containing both regions.30 Protocols have been established to enhance the neural stem cell properties of OM cells through neurosphere57,61 and monolayer culture.62(A) Organotypic-monolayer cultures: Non-dissociated adult human ON biopsy tissues, containing a portion of underlying LP, can be utilized for propagating OM cells in monolayer cultures in serum-containing media on uncoated or bronectin-coated plates30,60,62,63 or on a Matrigel substrate.22,64 In cultures
on bronectin-coated plates, epithelial cells rst grow out from biopsy tissue to form sheets, over which grow round, more rapidly proliferating cells after 2448 h.30,60
(B) Neurosphere cultures: Multiple groups have reported that cells collected from the human ON itself (with or without some underlying LP) can be cultured in neurospheres from dissociated biopsy tissue. In serum-containing media, when adult human ON tissue (without underlying LP) is rst dissociated, cells with the bipolar morphology of neurons, along with presumptive olfactory ensheathing cells, are present.61 Such
cells soon disappear in serum-containing media, and are replaced by a proliferating population, which form spheres and can be cultured for extended periods.61,6567 Alternatively,
adult human OM-derived cells can be dissociated and propagated on poly-L-lysine-coated plates in dened medium with EGF and FGF2 to form primary neurospheres, before being plated onto uncoated or bronectin-coated plates and maintained in serum-containing medium in monolayer culture.16,57
Additional approaches that have been employed in rodent studies include culture of adult rat ON-derived cells on a feeder layer of newborn rat glial cells,68 and co-culture of ON-derived cells with a 3T3 cell feeder layer at the airliquid culture interface.69
proteins characteristic of a number of OM cells types in vivo (Figure 2). These include NeuroD1, Gap43, TrkB, NCAM, -tubulin III, OMP, OcNc1 and OR3A1.25,30,61,63,66,74 Markers of sustentacular
and glial cells, such as Sus4, S100 and GFAP, and mesenchymal stem cells (Stro-1) have also been reported to be expressed in human OM culture cells (Figure 2).30,3638,49,66 Some ON-derived
cells are also capable of progressing from replication (indicated by BrdU incorporation) to terminal differentiation (OMP expression) in culture.30 At a functional level, some human ON cells cultured under these conditions demonstrate the odorant responsiveness specic to OSNs, with ~ 5.5% of cells in culture responding to helional, a ligand for the OR3A1 odorant receptor,63 and up to 10% of OMP-positive cells responding to a mixture of other odorants.71 Receptors for the key neurotransmitters serotonin (5-HT2 receptors), dopamine (D2 receptor) and glutamate (NMDA receptors), which have been identied in cultured ON cells, also display functional activity.63 Benitez-King and colleagues collected nasal exfoliates24,25 and reported that the cells harvested this way and propagated in monolayer culture expressed with varying frequencies a number of potential cell type-specic markers (Figure 2) and expressed action potentials.25
Neurosphere-forming cells cultivated from either the ON alone, or from both the ON and LP, express markers such as NCAM, -tubulin III, TrkA/B, nestin and Map2 (Figure 2).16,57,61,6567
Overall, the expression of ON neuronal lineage markers, and of other putative neuronal markers, suggests that many OM-derived culture cells may be of a neural lineage. To date, although multiple
OM-derived cell culture paradigms have been used in various studies, the relative expression of cell type-specic markers by cells cultured in these paradigms has not been directly compared. When indirectly compared, some similarities between paradigms exist in the expression of multiple markers, such as NCAM and nestin (Figure 2), suggesting a degree of consistency among paradigms employing serum-containing media. However, differences also exist, such as in tubulin III, which is expressed in 35% of ON-derived cells in the study of Matigian et al.,16 in the majority
of cells in multiple other studies57,61,67 and in ~100% of ON-
derived cells in the study of Benitez-King et al.25 Overall, further work to delineate the degree of consistency between culture paradigms, including characterization of cellular heterogeneity and marker expression, is needed.
The adult human olfactory LP alone can also be used to culture progenitors of ectomesenchymal origin, which are particularly suitable for the in vitro propagation of presumptive OECs.38,75
When cultured in serum-free media supplemented with NT3, almost all adult human LP-derived proliferating cells express OEC markers S100 and GFAP, ~80% express p75NGFR, whereas none express HNK1 (a marker for Schwann cells).59,75 These cells hold
promise for autologous transplantation in the treatment of spinal cord injury.50,59,76
In vitro culture of OM-derived cells from rodents and fetal humans The OM paradigm has been employed using in vitro cells from rodents, shedding light on the stem cell potential of OM-derived cells. For example, proliferation of neuronal precursors and successful genesis of mature OMP-positive OSNs from adult rat ON-derived cells in culture has been reported.68 When adult rodent ON-derived cells are co-cultured with a 3T3 cell feeder layer at the airliquid culture interface, cells with the molecular phenotypes of GBCs, HBCs, sustentacular cells and neurons can be generated, which can successfully reintegrate into the ON after transplantation.69 Monolayer culture of rat p75NGFR-expressing OM-derived cells, which were puried by uorescence-activated cell sorting, has been used for characterization of putative OECs in vitro.77 Cells from the mouse olfactory LP, when cultured in a monolayer, demonstrate the same OEC phenotype and cell type-specic marker expression as human LP-derived cells.36 When
cultured in spheres, mouse olfactory LP-derived cells have the capacity to integrate into lesioned hippocampus in vivo, differentiate into neurons and restore learning and memory.78
Numerous studies have used OM-derived cells from pre-natal/ neonatal rodents7988 and fetal humans,89,90 in part because such
cells can be propagated more readily. This is observed under controlled neurosphere assay conditions, in which ON-derived neurospheres are generated with progressively lower efciencies from E13.5 mice, P5 mice and adult mice.35 Importantly, neuro-spheres derived from E13.5 and adult animals contain cells with different morphologies, which express different cell type-specic markers and proliferate at different rates.91 As a result, the extent to which ndings in OM-derived cells from neonatal and early postnatal animals can be extrapolated to adults may be limited.
The effects of culture environment on neuronal identity of OM-derived cells
The properties of OM-derived cells, such as their proliferation, morphology and expression of cell type-specic markers, can be heavily inuenced by the in vitro environment, particularly culture medium57,60,61,65,66 (Table 1). In culture, BDNF (brain-derived
neurotrophic factor), NT3 and NT5 promote ON-derived cell survival,92 FGF2, FGF8, EGF and TGF- (transforming growth factor-alpha) have been used to stimulate progenitor proliferation,82,93,94 and serum deprivation, Db-cAMP, IGF-I (insu
lin-like growth factor-I), retinoic acid, forskolin and sonic hedgehog have been used to drive human ON culture cells toward a
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Figure 2. Expression of putative molecular markers of stem cells, neural progenitors, neurons and glia by various olfactory mucosa (OM) cell types, both in adult OM in vivo and in cells generated from adult OM tissues in vitro. Studies of OM explant tissues or dissociated cells immediately ex vivo are not included. Percentage values are approximate, and refer to the proportion of cells in culture which express each marker. Values are only shown for markers displaying consistency between studies, and only for cells grown in serum-containing media. *, studies using neonatally-derived rodent or fetally-derived human ON culture cells; &, markers whose signicance in OM culture cells is uncertain due to a discordance between human and rodent (for example, p75NGFR), low/uncertain levels of expression in adult ON in vivo (NF-M/H, Nestin, NeuN, MAP2) or a possible lack of cell type specicity (p75NGFR, Vimentin); #, markers whose expression increase when cells are cultured in base (unsupplemented dened) media rather than serum-containing media; ^, mesenchymal stem cell cluster of differentiation (MSC-CD) markers, used to conrm cell type, vary among studies. CK, cytokeratin; INP, intermediate neural progenitor; LP, lamina propria (indicates cultures derived solely from LP, without ON); LP-MSC, lamina propria mesenchymal stem cell; ND, not detected; NTR, neurotransmitter receptor; OEC, olfactory ensheathing cell; ON, olfactory neuroepithelium; OSN, olfactory sensory neuron.
neuronal phenotype.25,95,96 Proliferation of human LP-derived cells with an OEC phenotype is promoted by NT3, BDNF or NGF.75
Human LP-derived cells can be driven toward a neuronal phenotype with a cocktail of retinoic acid, forskolin and sonic hedgehog,97 or dexamethasone, insulin, 3-isobutyl-1-methylxanthine, indomethacin and ethanol,98 and toward a osteoblast phenotype with a cocktail of dexamethasone, L-ascorbic acid-2-phosphate, and -glycerophosphate98 or NaH2PO4.39 Other additional factors, many of which are relevant to the control of neurogenesis and differentiation in vivo, have also been shown to modulate OM-derived cell proliferation, morphology and expression of cell type-specic markers in vitro (Table 1).
THE USE OF OM-DERIVED CELLS IN PSYCHIATRY AND NEUROSCIENCE RESEARCH
Historically, the tools to study neural function and mechanisms of drug action in humans at a cellular level have proved elusive. OM-derived cells have emerged as a promising platform for such
investigations in a range of neuropsychiatric, neurodegenerative and developmental disorders.
The ability to investigate functional cellular characteristics represents a distinctive opportunity afforded by the OM-derived cell paradigm. The processes of cell proliferation, adhesion, motility, metabolism and apoptosis have been investigated by Mackay-Sim and colleagues, who showed that ON-derived cells from schizophrenia patients (but not bipolar disorder patients), when cultured in a monolayer from non-dissociated biopsy tissue, proliferate more rapidly than ON-derived cells from healthy controls.14,15,17 Such cells were also reported to adhere less
effectively to the culture plate than those from controls, both during establishment of the initial explant15 and during subsequent monolayer culture.18 This abnormal adhesion was accompanied by increased cell motility, decreased vinculin-positive adhesion complexes and faster disassembly of focal adhesions in schizophrenia ON-derived cells than controls.18
Metabolic activity of ON-derived cells has been investigated in schizophrenia and Parkinsons disease, and was reduced in
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Table 1. In vivo and in vitro factors inuencing the proliferation, differentiation and survival of cells within, or derived from, the olfactory mucosa (OM)
Factor Cell type impacted in vivo
Effect observed in vivo Effect on OM-, ON- or LP-derived cells in vitro
TGF- Stem cell Stimulates proliferation and progression of rodentHBCs99
Stimulates ON-derived cell (HBC) proliferation93
EGF Stem cell Stimulates proliferation of rodent HBCs99 Stimulates ON-derived cell (HBC) proliferation93 Np63 (p63) Stem cell Required for rodent HBC genesis and differentiation100
Sox2 Stem cell Increases Ascl1 expression and differentiation of rodentGBC/HBCs into neural progenitors94
Meis1 Stem cell Suppresses Ascl1 expression and stem cell differentiation94
Foxg1 Stem cell Promotes differentiation of rodent Sox2-positive stem cells into neural progenitors101
Retinoic acid Stem cell Stimulates basal cell proliferation and/or differentiation after lesion102
Promotes ON-derived cell (HBC) differentiation95
and LP-derived neuronal differentiation37
Uncx Early neural progenitor
Promotes proliferation of neural progenitors and survival of OSNs103
FGF2 Early neural progenitor
Enhances neural progenitor proliferation104 Stimulates OM-derived cell proliferation37,83,90,105
FGF8 Early neural progenitor
Promotes neurogenesis and progenitor proliferation106
by enhancing Sox2 and repressing Meis1 expression94
Ascl1 Early neural progenitor
Required for maturation of early neural progenitors into INPs107,108
Six1 Early neural progenitor
Required for maturation of early neural progenitors into INPs109
Ngn1 Early neural progenitor
Required for maturation of neural progenitors into INPs108
BMP2,4,7 Early neural progenitor, immature OSN
At lower concentrations, promotes survival of newly formed OSNs (BMP4 only),110 at higher concentrations inhibit neurogenesis by targeting Ascl1 for proteolysis111
FST Early neural progenitor
Promotes progenitor proliferation by antagonizing GDF11 and ACTB112,113
ACTB (activin) Early neural progenitor
Inhibits progenitor proliferation by promoting proteolysis113
Hes1 Early neural progenitor
Suppresses Ascl1-mediated neural progenitor maturation114,115
Hes5 (with Hes1)
Early neural progenitor
Suppresses Ngn1-mediated neural progenitor maturation114,115
MMP2, MTIMMP
Early neural progenitor, INP
Downregulated following bulbectomy in rodent GBCs116
Upregulated following bulbectomy in rodent GBCs116
GDF11 INP Inhibits proliferation of INPs by inducing reversible cell cycle arrest112,113
TIMP Early neural progenitor, INP
Runx1 INP Stimulates INP proliferation117 Stimulates ON-derived cell proliferation117 NPY INP Promotes, and is required for, INP proliferation andmaturation118121
Increases OM-derived cell proliferation via the Y1 receptor and PKC-dependent ERK1/2 phosphorylation119,121
Peptide YY INP Regulates INP differentiation118,119 Increases OM-derived cell proliferation118,119 Atf5 Immature OSN Promotes survival and maturation of immature OSNs122
Emxl2 Immature OSN Promotes survival and maturation of immature OSNs123
Lhx2 Immature OSN Promotes survival and maturation of immature
OSNs124,125
ZFP423/OAZ Immature OSN Inhibits maturation of immature OSNs126
LIF1 Immature OSN Promotes survival and inhibits maturation of immature
OSNs127,128
Increases OM- and ON-derived cell proliferation,93,129 decreases apoptosis, inhibits differentiation127,128
BDNF Multiple cell types May provide trophic support to maturing ON cells130132
Promotes LP-derived glial cell proliferation75 and
neuronal differentiation37
NT3,4 Multiple cell types May provide trophic support to maturing ON cells131 (NT3) promotes LP-derived glial cell proliferation75
NGF Multiple cell types Expressed in neuronal ON layers, may provide trophic support to maturing ON cells131
Promotes LP-derived glial cell proliferation75
PACAP Promotes proliferation of OM-derived cells133
IGF-I Decreases OM-derived cell proliferation and promotes cell differentiation96
Nitric oxide Inhibits ON-derived cell proliferation and promotes differentiation134
Serum deprivation
Decreases ON-derived cell proliferation and increases neuronal differentiation66,82
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Table. 1. (Continued)
Factor Cell type impacted in vivo
Effect observed in vivo Effect on OM-, ON- or LP-derived cells in vitro
Db-cAMP Promotes ON-derived cell differentiation25,61
Forskolin Promotes ON-derived cell differentiation95
Sonic hedgehog
Promotes ON-derived cell (HBC) differentiation95
Abbreviations: BDNF, brain-derived neurotrophic factor; GBC, globose basal cell; HBC, horizontal basal cell; IGF-I, insulin-like growth factor-I; INP, intermediate neuronal precursor; LP, lamina propria; NGF, nerve growth factor; ON, olfactory neuroepithelium; OSN, olfactory sensory neuron; TGF-, transforming growth factor-alpha.
Parkinsons disease.16 Increased cell death has been observed in ON-derived cells from bipolar disorder patients (but not schizophrenia patients), relative to healthy controls,14 although a subsequent study reported increased caspase-3/7 activity in ON-derived cells from schizophrenia patients, suggesting increased apoptotic activity.16
Cellular processes governing microtubule formation and cytoskeletal structure have also been investigated in OM-derived cells from individuals with psychiatric illness. Dynamic structural processes have been monitored across time in ON-derived cells, and increased microtubule stability in ON-derived cells from individuals with schizophrenia than in ON-derived cells from controls has been reported.135 In addition, in a paradigm employing exfoliation and monolayer culture, ON-derived cells from schizophrenia patients were described by Benitez-King and colleagues to display abnormal microtubule organization, whereas ON-derived cells from bipolar disorder patients displayed decreased microtubule length and cytoplasmic -tubulin III protein expression.24,25 Decreased L-type voltage-activated calcium currents in the ON-derived cells from individuals with schizophrenia were also reported in this study.26 Overall, these studies suggest the potential utility of OM-derived cells for the investigation of cellular function in neuropsychiatric disorders. The reported ndings in OM-derived cells implicate processes and pathways, which have been independently implicated in schizophrenia and bipolar disorder by postmortem studies, such as in neurogenesis,136,137 neuronal migration137,138 and apoptosis.139
However, they also hold the promise of revealing aspects of neural cell function in psychiatric illness that are difcult to investigate using traditional approaches.
The OM-derived cell culture approach also provides a powerful platform to explore mechanisms of drug action, and to target pharmacologically the molecular pathways of interest in psychiatric disorders. Studies have been performed investigating the effects of pharmacological agents on intracellular signaling, molecular expression and cell function in ON-derived cells derived from schizophrenia, bipolar disorder and major depressive disorder patients. Feron et al.140 demonstrated that dopamine increases apoptosis in control ON-derived cells, but decreases apoptosis in schizophrenia cells. Cellular effects of lithium, a rst-line bipolar disorder treatment, have also been explored in ON-derived cells, revealing amelioration by lithium of ionic stress-induced apoptosis and TRPM2 overexpression.141 In major
depressive disorder, there is evidence that ON-derived cells display decreased nuclear translocation of the glucocortocoid receptor, GR, in response to glucocorticoids relative to controls.142
This diminished receptor translocation was accompanied by greater cytoplasmic association of GR with the immunophillin FKBP51.142 These studies suggest the potential utility of ON-derived cells for understanding current treatments and developing novel pharmacological agents in psychiatry.
The experimental potential of OM-derived cells from psychiatric patients has also been highlighted by studies of subcellular molecular abnormalities. As in work with ex vivo ON tissue, many
molecular and cellular studies using ON-derived cells have demonstrated a concordance with postmortem brain studies. ON-derived cells from Alzheimers patients exhibited increased amyloid precursor protein levels,143 along with other protein alterations,23 consistent with changes in multiple brain regions in the illness.52 Dysregulation of gene expression in multiple cellular pathways, including cell cycle regulation,14,17 oxidative stress response,16,144 focal adhesion16 and axon guidance16 have been reported in ON-derived cells from schizophrenia patients, some of which may be meditated by altered histone H3 lysine 4 trimethylation.144 These ndings are consistent with postmortem brain studies in schizophrenia showing aberrant histone H3 lysine 4 trimethylation within the GAD1 gene145 and dysregulated expression of immune genes.146,147 Protein changes in oxidative
stress and cell adhesion pathways have also been recently reported in a preliminary human iPSC study.148 As described
earlier, increased expression of miR382 has been reported in ON-derived cells from individuals with schizophrenia,21 mirroring ndings in ex vivo microdissected ON tissue21 and postmortem prefrontal cortex.53 In ON-derived cells from bipolar disorder patients, gene expression changes within the phosphatidylinositol signaling pathway14 have been reported. In Alzheimers and Parkinsons diseases, OM-derived cells have also demonstrated altered gene and protein expression proles, implicating mitochondrial function, oxidative stress and xenobiotic metabolism in these illnesses.16,22 Finally, OM-derived cells have also yielded insights into molecular pathologies in developmental disorders. In an early study interpreted as suggesting bloodbrain concordance of FMRP decits in fragile X syndrome, decreased FMRP protein was observed in ON-derived cells from affected individuals.149 LP
MSCs from individuals with familial dysautonomia, which is caused by mutations within the IKBKAP gene, display lower IKBKAP mRNA expression, decreased IKAP/hELP1 protein, altered IKAP/hELP1 subcellular localization and dysregulation of genes involved in cell migration and cytoskeletal reorganization.97 Overall, these studies indicate the potential for OM-derived cells to shed light on molecular abnormalities associated with brain disorders, and the concordance of many OM-derived cell studies with postmortem ndings highlights the potential for OM-derived cells to reect pathophysiological mechanisms in the brain.
CONCLUSIONSAdvantages and challenges for olfactory paradigms in the study of neuropsychiatric illnesses
Olfactory mucosal biopsy studies afford a unique opportunity in neuropsychiatric research, offering ex vivo and in vitro neural cells from living individuals, which harbor biological characteristics that may be more relevant to neuropsychiatric disease than blood cells150 or skin broblasts. Figure 3 provides a summary of the previous and potential uses of OM-derived cells in psychiatric and neuroscience research, as described throughout this review.
The OM model has a number of strengths. First, ex vivo OM tissues were previously exposed to the in vivo neurohormonal
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Figure 3. Examples of the use of ex vivo olfactory tissues and in vitro OM-derived cells in psychiatric research.
milieu and contain neurobiological signatures of the in vivo condition. As such, they can serve as a point of reference for in vivo and in vitro OM ndings, bridging the gap between these two approaches. Second, OM biopsies, though limited by the quantity of tissue available, can be safely obtained from the same subjects multiple times (monthly, up to three consecutive times in our human subjects protocols) and can be tted with longitudinal clinical study designs. In this approach, OM biopsies are obtained in specic phases of the illness, while patients clinical conditions are carefully characterized. Interpretation of neurobiological parameters in the context of clinical changes is a rare opportunity in neuropsychiatric research. Examples of such use include but are not limited to the collection and examination of OM biopsies from(1) individuals at risk for schizophrenia and unaffected relatives before and after the onset of illness, (2) mood disorder patients before, during and after disease episode (such as in depressive disorders or bipolar disorder), (3) any neurodevelopmental or psychiatric disease before, during or after treatment. Finally, one promising aspect of using ex vivo OM tissues in psychiatric illness involves their relation with the broader olfactory neurocircuitry: olfactory mucosa olfactory bulb olfactory cortex. Disease-associated alterations within the ON may not occur in isolation, and may reect brain abnormalities within the broader olfactory neurocircuitry. Indeed, numerous structural and functional decrements associated with neuropsychiatric diseases are reported in many of these brain regions. Olfactory circuitry can now be interrogated by psychophysical testing, electroolfactogram and
evoked potentials. Combining these measures with molecular and cellular measures derived from OM biopsy tissues can help us to link molecules to cellular changes and then to circuit activity.
An important consideration in using ex vivo tissues is that the human ON is patchy and thus some biopsy tissues may not contain OSNs. Approximately 70 to 80% of ON biopsy tissues obtained from nasal septum or middle turbinate yield olfactory receptor neurons when dissociated immediately from the biopsy (unpublished observations). This is an important consideration, however, particularly for histologic examination of OE tissues. As the neuroepithelial patches are intermixed with respiratory epithelium, quantitative assessment of protein immunoreactivity or mRNA needs to be conducted by delimiting neuroepithelial versus respiratory patches in histologic sections. Under in vitro conditions, however, such limitations do not appear to gure greatly into the overall cellular composition of resultant culture cells. Nonetheless, quantitative assessment of cell type-specic proteins, whether neuronal, glial or epithelial, should be a guide for including cell lines for between-group comparison studies.
As noted in the preceding sections, the in vitro ON paradigm is particularly informative in studies of active cellular processes and pathways, where pharmacologic manipulations are to be tested or where larger numbers of cells from an individual are required. Importantly, as these cells are of neuronal and glial lineages they better represent the molecular dysregulations of neuropsychiatric disease than those of other cell types used in in vitro studies, such as blood cells. Indeed gene expression proles of cells derived
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Table 2. General comparison of some characteristics of olfactory mucosa (OM)-derived, induced pluripotent (iPS) and induced neural (iN) stem cells relevant to the use of these cells in the study of psychiatric, neurodevelopmental and neurodegenerative disorders
Characteristic Adult human OM-derived cells Adult human iPS cells Adult human iN cells
Ease of collection Moderatea High High Current cost to preparecells for assay
Lowmoderate High Moderate
Extent of in vitro modication
Little modication: only the inuence of in vitro conditions
Substantial modication: reprogramming for pluripotency plus the inuence of in vitro conditions
Substantial modication: reprogramming for somatic conversion plus the inuence of in vitro conditions
Availability of ex vivo neural reference tissue
Yes No No
Proliferative capacity of cells used for assays
Moderatehigh Moderate None
Capacity to be driven to neuronal differentiation
Varying between protocols Robust, high efciency Robust, moderate efciency
Cellular phenotype commonly used in assays
Proliferative putative neural progenitor cell
Differentiated neuron Differentiated neuron
Action potential generation
To be further demonstrated25 Yes Yes
Detailed discussion of iPS and iN cell paradigms is beyond the scope of this review, but these paradigms have been reviewed extensively elsewhere.1,2 See text
for details and references for OM-derived cell studies. aCollection of OM tissues is performed by a qualied otorhinolaryngologist using local anesthetic.
from OM biopsy more closely resemble brain tissue, stem cells and neurons derived from stem cells than blood cells (see Horiuchi et al.150 for a comparison of gene expression proles of brain tissue, OM cells, stem cells and iPSCs). While iPS and induced neuronal cell paradigms offer unparalleled opportunities to generate differentiated neurons in vitro from living individuals, there has been considerably less study of the capacity of OM cells to generate differentiated neurons. Nevertheless, OM cells have distinct advantages (Table 2). The timeline from OM biopsy to availability of proliferated precursor cells is ~4 weeks, whereas iPSC reprogramming protocol timelines are considerably longer, varying on the basis of source cells and methods. In addition, OM cells are readily proliferative and can be rapidly expanded over multiple passages. Thus OM studies have lower costs and require less time and labor (Table 2).
Most importantly, OM tissues are obtained as neural tissues (Table 2) and thus they do not require genomic reprogramming as with iPS or induced neuronal cells. A challenge in the iPS and induced neuronal paradigms is to determine to what degree phenotypes of these cells have been modied compared with those of brain cells. In the OM approach, however, neurobiological properties, such as epigenomic proles, may be less modied than in the cells genetically reprogrammed. Given that the ex vivo tissues are available as the source of in vitro cells, the extent of modication could at least be evaluated in OM cells.
There are a number of challenges for researchers working with in vitro OM cells. Differences in protocols between research groups must be understood and controlled for, in particular during studies with OM-derived cells since such cells may be susceptible to variation in the means of cell harvest and the culture environment. To what degree OM-derived cells assume characteristics of differentiated neurons or glia is not entirely clear at present. As discussed earlier, for these olfactory paradigms to provide consistent insight into the pathophysiology and treatment of neuropsychiatric illnesses, additional characterization of OM-derived cells in different protocols is required to determine the characteristics of their molecular phenotypes and the similarities and differences between protocols. Ultimately, the key question surrounding the use of OM tissues and OM-derived cells in the study of psychiatric illness will be in what way ndings from these studies can shed light on abnormalities in other important brain regions and neural circuits in psychiatric illness. This will involve the examination of olfactory-derived tissues and
function at multiple levels of the olfactory circuit, as well as direct parallels between OM ndings and brain pathology.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGMENTS
This work was supported by funding from K23MH079498 (KB-W), MH059852 and MH080193 (C-GH), T32MH019112-24 (SLW) and funding from the NHMRC (Australia) CJ Martin Fellowship APP1072878 (DS).
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Copyright Nature Publishing Group Mar 2015
Abstract
The olfactory mucosa (OM) is a unique source of regenerative neural tissue that is readily obtainable from living human subjects and thus affords opportunities for the study of psychiatric illnesses. OM tissues can be used, either as ex vivo OM tissue or in vitro OM-derived neural cells, to explore parameters that have been difficult to assess in the brain of living individuals with psychiatric illness. As OM tissues are distinct from brain tissues, an understanding of the neurobiology of the OM is needed to relate findings in these tissues to those of the brain as well as to design and interpret ex vivo or in vitro OM studies. To that end, we discuss the molecular, cellular and functional characteristics of cell types within the olfactory mucosa, describe the organization of the OM and highlight its role in the olfactory neurocircuitry. In addition, we discuss various approaches to in vitro culture of OM-derived cells and their characterization, focusing on the extent to which they reflect the in vivo neurobiology of the OM. Finally, we review studies of ex vivo OM tissues and in vitro OM-derived cells from individuals with psychiatric, neurodegenerative and neurodevelopmental disorders. In particular, we discuss the concordance of this work with postmortem brain studies and highlight possible future approaches, which may offer distinct strengths in comparison to in vitro paradigms based on genomic reprogramming.
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