- Research article
- Open Access
Secreted factors from olfactory mucosa cells expanded as free-floating spheres increase neurogenesis in olfactory bulb neurosphere cultures
© Barraud et al; licensee BioMed Central Ltd. 2008
- Received: 26 October 2007
- Accepted: 18 February 2008
- Published: 18 February 2008
The olfactory epithelium is a neurogenic tissue comprising a population of olfactory receptor neurons that are renewed throughout adulthood by a population of stem and progenitor cells. Because of their relative accessibility compared to intra-cranially located neural stem/progenitor cells, olfactory epithelium stem and progenitor cells make attractive candidates for autologous cell-based therapy. However, olfactory stem and progenitor cells expand very slowly when grown as free-floating spheres (olfactory-spheres) under growth factor stimulation in a neurosphere assay.
In order to address whether olfactory mucosa cells extrinsically regulate proliferation and/or differentiation of immature neural cells, we cultured neural progenitor cells derived from mouse neonatal olfactory bulb or subventricular zone (SVZ) in the presence of medium conditioned by olfactory mucosa-derived spheres (olfactory-spheres). Our data demonstrated that olfactory mucosa cells produced soluble factors that affect bulbar neural progenitor cell differentiation but not their proliferation when compared to control media. In addition, olfactory mucosa derived soluble factors increased neurogenesis, especially favouring the generation of non-GABAergic neurons. Olfactory mucosa conditioned medium also contained several factors with neurotrophic/neuroprotective properties. Olfactory-sphere conditioned medium did not affect proliferation or differentiation of SVZ-derived neural progenitors.
These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the olfactory epithelial stem/progenitor cell population.
- Glial Fibrillary Acidic Protein
- Olfactory Bulb
- Soluble Factor
- Olfactory Epithelium
- Neural Progenitor Cell
The olfactory epithelium is a neurogenic tissue containing a population of olfactory receptor neurons (ORNs) that are renewed throughout adulthood . Precursors of the ORNs reside in the olfactory epithelium as a population of transit amplifying cells called globose basal cells (GBCs) [2, 3]. Adjacent to the GBCs and in contact with the basement membrane of the olfactory epithelium, are the horizontal basal cells (HBCs). In normal conditions, HBCs are relatively quiescent, but following injury and ORN degeneration they proliferate and can give rise to GBCs and non-neuronal lineage cells, and thereby regenerate the olfactory epithelium [4, 5]. In contrast to the subventricular zone (SVZ), hippocampus and the olfactory bulb (all of which are located within the cranium), the olfactory epithelium represents an accessible source of stem/progenitor cells for autologous transplantation for central nervous system (CNS) repair that can be isolated by simple biopsy without profoundly altering the sense of smell [6, 7]. However, few methods to expand olfactory stem and progenitor cells under serum-free conditions have been described and as a result, the therapeutic values of olfactory stem and progenitor cells remains uncertain.
Attempts to expand olfactory stem and progenitor cells under growth factor stimulation in serum-free condition according to the well-established free-floating cell aggregate CNS neurosphere culture method  have been described in two studies [9, 10]. Although olfactory stem cells from the developing mouse generate primary spheres, they fail to generate secondary spheres after cell dissociation or passage . Whether this is due to intrinsic factors, inappropriate extrinsic factors or a combination of both is not known. We have reasoned that if extrinsic factors were involved then they should be present in olfactory-sphere conditioned medium and that these factors would affect the proliferative properties of a neural stem/progenitor cell known to readily generate secondary spheres. In order to address this, we cultured neonatal mouse neural progenitor cells isolated from the olfactory bulb and the SVZ as neurospheres  in a medium supplemented with either olfactory mucosa cell-conditioned medium or with fresh bFGF/EGF-containing medium. Our data demonstrate that olfactory mucosa cells produce soluble factors that affect olfactory bulb neural progenitor cell differentiation toward a neuronal cell fate without affecting their proliferation but have no effect on SVZ neurosphere cell proliferation and differentiation. We found that soluble factors from olfactory mucosa cells increased neurogenesis, but not gliogenesis (although were able to decrease expression of the astrocyte marker glial fibrillary acidic protein (GFAP)) from olfactory bulb-derived spheres. These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the stem/progenitor cell population.
Olfactory mucosa conditioned medium does not affect proliferation of olfactory bulb- or subventricular zone-derived neural progenitor cells
In order to examine the effects of OM-CM on neural progenitor cell proliferation we counted total cell numbers using the trypan blue exclusion method after the first, second and third passage and compared cell numbers from the OB-ns and the SVZ-ns expanded in the presence of OM-CM with control cultures (Fig 1E,F). We found no significant differences in the cell numbers between OM-CM and control cultures at passages 1, 2 or 3, indicating OM-CM did have an additive affect on neural progenitor cell proliferation.
To address whether soluble factors present in the OM-CM affected expression neural stem/progenitor cell markers Sox2, nestin, Notch1 and the Notch regulators Hes1 and Hes5, we looked at RNA expression of these markers in OB-ns cells expanded in OM-CM and control cultures using semi-quantitative RT-PCR [12–15]. We found minor differences in the expression level of nestin, Notch1, Hes1 and Hes5 between the two control cultures: Hes5 transcripts were only detected in control 2 but not in control 1 and expression levels of nestin, Notch1 and Hes1 was lower in control 2 compared to control 1 (Fig 1G). However, similar expression levels of all these neural stem/progenitor cell genes were detected between control 1 and OM-CM cultures indicating that olfactory mucosa soluble factors did not contain factors affecting expression levels of these markers (Fig 1G).
These data indicate that factors present in OM-CM do not influence proliferation of neural progenitor cells nor do they affect expression level of gene encoding neural stem/progenitor cell markers. OM-CM therefore does not contain inhibitors of progenitor proliferation which might account for the slow expansion of growth properties of olfactory-spheres.
Olfactory mucosa cells produce soluble factors that affect neurogenesis from olfactory bulb-derived progenitors but not from subventricular zone-derived progenitors
OM-CM decrease the number of GABAergic but not calretinin-containing neurons from the olfactory bulb
Neurotrophic/neuroprotective factors produced by olfactory-spheres
In the mature olfactory epithelium the generation of new ORNs is under genetic and autocrine/paracrine controls [30–32]. In this study we investigated whether soluble factors released by olfactory-spheres that are composed of HBCs, GBCs, olfactory ensheathing cells, and sustentacular cells  affected neural progenitor cell proliferation and/or differentiation. Using olfactory bulb as a source of neural stem/progenitor cells and a neurosphere assay, we found that factors produced by olfactory-spheres 1) increased adherence of neural stem/progenitor cells without affecting their proliferation and 2) increased neurogenesis while decreasing the proportion of GFAP-expressing cells. When the SVZ was used as source of neural stem and progenitor cells, factors secreted by olfactory mucosa cells had no effects on their proliferation or differentiation.
Transcripts encoding factors known to have mitogenic effects on neural progenitor cells like VEGF and Galectin-1 [33–36] were detected in our olfactory-spheres indicating that neural stem/progenitor mitogens are likely to be present in the OM-CM. The fact that we did not observe any significant differences in total cell numbers (between control and OM-CM cultures) could be explained by the following assumptions: 1) mitogens are present at a too low concentration to readily affect progenitor proliferation, 2) their activity is dependent of the status of the proteins (e.g. monomeric or dimeric form, oxidized) and therefore mitogens present in the OM-CM may be inactive, or 3) olfactory bulb and olfactory epithelial neural progenitor cells may require distinct factors cooperating with bFGF and/or EGF to stimulate their proliferation.
During the sphere expansion phase we found that OM-CM stimulated neurospheres to adhere to the culture flask. Several proteins have been reported to promote neural stem/progenitor cell adhesion without altering their proliferation and/or promoting their differentiation when expanding under growth factor stimulation. Such proteins include poly-ornithine, fibronectin and laminin [37–39]. Poly-ornithine and laminin have been used to expand under serum-free conditions neural stem/progenitor cells from fetal and adult mouse brains [37, 39]. Furthermore, disruption of β1-integrin expression – a receptor for both laminin and fibronectin – on neural stem/progenitor cells dramatically decreased their adherence in addition to their proliferation when stimulated by both bFGF and EGF . All these data together indicates that extracellular proteins like laminin and/or fibronectin may be produced by olfactory-spheres and such proteins may increase adherence of olfactory bulb-derived neurospheres without altering their proliferation.
In addition to increase cell adhesion during expansion, OM-CM affected neurogenesis when neurospheres were subjected to differentiation. Upon differentiation for 3 div, expression level of the neuronal marker Dlx5 was increased when cultures were exposed to OM-CM during their expansion compared to control cultures. Moreover, when neurospheres were differentiated for 7 div, total number of βIII-tubulin+ cells increased when the cells were exposed to OM-CM during their expansion compared to control cultures. It is probable that during the expansion phase and in the early phase of differentiation (3 days after differentiation was induced), a component contained in OM-CM may have affected survival and/or stimulated proliferation of a small subset of proliferating neuronal precursor cells. Supporting this conjecture is the presence of mRNA encoding Dlx5 (a transcription factor involved in the generation of bulbar calretinin-containing neurons and a subset of GABAergic interneurons; see [17–20]) that was expressed at a higher level in differentiating OM-CM cultures than differentiating control cultures. In our study we found that the proportion of βIII-tubulin+/GABA+ cells decreased when neurospheres were primed with OM-CM prior to differentiation compared to unprimed cultures (control cultures) whereas proportion of βIII-tubulin+/calretinin+ cells were similar in all three culture conditions. However, since there is a two fold increase in βIII-tubulin+ cells in OM-CM cultures compared to controls these data indicate that soluble factors present in OM-CM do not affect differentiation toward GABAergic neurons but favours the calretinin-containing neurons. Tyrosine hydroxylase (a limiting enzyme in the biosynthesis of dopamine) and calbindin were not detected in our cultures by immunocytochemistry and we cannot conclude whether OM-CM affects the dopaminergic and/or calbindin-containing subclasses of periglomerular cells.
In this study we found that olfactory bulb neurospheres expanded in the presence of OM-CM during their expansion for 7 div 1) attached to the culture flask, 2) gave rise to more neurons than control cultures, 3) contained less GABAergic neurons than control cultures and 4) contained less GFAP-expressing cells than control cultures. These data are similar to the data obtained from Tarasenko and colleagues . These authors generated neurospheres from human fetal forebrain and cultured them in a serum free-medium supplemented with heparin, bFGF and laminin prior to differentiation. Addition of laminin while neurospheres proliferated, increased their adherence to the flask, increased neurogenesis, decreased expression of GFAP and finally decreased total numbers of GABA+/βIII-tubulin+ cells . Given similarities between our results and Tarasenko et al.'s data, it is tempting to propose that laminin may be one of the candidate proteins that affect cell adhesion and neurogenesis in both olfactory bulb neurospheres and olfactory mucosa spheres. In our previous study we found that olfactory mucosa cells attached to the bottom of the flask whereas olfactory bulb neurospheres cultured in the same expansion medium did not adhere to the flask. Moreover, olfactory mucosa cells were expanded as free-floating spheres when plated on culture flask coated with a polymer in order to prevent their adhesion (; see material and methods). Recently, it has been reported that anti-GBC-3, an antibody raised to a cell surface antigen specifically expressed by GBCs, recognized a non-integrin precursor of a receptor for laminin . In the olfactory system, laminin in addition to galectin-1 are abundant along the olfactory nerve pathway . Laminin and galectin-1 are both secreted by olfactory ensheathing cells and both create a permissive environment for the olfactory receptor axons to project to specific areas of the bulb .
Based on our previous data  and considering results from other groups [40–42], we propose the following scenario: olfactory mucosa cells expanded as olfactory-spheres may secrete laminin into the medium. Secreted laminin may increase adherence of the olfactory-spheres to the bottom of the flask and even promote differentiation of the GBCs given that a large number of cells were positive for the neural cell adhesion molecule (NCAM) a marker for differentiating neurons. As a result laminin may decrease proliferation potential of olfactory stem/progenitor cells when expanded as free-floating spheres.
Although olfactory-spheres differed from neurospheres for their limited potential to self-renew (i.e. to generate new spheres after subsequent dissociation or passage), they demonstrated potential to produce soluble factors with neuroprotective effects. In this study, mRNA encoding Galectin-1, VEGF and NGF were detected in both olfactory-spheres and neurospheres indicating that olfactory mucosa cells expanded under bFGF/EGF stimulation can secrete factors known to have effects on neural cell survival, axonal growth and even axonal regeneration (for review see [43–45]). We therefore provide in this study the first evidence that olfactory stem/progenitor cells expanded as free-floating spheres can potentially promote axonal regeneration and/or neuroprotection by soluble factors they secreted.
What implications do our data gathered from mouse tissue have for human-derived cells? Recent studies performed in rodent and human olfactory mucosa indicate that basal cells exhibit distinct differences between the two species. These include basal cell morphology, marker expression and response to growth factors. In rodent HBCs and GBCs are morphologically distinct and HBCs express cytokeratin 14 whereas in human HBCs and GBCs are not morphologically distinguishable and express p75NGFR, an OEC marker [1, 2, 46, 47]. It is claimed that human olfactory mucosa-derived spheres can be maintained in long-term in culture (up to 200 passages) whereas rodent olfactory mucosa-derived spheres have limited self-renewal potential and do not generate secondary spheres following passage [9, 10, 48]. EGF has predominantly survival effects on rodent HBCs and GBCs but no significant effect on human olfactory mucosa cell viability [9, 49–52]. Thus, these differences caution against a too literal extrapolation from data obtained using rodent to the situation that will pertain in humans [53, 54]. Nevertheless, we believe the broad principles established to be of translational value.
By using media conditioned by olfactory-spheres made from the olfactory mucosa we have been able to test the ability of mucosa derived soluble factors to alter the properties of neural progenitor cells. Using neural progenitors from the olfactory bulb (a CNS structure containing progenitors of SVZ origin) we have shown that the olfactory mucosa does not produce factor that inhibit progenitor proliferation but neither does it contain factors that stimulate their proliferation compared to controls. However, we have shown that olfactory mucosa-derived factors stimulate neuronal differentiation of neural progenitors, favouring calretinin-containing rather than GABAergic phenotypes, and suppresses expression of GFAP by neural progenitor-derived astrocytes. Furthermore, the olfactory mucosa produces several factors with neuroprotective and neurotrophic properties. These data suggest that the difficulties encountered in expanding and propagating olfactory-spheres may be attributable to intrinsic properties of the olfactory epithelial stem/progenitor cells.
All the experiments were performed using neonatal CD1 mice (post-natal day 0 or P0 to post-natal day 3 or P3). All animal-related procedures were conducted in accordance with local ethical guidelines and approved animal care protocols.
Olfactory mucosa conditioned medium preparation
Newborn mice were killed by decapitation and olfactory mucosae were immediately dissected into ice cold phosphate-buffered saline (PBS). Tissues were incubated for 20 min at 37°C into 0.25% Collagenase IA (Sigma) and 0.25% DNAse I (Roche) containing Dulbecco's Modified Eagle Medium (DMEM; Gibco) prior to mechanical dissociation to a cell suspension. Remaining cell aggregates were removed after filtration through a 40 μm cell strainer (BD Falcon) and cell numbers were estimated using the Trypan Blue exclusion method. Cell density was adjusted at ≈ 105 cells/ml. Cells were transferred into a poly 2-hydroxyethyl Methacrylate (PolyHEMA; Sigma) coated 25-cm2 flask (Nunc) to avoid cell attachment in the "expansion medium" containing 1:3 mixture of DMEM/F12, 2% B27, 1% penicillin/streptomycin (P/S; all from Gibco), heparin (4 ng/ml, Sigma), supplemented with basic fibroblast growth factor (human recombinant bFGF; 20 ng/ml; R&D Systems), and epidermal growth factor (human recombinant EGF; 20 ng/ml; R&D Systems). Cultures were maintained for 14 days in vitro (div) at 37°C in a humid atmosphere with 95% oxygen and 5% CO2. Fresh medium was added to the cultures every third days. After 14 div, olfactory mucosa conditioned medium (OM-CM) was sterilized by filtration through a 0.22 μm membrane and stored at -20°C.
Bulk olfactory bulb and subventricular zone neurosphere preparation
Newborn mice were killed by decapitation and the brains were immediately isolated into ice cold PBS. Brains were transferred into a new dissection dish containing clean ice cold PBS, meninges were removed prior to olfactory bulb and subventricular zone (SVZ) dissection. Bulbs and SVZ were mechanically dissociated to a cell suspension. Cell numbers were estimated using Trypan Blue exclusion method and cell density was adjusted at ≈ 105 cells/ml. Cells were transferred onto uncoated 25-cm2 flask (Nunc) for 7 div in three different culture conditions. One culture condition consisted in 50% "expansion medium" (see composition above) – resulting in a final concentration of 10 ng/ml fresh bFGF and 10 ng/ml fresh EGF, 1% fresh B27 as for control 2 – and 50% OM-CM. Two control cultures were prepared named "control 1" in which the cells were plated in "expansion medium" (containing 20 ng/ml fresh bFGF, 20 ng/ml fresh EGF, 2% fresh B27), and a second control named "control 2" composed of 50% "expansion medium" and 50% DMEM/F12 – resulting in a final concentration of 10 ng/ml fresh bFGF, 10 ng/ml fresh EGF, and 1% fresh B27.
To induce differentiation, spheres from control cultures and OM-CM cultures were dissociated mechanically to a cell suspension prior to plating either onto laminin/collagen-coated 8 chamber slides (for immunofluorescence) or culture flask (for western blots or RT-PCR) at the density ≈ 105 cells/ml. Differentiation was induced by plating spheres expanded in OM-CM or control cultures in a medium supplemented with 1% fetal bovine serum (Sigma) instead of the growth factors and OM-CM. Cultures were maintained either for 3 div (to measure expression level of gene transcripts by RT-PCR) or for 7 div (for immunocytochemistry, RT-PCR, or western blots). After 7 div, cells were fixed for 15 min with 4% paraformaldehyde (PFA) containing PBS at room temperature (RT), rinsed three times in PBS and processed for immunocytochemistry.
For double-immunolabelling differentiated neurosphere cultures were pre-incubated in 5% normal serum and 0.025% Triton-× in PBS ("incubation solution"), 1 h at RT prior to incubation overnight at RT with primary antibodies diluted in the incubation solution. The primary antibodies used were: mouse anti-Nestin (1:1000, Chemicon), mouse IgG2b anti-βIII-tubulin (1:250, Sigma), rabbit IgG anti-glial fibrillary acidic protein (GFAP; 1:500, DAKO), rabbit polyclonal anti-γ aminobutyric acide (GABA; 1:1000, Sigma), rabbit polyclonal anti-calretinin (1:500, Swant). After three rinses in PBS, cells were incubated with FITC- or Cy3-conjugated secondary antibodies (1:200, Jackson Immunoresearch) 2 h at RT. Cultures were mounted in 4',6-diamidino-2-phenylindole (DAPI) containing VECTASHIELD (Vector Laboratories).
List of primers for neural stem progenitor cell markers
modified from 
List of primers encoding markers of bulbar interneuron proliferation and differentiation
List of primers for neurotrophic/growth factors
Olfactory bulb neurospheres (controls 1 and 2, and OM-CM cultures) differentiated for 7 div were collected by centrifugation and incubated for 15 min in lysis buffer (Sigma) containing a cocktail of protease inhibitor (Sigma). Samples were then centrifuged at maximum speed for 10 min at 4°C and the supernatant was collected. Protein concentration was measured using a spectrophotometer. Five μg proteins were diluted in loading buffer containing DTT (20 mM) and denatured at 95°C for 10 min prior to loading on a 12% acrylamide gel (Bio-Rad). Electrophoresis was performed in running buffer (0.5 M Tris Base, 1.92 M glycine, 0.5% SDS) for 1 h at 100 V. Proteins were transferred on a nylon membrane in transfer buffer (25 mM Tris Base, 150 mM glycine, 0.05% SDS, 20% methanol) at 100 V for 3 h on ice. The membrane was then rinsed in TBS-Tween buffer (10 mM Tris Base, 150 mM NaCl, 0.1% Tween20) before blocking in TBS-Tween containing 5% powdered milk at RT for 1 h. Membrane was rinsed three times in TBS-Tween and then incubated at 4°C overnight with rabbit IgG anti-GFAP (1:1000, DAKO) and mouse IgG anti-GAPDH (1:5000; Cell signaling technology) in TBS-Tween-Milk. Membrane was rinsed in TBS-Tween and then incubated for 1 h at RT with anti-mouse immunoglobulins conjugated with HRP (Dako). After additional washes, protein bands on the membrane were detected using ECL revelation kit (Amersham Bioscience).
Cell counting and statistical analysis
Photomicrographs (magnification times 20) were taken in approximately 7 random visual fields per well. For each marker, cell quantifications were performed in 3 to 4 wells per cell cultures from 3 to 4 separate cultures (generated from distinct dissections). Total numbers of DAPI-stained nuclei in addition to total numbers of cells expressing the phenotypic markers (GFAP, βIII-tubulin, GABA and Calretinin) or cells co-expressing two markers (βIII-tubulin/GABA and βIII-tubulin/Calretinin) were counted directly on photomicrographs. Differences between culture conditions were assessed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. The standard error means were considered significantly different when the value of the variance P was <0.05. Significance was accepted at the 95% confidence level.
This work was supported by a Strategic Stem Cell grant from the UK Medical Research Council (67395).
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