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Olfactory sensory neurons (OSNs) undergo constant turnover under physiological conditions but also regenerate efficiently following tissue injury. Maintenance and repair neurogenesis in the olfactory epithelium (OE) have been attributed to the selective activity of globose (GBCs) and horizontal basal cells (HBCs), respectively. In zebrafish, cells with GBC-like properties are localized to the peripheral margins of the sensory OE and contribute to OSN neurogenesis in the intact OE, while cells that resemble HBCs at the morphological and molecular level are more uniformly distributed. However, the contribution of these cells to the restoration of the injured OE has not been demonstrated. Here, we provide a detailed cellular and molecular analysis of the tissue response to injury and show that a dual progenitor cell system also exists in zebrafish. Zebrafish HBCs respond to the structural damage of the OE and generate a transient population of proliferative neurogenic progenitors that restores OSNs. In contrast, selective ablation of OSNs by axotomy triggers neurogenic GBC proliferation, suggesting that distinct signaling events activate GBC and HBC responses. Molecular analysis of differentially expressed genes in lesioned and regenerating OEs points toward an involvement of the canonical Wnt/β-catenin pathway. Activation of Wnt signaling appears to be sufficient to stimulate mitotic activity, while inhibition significantly reduces, but does not fully eliminate, HBC responses. Zebrafish HBCs are surprisingly active even under physiological conditions with a strong bias toward the zones of constitutive OSN neurogenesis, suggestive of a direct lineage relationship between progenitor cell subtypes.

The horizontal basal cells (HBCs) of olfactory epithelium (OE) serve as reservoirs for stem cells during OE regeneration, through proliferation and differentiation, which is important in recovery of olfactory function. However, the molecular mechanism of regulation of HBC proliferation and differentiation after injury remains unclear. Here, we found that yes-associated protein (YAP) was upregulated and activated in HBCs after OE injury. Deletion of YAP in HBCs led to impairment in OE regeneration and functional recovery of olfaction after injury. Mechanically, YAP was activated by S1P/S1PR2 signaling, thereby promoting the proliferation of HBCs and OE regeneration after injury. Finally, activation of YAP signaling enhanced the proliferation of HBCs and improved functional recovery of olfaction after OE injury or in Alzheimer's disease model mice. Taken together, these results reveal an S1P/S1PR2/YAP pathway in OE regeneration in response to injury, providing a promising therapeutic strategy for OE injury.

International audience; The olfactory mucosa (OM) and olfactory bulb (OB) are neuronal tissues that contribute to the early processing of olfactory information. They contain significant amounts of n-3 and n-6 polyunsaturated fatty acids (PUFAs), which are crucial for neuronal tissue development. In this study, we evaluated the impact of feeding mice diets that are either deficient in α-linolenic acid (ALA) or supplemented with n-3 long-chain PUFAs from gestation to adolescence on the phospholipid and ganglioside composition of these tissues. Both diets modified the levels of some phospholipid classes, notably the phosphatidylserine and phosphatidylethanolamine levels. In addition, the low-ALA diet enriched n-6 PUFAs in the main phospholipid classes of both tissues, while the diet supplemented with n-3 PUFAs enhanced the n-3 PUFA-containing phospholipid species level, mainly in OM. The diets also modulated the levels and profiles of several ganglioside classes in OM and OB. These modifications may have repercussions on the olfactory sensitivity.

Olfactory ensheathing cells (OECs) from the olfactory bulb (OB) and the olfactory mucosa (OM) have the capacity to repair nerve injury. However, the difference in the therapeutic effect between OB-derived OECs and OM-derived OECs remains unclear. In this study, we extracted OECs from OB and OM and compared the gene and protein expression profiles of the cells using transcriptomics and non-quantitative proteomics techniques. The results revealed that both OB-derived OECs and OM-derived OECs highly expressed genes and proteins that regulate cell growth, proliferation, apoptosis and vascular endothelial cell regeneration. The differentially expressed genes and proteins of OB-derived OECs play a key role in regulation of nerve regeneration and axon regeneration and extension, transmission of nerve impulses and response to axon injury. The differentially expressed genes and proteins of OM-derived OECs mainly participate in the positive regulation of inflammatory response, defense response, cytokine binding, cell migration and wound healing. These findings suggest that differentially expressed genes and proteins may explain why OB-derived OECs and OM-derived OECs exhibit different therapeutic roles. This study was approved by the Animal Ethics Committee of the General Hospital of Ningxia Medical University (approval No. 2017-073) on February 13, 2017.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

BACKGROUND: In the olfactory epithelium, mitotically active globose basal cells (GBCs) continuously replenish olfactory sensory neurons lost throughout life. Although an essential role of the transcription factor Sox2 in expanding olfactory progenitors/stem cells has been shown, its precise role in olfactory GBCs remain incompletely understood. METHODS: We characterized the Sox2 expression in olfactory GBCs in normal conditions and in a lesion-regeneration model using a Lgr5EGFP-IRES-creERT2 strain. During GBC-mediated regeneration, genetic deletion of sox2 and lineage tracing experiments were performed to examine the function of Sox2 in the progeny of Lgr5-EGFP(+) GBCs. RESULTS: Over 95% of Lgr5-EGFP(+) GBCs express Sox2 in normal or regeneration conditions. Loss of Sox2 dramatically reduces the cell number in each lineage traced cluster. In the progeny of Lgr5-EGFP(+) GBCs, loss of Sox2 significantly decreased the portion of OMP(+) olfactory sensory neurons. However, the generation of sustentacular cells was unchanged. CONCLUSIONS: Our observations support an essential role of Sox2 in adult olfactory regeneration, likely acting on neuronal-lineage GBCs.

Olfactory sensory neurons express a large family of odorant receptors (ORs) and a small family of trace amine-associated receptors (TAARs). While both families are subject to so-called singular expression (expression of one allele of one gene), the mechanisms underlying TAAR gene choice remain obscure. Here, we report the identification of two conserved sequence elements in the mouse TAAR cluster (T-elements) that are required for TAAR gene expression. We observed that cell-type-specific expression of a TAAR-derived transgene required either T-element. Moreover, deleting either element reduced or abolished expression of a subset of TAAR genes, while deleting both elements abolished olfactory expression of all TAARs in cis with the mutation. The T-elements exhibit several features of known OR enhancers but also contain highly conserved, unique sequence motifs. Our data demonstrate that TAAR gene expression requires two cooperative cis-acting enhancers and suggest that ORs and TAARs share similar mechanisms of singular expression., How olfactory sensory neurons express one allele of one TAAR gene is not well understood. Here the authors identify two cooperative cis-acting enhancers that govern TAAR gene choice and that share both similarities and differences with known olfactory enhancers.

The adult olfactory epithelium (OE) regenerates sensory neurons and nonsensory supporting cells from resident stem cells after injury. How supporting cells contribute to OE regeneration remains largely unknown. In this study, we elucidated a novel role of Ym2 (also known as Chil4 or Chi3l4), a chitinase-like protein expressed in supporting cells, in regulating regeneration of the injured OE in vivo in both male and female mice and cell proliferation/differentiation in OE colonies in vitro. We found that Ym2 expression was enhanced in supporting cells after OE injury. Genetic knockdown of Ym2 in supporting cells attenuated recovery of the injured OE, while Ym2 overexpression by lentiviral infection accelerated OE regeneration. Similarly, Ym2 bidirectionally regulated cell proliferation and differentiation in OE colonies. Furthermore, anti-inflammatory treatment reduced Ym2 expression and delayed OE regeneration in vivo and cell proliferation/differentiation in vitro, which were counteracted by Ym2 overexpression. Collectively, this study revealed a novel role of Ym2 in OE regeneration and cell proliferation/differentiation of OE colonies via interaction with inflammatory responses, providing new clues to the function of supporting cells in these processes. SIGNIFICANCE STATEMENT The mammalian olfactory epithelium (OE) is a unique neural tissue that regenerates sensory neurons and nonsensory supporting cells throughout life and postinjury. How supporting cells contribute to this process is not entirely understood. Here we report that OE injury causes upregulation of a chitinase-like protein, Ym2, in supporting cells, which facilitates OE regeneration. Moreover, anti-inflammatory treatment reduces Ym2 expression and delays OE regeneration, which are counteracted by Ym2 overexpression. This study reveals an important role of supporting cells in OE regeneration and provides a critical link between Ym2 and inflammation in this process.

Functional characterization of mammalian olfactory receptors (ORs) remains a major challenge to ultimately understanding the olfactory code. Here, we compare the responses of the mouse Olfr73 ectopically expressed in olfactory sensory neurons using AAV gene delivery in vivo and expressed in vitro in cell culture. The response dynamics and concentration-dependence of agonists for the ectopically expressed Olfr73 were similar to those reported for the endogenous Olfr73, however the antagonism previously reported between its cognate agonist and several antagonists was not replicated in vivo. Expressing the OR in vitro reproduced the antagonism reported for short odor pulses, but not for prolonged odor exposure. Our findings suggest that both the cellular environment and the stimulus dynamics shape the functionality of Olfr73 and argue that characterizing ORs in ‘native’ conditions, rather than in vitro, provides a more relevant understanding of ligand-OR interactions.

Individual olfactory sensory neurons (OSNs) in the mouse main olfactory epithelium express a single odorant receptor (OR) gene from the repertoire of either class I or class II ORs. The transcription factor Bcl11b determines the OR class to be expressed in OSNs. The septal organ (SO), a small neuroepithelium located at the ventral base of the nasal septum, is considered as an olfactory subsystem because it expresses a specific subset of ORs. However, the mechanisms underlying the generation and differentiation of SO-OSN remain unknown. In the present study, we show that the generation and differentiation of SO-OSN employ the same genetic pathway as in the OSN lineage, which is initiated by the neuronal fate determinant factor Ascl1. Additionally, the key role of Bcl11b in the SO is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: significant reduction in the expression of OR genes and in the number of mature SO-OSNs. Although SO-OSNs are specified to express a subset of class II OR genes in wild-type mice, the Bcl11b deletion led to the expression of class I OR genes, while the expression of class II OR genes was significantly decreased, with one exception of Olfr15. These results indicate that Bcl11b is necessary for proper OR expression in SO-OSNs.

Olfactory dysfunctions, including hyposmia and anosmia, affect ~100 million people around the world and the underlying causes are not fully understood. Degeneration of olfactory sensory neurons and incapacity of globose basal cells to generate olfactory sensory neurons are found in elder people and patients with smell disorders. Thus, olfactory stem cell may function as a promising tool to replace inactivated globose basal cells and to generate sensory neurons. Methods: We established clonal expansion of cells from the murine olfactory epithelium as well as colony growth from human olfactory mucosa using Matrigel-based three-dimensional system. These colonies were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Chemical addition was optimized to promote Lgr5 expression, colony growth and sensory neuron generation, tested by quantitative PCR and immunostaining against progenitor and neuronal markers. The differential transcriptomes in multiple signaling pathways between colonies under different base media and chemical cocktails were determined by RNA-Seq. Results: In defined culture media, we found that VPA and CHIR99021 induced the highest Lgr5 expression level, while LY411575 resulted in the most abundant yield of OMP+ mature sensory neurons in murine colonies. Different base culture media with drug cocktails led to apparent morphological alteration from filled to cystic appearance, accompanied with massive transcriptional changes in multiple signaling pathways. Generation of sensory neurons in human colonies was affected through TGF-β signaling, while Lgr5 expression and cell proliferation was regulated by VPA. Conclusion: Our findings suggest that targeting expansion of olfactory epithelium/mucosa colonies in vitro potentially results in discovery of new source to cell replacement-based therapy against smell loss.

In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of βIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.

Olfactory sensory neurons (OSNs) are bipolar neurons, unusual because they turn over continuously and have a multiciliated dendrite. The extensive changes in gene expression accompanying OSN differentiation in mice are largely known, especially the transcriptional regulators responsible for altering gene expression, revealing much about how differentiation proceeds. Basal progenitor cells of the olfactory epithelium transition into nascent OSNs marked by Cxcr4 expression and the initial extension of basal and apical neurites. Nascent OSNs become immature OSNs within 24–48 h. Immature OSN differentiation requires about a week and at least 2 stages. Early-stage immature OSNs initiate expression of genes encoding key transcriptional regulators and structural proteins necessary for further neuritogenesis. Late-stage immature OSNs begin expressing genes encoding proteins important for energy production and neuronal homeostasis that carry over into mature OSNs. The transition to maturity depends on massive expression of one allele of one odorant receptor gene, and this results in expression of the last 8% of genes expressed by mature OSNs. Many of these genes encode proteins necessary for mature function of axons and synapses or for completing the elaboration of non-motile cilia, which began extending from the newly formed dendritic knobs of immature OSNs. The cilia from adjoining OSNs form a meshwork in the olfactory mucus and are the site of olfactory transduction. Immature OSNs also have a primary cilium, but its role is unknown, unlike the critical role in proliferation and differentiation played by the primary cilium of the olfactory epithelium’s horizontal basal cell.

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 105 cells/ml and 4.9 × 104 cells/ml for plastic and plastic plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.

Cell therapies represent promising strategies to improve neurological functions after spinal cord injury (SCI). Olfactory mucosa (OM) might be an attractive source of multipotent cells for neuroregeneration because olfactory stem cells (OSCs) are resident. The regenerative capacity of OSCs has been demonstrated in animal models and some clinical case reports. Up to now, there are no standard methods for purification, characterization, and delivery of OSCs to the injury site. However, purification and characterization of the grafted cells are prerequisites for clinical use to ensure maximum safety for the patients. In this study, we isolated and purified OSCs from human OM using the neurosphere assay. Subsequently, the cells were characterized, and the behavior of purified OSCs in a plasma clot was investigated. Our study demonstrated that isolated cells from OM form neurospheres, which cells are positive for CD105 (98%) and CD90 (99%) and negative for Epcam (

Generally, the olfactory organ of vertebrates consists of the olfactory epithelium (OE) and the vomeronasal organ (VNO). The OE contains ciliated olfactory receptor neurons (ORNs), while the VNO contains microvillous ORNs. The ORNs in the OE express odorant receptors (ORs), while those in the VNO express type 1 and type 2 vomeronasal receptors (V1Rs and V2Rs). In turtles, the olfactory organ consists of the upper (UCE) and lower chamber epithelia (LCE). The UCE contains ciliated ORNs, while the LCE contains microvillous ORNs. Here we investigated the distribution of cells expressing vomeronasal receptors in the olfactory organ of turtles. The turtle vomeronasal receptors were encoded by two V1R genes and two V2R genes. Among them, V2R1 and V2R26 were mainly expressed in the LCE, while V1R3 was expressed both in the UCE and LCE. Notably, vomeronasal receptors were expressed by a limited number of ORNs, which was confirmed by the expression of the gene encoding TRPC2, an ion channel involved in the signal transduction of vomeronasal receptors. Furthermore, expression of ORs by the majority of ORNs was suggested by the expression of the gene encoding CNGA2, an ion channel involved in the signal transduction of ORs. Thus, olfaction of turtle seems to be mediated mainly by the ORs rather than the vomeronasal receptors. More importantly, the relationship between the fine structure of ORNs and the expression of olfactory receptors are not conserved among turtles and other vertebrates.

Cancer is often treated with broad-spectrum cytotoxic drugs that not only eradicate cancerous cells but also have detrimental side effects. One of these side effects, disruption of the olfactory system, impedes a patient’s ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from cancer. Recent studies reported that the chemotherapy drug, cyclophosphamide (CYP), can damage gustatory epithelia and disrupt cell proliferation in olfactory epithelia. In this study, we asked if CYP altered globose and horizontal basal cell proliferation in the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We used antibodies for Ki67, a marker strictly associated with cell proliferation, and Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Our results revealed a significant CYP-induced decrease in the number of proliferative cells in both epithelia, especially globose basal cells in the MOE, within the first 1–2 days postinjection. Recovery of cell renewal was apparent 6 days after injection. The immunohistochemical markers showed significantly higher levels of globose and horizontal basal cell proliferation in CYP-injected mice at 14 and 30 days postinjection compared with control mice. The prolonged proliferative activation of globose and horizontal basal cells suggests that, besides altering proliferation of olfactory epithelia, the epithelial substrate needed for successful cell renewal may be adversely affected by CYP.

Studies have found increased rates of dysosmia in patients with Novel Coronavirus disease 2019 (COVID-19). However, the mechanism that causes olfactory loss is unknown. The primary objective of this study was to explore local proinflammatory cytokine levels in the olfactory epithelium in patients with COVID-19. Biopsies of the olfactory epithelium were taken from patients with confirmed COVID-19 as well as uninfected controls. Levels of tumor necrosis factor α (TNF-α) and interleukin-1-beta (IL-1β) were assessed using ELISA and compared between groups. Average TNF-α levels were significantly increased in the olfactory epithelium of the COVID-19 group compared to the control group (P < 0.05). However, no differences in IL-1β were seen between groups. Elevated levels of the proinflammatory cytokine TNF-α were seen in the olfactory epithelium in patients with COVID-19. This suggests that direct inflammation of the olfactory epithelium could play a role in the acute olfactory loss described in many patients with COVID-19.

Microglial cells are the first line immune cells that initiate inflammatory responses following cerebral ischemia/reperfusion(I/R) injury. Microglial cells are also associated with a novel subtype of pro-inflammatory programmed cell death known as pyroptosis. Research has been directed at developing treatments that modulate inflammatory responses and protect against cell death caused by cerebral I/R. Key among such treatments include mesenchymal stem cell (MSC) therapy. A unique type of MSC termed olfactory mucosa mesenchymal stem cell (OM-MSC) confers neuroprotection by promoting the secretion of paracrine factors, and neuroprotection. This study investigated whether hypoxic OM-MSCs could inhibit microglial cell death upon I/R insult in vitro. A traditional oxygen-glucose deprivation/reperfusion (OGD/R) model, analogous to I/R, was established. Results showed that OGD/R induced apoptosis and pyroptosis in microglial cells while hypoxia in OM-MSCs significantly attenuated these effects. Moreover, the effects of OM-MSCs were mediated by Hypoxia-inducible factor 1-alpha (HIF-1α). Taken together, these findings reveal that hypoxia-preconditioned OM-MSC inhibits pyroptotic and apoptotic death of microglial cell in response to cerebral ischemia/reperfusion insult by activating HIF-1α in vitro.

The cellular and molecular basis of metaplasia and declining neurogenesis in the aging olfactory epithelium (OE) remains unknown. The horizontal basal cell (HBC) is a dormant tissue-specific stem cell presumed to only be forced into self-renewal and differentiation by injury. Here we analyze male and female mice and show that HBCs also are activated with increasing age as well as non-cell-autonomously by increased expression of the retinoic acid-degrading enzyme CYP26B1. Activating stimuli induce HBCs throughout OE to acquire a rounded morphology and express IP3R3, which is an inositol-1,4,5-trisphosphate receptor constitutively expressed in stem cells of the adjacent respiratory epithelium. Odor/air stimulates CYP26B1 expression in olfactory sensory neurons mainly located in the dorsomedial OE, which is spatially inverse to ventrolateral constitutive expression of the retinoic acid-synthesizing enzyme (RALDH1) in supporting cells. In ventrolateral OE, HBCs express low p63 levels and preferentially differentiate instead of self-renewing when activated. When activated by chronic CYP26B1 expression, repeated injury, or old age, ventrolateral HBCs diminish in number and generate a novel type of metaplastic respiratory cell that is RALDH−and secretes a mucin-like mucus barrier protein (FcγBP). Conversely, in the dorsomedial OE, CYP26B1 inhibits injury-induced and age-related replacement of RALDH−supporting cells with RALDH1+ciliated respiratory cells. Collectively, these results support the concept that inositol-1,4,5-trisphosphate type 3 receptor signaling in HBCs, together with altered retinoic acid metabolism within the niche, promote HBC lineage commitment toward two types of respiratory cells that will maintain epithelial barrier function once the capacity to regenerate OE cells ceases.SIGNIFICANCE STATEMENTLittle is known about signals that activate dormant stem cells to self-renew and regenerate odor-detecting neurons and other olfactory cell types after loss due to injury, infection, or toxin exposure in the nose. It is also unknown why the stem cells do not prevent age-dependent decline of odor-detecting neurons. We show that (1) stem cells are kept inactive by the vitamin A derivative retinoic acid, which is synthesized and degraded locally by olfactory cells; (2) old age as well as repeated injuries activate the stem cells and exhaust their potential to produce olfactory cells; and (3) exhausted stem cells alter the local retinoic acid metabolism and maintain the epithelial tissue barrier by generating airway cells instead of olfactory cells.

The mammalian main olfactory epithelium (MOE) is exposed to a wide spectrum of external chemicals during respiration and relies on adaptive plasticity to maintain its structural and functional integrity. We previously reported that the chemo-responsive and cholinergic transient receptor potential channel M5 (TRPM5)-expressing-microvillous cells (MCs) in the MOE are required for maintaining odor-evoked electrophysiological responses and olfactory-guided behavior during two-week exposure to an inhaled chemical mixture. Here, we investigated the underlying factors by assessing the potential modulatory effects of TRPM5-MCs on MOE morphology and cell proliferation and apoptosis, which are important for MOE maintenance. In the posterior MOE of TRPM5-GFP mice, we found that two-week chemical-exposure induced a significant increase in Ki67-expressing proliferating basal stem cells without a significant reduction in the thickness of the whole epithelium or mature olfactory sensory neuron (OSN) layer. This adaptive increase in stem cell proliferation was missing in chemical-exposed transcription factor Skn-1a knockout (Skn-1a(−/−)) mice lacking TRPM5-MCs. In addition, a greater number of isolated OSNs from chemical exposed Skn-1a(−/−) mice displayed unhealthily high levels of resting intracellular Ca(2+). Intriguingly, in the anterior MOE where we found a higher density of TRPM5-MCs, chemical-exposed TRPM5-GFP mice exhibited a time-dependent increase in apoptosis and a loss of mature OSNs without a significant increase in proliferation or neurogenesis to compensate for OSN loss. Together, our data suggest that TRPM5-MC-dependent region-specific upregulation of cell proliferation in the majority of the MOE during chemical exposure contributes to the adaptive maintenance of OSNs and olfactory function.

The novel coronavirus SARS-CoV-2, which was identified after a recent outbreak in Wuhan, China, in December 2019, has kept the whole world in tenterhooks due to its severe life-threatening nature of the infection. The virus is unlike its previous counterparts, SARS-CoV and MERS-CoV, or anything the world has encountered before both in terms of virulence and severity of the infection. If scientific reports relevant to the SARS-CoV-2 virus are noted, it can be seen that the virus owes much of its killer properties to its unique structure that has a stronger binding affinity with the human angiotensin-converting enzyme 2 (hACE2) protein, which the viruses utilize as an entry point to gain accesses to its hosts. Recent reports suggest that it is not just the lung that the virus may be targeting; the human brain may soon emerge as the new abode of the virus. Already instances of patients with COVID-19 have been reported with mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if that is so, then it gives us more reasons to be frightened of this killer virus. Keeping in mind that the situation does not worsen from here, immediate awareness and more thorough research regarding the neuroinvasive nature of the virus is the immediate need of the hour. Scientists globally also need to up their game to design more specific therapeutic strategies with the available information to counteract the pandemic. In this Viewpoint, we provide a brief outline of the currently known neurological manifestations of COVID-19 and discuss some probable ways to design therapeutic strategies to overcome the present global crisis.

The novel SARS-CoV-2 virus has very high infectivity, which allows it to spread rapidly around the world. Attempts at slowing the pandemic at this stage depend on the number and quality of diagnostic tests performed. We propose that the olfactory epithelium from the nasal cavity may be a more appropriate tissue for detection of SARS-CoV-2 virus at the earliest stages, prior to onset of symptoms or even in asymptomatic people, as compared to commonly used sputum or nasopharyngeal swabs. Here we emphasize that the nasal cavity olfactory epithelium is the likely site of enhanced binding of SARS-CoV-2. Multiple non-neuronal cell types present in the olfactory epithelium express two host receptors, ACE2 and TMPRSS2 proteases, that facilitate SARS-CoV-2 binding, replication, and accumulation. This may be the underlying mechanism for the recently reported cases of smell dysfunction in patients with COVID-19. Moreover, the possibility of subsequent brain infection should be considered which begins in olfactory neurons. In addition, we discuss the possibility that olfactory receptor neurons may initiate rapid immune responses at early stages of the disease. We emphasize the need to undertake research focused on additional aspects of SARS-CoV-2 actions in the nervous system, especially in the olfactory pathway.

Summary The neural stem cells (NSCs) residing in the olfactory epithelium (OE) regenerate damaged olfactory sensory neurons throughout adulthood. The accessibility and availability of these NSCs in living individuals, including humans, makes them a promising candidate for harvesting their potential for cell replacement therapies. However, this requires an in-depth understanding of their developmental potential after grafting. Here, we investigated the developmental potential and plasticity of mouse OE-derived NSCs after grafting into the adult subventricular zone (SVZ) neurogenic niche. Our results showed that OE-derived NSCs integrate and proliferate just like endogenous SVZ stem cells, migrate with similar dynamics as endogenous neuroblasts toward the olfactory bulb, and mature and acquire similar electrophysiological properties as endogenous adult-born bulbar interneurons. These results reveal the developmental potential and plasticity of OE-derived NSCs in vivo and show that they can respond to heterotopic neurogenic cues to adapt their phenotype and become functional neurons in ectopic brain regions., Graphical Abstract, Highlights • OE-derived NSCs integrate in the SVZ after heterotopic transplantation • OE-derived NSCs respond to SVZ niche factors and change their developmental program • The development of OE-derived and SVZ NSCs are indistinguishable • OE-derived NSCs grafted into the SVZ become functional bulbar interneurons, In this article, Saghatelyan and colleagues show that olfactory epithelium-derived neural stem cells adapt their developmental program in response to heterotopic neurogenic niche factors after grafting in vivo. They migrate and differentiate into ectopic brain region neurons that are indistinguishable in their morpho-functional properties from endogenous adult-born neurons.

Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells. Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation. Materials and methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors. Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample. Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells. Resumen Introducción. El recambio celular del neuroepitelio olfatorio ocurre durante la vida del individuo gracias a precursores olfatorios. Además, las células mesenquimales del estroma también contribuyen a la homeostasis de la mucosa. Cuando un explante de una biopsia de mucosa se cultiva en un medio esencial mínimo, se genera una población predominante de células adherentes que expresan proteínas típicas de las células mesenquimales del estroma. La coexpresión de marcadores fenotípicos de precursores olfatorios y de células del recubrimiento del nervio olfatorio constituiría una propiedad única de las células mesenquimales del estroma. Objetivo. Determinar si la diferenciación celular de las células mesenquimales hacia fenotipos neurales modula la expresión de los marcadores mesenquimales característicos. Materiales y métodos. Se compararon las células aisladas de la mucosa olfatoria en un medio de cultivo con suplemento de 10 % de suero fetal bovino con esferas generadas en un medio sin suero más factores de crecimiento. Resultados. Se determinó la expresión de proteínas de las células mesenquimales del estroma (CD29+, CD73+, CD90+, CD45-), de las basales horizontales (ICAM-1/CD54+, p63+, p75NGFr+), y de las del recubrimiento del nervio olfatorio (nestin+, GFAP+) en la misma población cultivada. La determinación de Oct 3/4, Sox-2 y Mash-1, así como de las neurotrofinas BDNF, NT3 y NT4, sugirió que las células del estroma son funcionales. La expresión de las proteínas de las células mesenquimales y los precursores olfatorios, disminuyó en las células de las mesenesferas inducidas por ausencia de suero en el medio de cultivo. Conclusión. Las células mesenquimales del estroma de la mucosa olfatoria presentan una tendencia dominante hacia la diferenciación neural.

Rabbits are sometimes used for intranasal toxicology studies. We investigated the postnatal development of the nasal passage in juvenile Japanese white rabbits from just after birth to 6-week-old to provide information for conducting intranasal toxicological evaluation using juvenile animals. On postnatal day (PND) 1, the nasal passage consisted of the septum with mostly cartilaginous nasal wall and turbinates. The lining squamous, transitional, respiratory, and olfactory epithelia were already distributed similar to adults and were still underdeveloped. The nasal passage gradually expanded with age, as did the nasal wall, including the turbinates formed by endochondral ossification. The maxilloturbinate elongated, during which it branched complexly. The respiratory epithelium takes the form of columnar epithelium together with a reduction in goblet cells. In addition, the olfactory epithelium had clear cytoplasm in the ethmoturbinate, the olfactory nerve bundles thickened, and Bowman’s gland acini increased in size and number. Other tissues, including the vomeronasal organ, nasal-associated lymphoid tissue, and nasolacrimal duct, also developed histologically with age. This investigation characterized the postnatal histological development of the nasal passage in Japanese white rabbits, providing basic knowledge regarding the histological examination and rationale for appropriate study design of intranasal toxicology studies in juvenile rabbits.

Olfactory epithelial cells are in direct contact with myriad environmental contaminants which may consequently disrupt their structure and function. Copper ions (Cu2+) and copper nanoparticles (CuNPs) are two types of olfactory neurotoxicants. However, their effects on the structure of olfactory epithelium are largely uninvestigated. The density of olfactory goblet cells in CuNP- and Cu2+ - exposed rainbow trout was assessed using light microscopy throughout time. In both copper (Cu) treatments, the number of goblet cells increased initially over the 24 h exposure and then recovered to normal throughout the 96 h exposure. These data suggested the 96 h exposure to Cu contaminants interfered with protective barrier provided by goblet cells. Nonetheless, lamellar and epithelial thickness of olfactory rosette did not change in the Cu-exposed fish. The gene transcript profile of olfactory mucosa studied by RNA-seq indicated Cu2+ and CuNPs differentially targeted the molecular composition of cell junctions. In the Cu2+ treatment, reduced mRNA abundances of tight junctions, adherens junction, desmosomes and hemidesmosomes, suggest that Cu2+-exposed olfactory mucosal cells had weak junctional complexes. In the CuNP treatment, on the other hand, the transcript abundances of cell junction compositions, except adherens junction, were upregulated. Transcripts associated with gap junctional channels were increased in both Cu treatments. The elevated transcript levels of gap junctions in both Cu treatments suggested that the demand for intercellular communication was increased in the Cu-exposed olfactory mucosa. Overall, our findings suggested that Cu2+ induced greater adverse effects on the molecular composition of olfactory cell junctions relative to CuNPs. Impairment of junctional complexes may disrupt the structural integrity of olfactory mucosa.

Amyloid precursor protein (APP) is expressed in many tissues in human, mice and in zebrafish. In zebrafish, there are two orthologues, Appa and Appb. Interestingly, some cellular processes associated with APP overlap with cilia-mediated functions. Whereas the localization of APP to primary cilia of in vitro-cultured cells has been reported, we addressed the presence of APP in motile and in non-motile sensory cilia and its potential implication for ciliogenesis using zebrafish, mouse, and human samples. We report that Appa and Appb are expressed by ciliated cells and become localized at the membrane of cilia in the olfactory epithelium, otic vesicle and in the brain ventricles of zebrafish embryos. App in ependymal cilia persisted in adult zebrafish and was also detected in mouse and human brain. Finally, we found morphologically abnormal ependymal cilia and smaller brain ventricles in appa−/−appb−/− mutant zebrafish. Our findings demonstrate an evolutionary conserved localisation of APP to cilia and suggest a role of App in ciliogenesis and cilia-related functions.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3′-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer. Graphical Abstract

Transplantation of olfactory ensheathing cells, the glia of the primary olfactory nervous system, has been trialed for spinal cord injury repair with promising but variable outcomes in animals and humans. Olfactory ensheathing cells can be harvested either from the lamina propria beneath the neuroepithelium in the nasal cavity, or from the olfactory bulb in the brain. As these areas contain several other cell types, isolating and purifying olfactory ensheathing cells is a critical part of the process. It is largely unknown how contaminating cells such as fibroblasts, other glial cell types and supporting cells affect olfactory ensheathing cell function post-transplantation; these cells may also cause unwanted side-effects. It is also, however, possible that the presence of some of the contaminant cells can improve outcomes. Here, we reviewed the last decade of olfactory ensheathing cell transplantation studies in rodents, with a focus on olfactory ensheathing cell purity. We analyzed how purification methods and resultant cell purity differed between olfactory mucosa- and olfactory bulb-derived cell preparations. We analyzed how the studies reported on olfactory ensheathing cell purity and which criteria were used to define cells as olfactory ensheathing cells. Finally, we analyzed the correlation between cell purity and transplantation outcomes. We found that olfactory bulb-derived olfactory ensheathing cell preparations are typically purer than mucosa-derived preparations. We concluded that there is an association between high olfactory ensheathing cell purity and favourable outcomes, but the lack of olfactory ensheathing cell-specific markers severely hampers the field.

Adult neurogenesis, analogous to early development, is comprised of several, often concomitant, processes including proliferation, differentiation, and formation of synaptic connections. However, due to continual, asynchronous turn-over, newly-born adult olfactory sensory neurons (OSNs) must integrate into existing circuitry. Additionally, OSNs express high levels of cellular prion protein (PrPC), particularly in the axon, which implies a role in this cell type. The cellular prion has been shown to be important for proper adult OSN neurogenesis primarily by stabilizing mature olfactory neurons within this circuitry. However, the role of PrPC on each specific adult neurogenic processes remains to be investigated in detail. To tease out the subtle effects of prion protein expression level, a large population of regenerating neurons must be investigated. The thyroid drug methimazole (MTZ) causes nearly complete OSN loss in rodents and is used as a model of acute olfactory injury, providing a mechanism to induce synchronized OSN regeneration. This study investigated the effect of PrPC on adult neurogenesis after acute nasotoxic injury. Altered PrPC levels affected olfactory sensory epithelial (OSE) regeneration, cell proliferation, and differentiation. Attempts to investigate the role of PrPC level on axon regeneration did not support previous studies, and glomerular targeting did not recover to vehicle-treated levels, even by 20 weeks. Together, these studies demonstrate that the cellular prion protein is critical for regeneration of neurons, whereby increased PrPC levels promote early neurogenesis, and that lack of PrPC delays the regeneration of this tissue after acute injury.

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for the regeneration of damaged neurons. While they maintain tissue homeostasis in the olfactory mucosa (OM) and olfactory bulb (OB), their regenerative properties also support the normal sense of smell by enabling continual turnover and axonal regrowth of olfactory sensory neurons (OSNs). However, the molecular physiology of OECs is not fully understood, especially that of OECs from the mucosa. Here, we carried out whole-cell patch-clamp recordings from individual OECs cultured from the OM and OB of the adult rat, and from the human OM. A subset of OECs from the rat OM cultured 1-3 days in vitro had large weakly rectifying K+ currents, which were sensitive to Ba2+ and desipramine, blockers of Kir4-family channels. Kir4.1 immunofluorescence was detectable in cultured OM cells colabeled for the OEC marker S100, and in S100-labeled cells found adjacent to OSN axons in mucosal sections. OECs cultured from rat OB had distinct properties though, displaying strongly rectifying inward currents at hyperpolarized membrane potentials and strongly rectifying outward currents at depolarized potentials. Kir4.1 immunofluorescence was not evident in OECs adjacent to axons of OSNs in the OB. A subset of human OECs cultured from the OM of adults had membrane properties comparable to those of the rat OM that is dominated by Ba2+ -sensitive weak inwardly rectifying currents. The membrane properties of peripheral OECs are different to those of central OECs, suggesting they may play distinct roles during olfaction.

The bHLH transcription factor Olig2 is required for sequential cell fate determination of both motor neurons and oligodendrocytes and for progenitor proliferation in the central nervous system. However, the role of Olig2 in peripheral sensory neurogenesis remains unknown. We report that Olig2 is transiently expressed in the newly differentiated olfactory sensory neurons (OSNs) and is downregulated in the mature OSNs in mice from early gestation to adulthood. Genetic fate mapping demonstrates that Olig2-expressing cells solely give rise to OSNs in the peripheral olfactory system. Olig2 depletion does not affect the proliferation of peripheral olfactory progenitors and the fate determination of OSNs, sustentacular cells, and the olfactory ensheathing cells (OECs). However, the terminal differentiation and maturation of OSNs are compromised in either Olig2 single or Olig1/Olig2 double knockout mice, associated with significantly diminished expression of multiple OSN maturation and odorant signaling genes, including Omp, Gnal, Adcy3, and Olfr15. We further demonstrate that Olig2 binds to the E-box in the Omp promoter region to regulate its expression. Taken together, our results reveal a distinctly novel function of Olig2 in the periphery nervous system to regulate the terminal differentiation and maturation of olfactory sensory neurons.

Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.

Olfaction is normally taken for granted in our lives, not only assisting us to escape from dangers, but also increasing our quality of life. Although olfactory neuroepithelium (ON) can reconstitute its olfactory receptor neurons (ORNs) after injury, no adequate treatment for olfactory loss has yet emerged. The present study investigates the role of glycosaminoglycans (GAGs) in modulating olfactory neuronal homeostasis and elucidates the regulatory mechanism. This work isolates and cultures human olfactory neuroepithelial cells (HONCs) with various GAGs for 7 days, and find that chitosan promotes ORN maturation, expressing olfactory marker protein (OMP) and its functional components. Growth factor protein array, ELISA and western blot analysis reveal that insulin-like growth factor binding protein 2 (IGFBP2) shows a higher level in chitosan-treated HONCs than in controls. Biological activity of insulin-like growth factor-1 (IGF-1), IGF-2 and IGF-1 receptor (IGF1R) is further investigated. Experimental results indicate that IGF-1 and IGF-2 enhance the growth of immature ORNs, expressing βIII tubulin, but decrease mature ORNs. Instead, down-regulation of phosphorylated IGF1R lifts the OMP expression, and lowers the βIII tubulin expression, by incubation with the phosphorylated inhibitor of IGF1R, OSI-906. Finally, the effect of chitosan on ORN maturity is antagonized by concurrently adding IGFBP2 protease, matrix metallopeptidase-1. Overall, our data demonstrate that chitosan promotes ORN differentiation by raising the level of IGFBP2 to sequestrate the IGFs-IGF1R signaling. Statement of Significance Olfactory dysfunction serves as a crucial alarm in neurodegenerative diseases, and one of its causes is lacking of sufficient mature olfactory receptor neurons to detect odorants in the air. However, the clinical treatment for olfactory dysfunction is still controversial. Chitosan is the natural linear polysaccharide and exists in rat olfactory neuroepithelium. Previously, chitosan has been demonstrated to mediate the differentiation of olfactory receptor neurons in an in vitro rat model, but the mechanism is unknown. The study aims to evaluate the role and mechanism of chitosan in an in vitro human olfactory neurons model. Overall, these results reveal that chitosan is a potential agent for treating olfactory disorder by the maintenance of olfactory neural homeostasis. This is the first report to demonstrate that chitosan promotes differentiation of olfactory receptor neurons through increasing IGFBP2 to sequestrate the IGFs-IGF1R.

The anatomical structure of the olfactory organ and the organization of various cells lining the olfactory mucosa of Clupisoma garua (Siluriformes; Schilbeidae) were investigated with light and scanning electron microscopy. The olfactory organ was composed of numerous lamellae of various sizes, radiating outward from both sides of the narrow midline raphe, forming an elongated rosette. Each lamella consisted of the olfactory epithelium and a central lamellar space, the central core. The epithelium covering the surface of the rosette folds was differentiated into zones of sensory and indifferent epithelia. The sensory part of epithelium was characterized by three types of morphologically distinct receptor neurons: ciliated receptor cells, microvillous receptor cells, and rod receptor cells for receiving olfactory sensation from the aquatic environment. The indifferent epithelium comprising a large surface area of the lamella, was covered with compact non-sensory cilia. The non-sensory epithelium contained stratified epithelial cells with microridges, mucin secreting mucous cells, labyrinth cells, and basal cells, which were arranged in a layer at the base of the epithelium. Various cells on the olfactory epithelium were correlated with the functional significance of the fish concerned.

Cell transplantation has become a possible therapeutic approach in the treatment of neurodegenerative diseases of the nervous system by replacing lost cells. The current study aimed to make a comparison between the differentiation capacity of the olfactory bulb neural stem cells (OB-NSCs) and olfactory ectomesenchymal stem cells (OE-MSCs) into dopaminergic-like neurons under the inductive effect of transforming growth factor β (TGF-β). After culturing and treating with TGF-β, the differentiation capacities of both types of stem cells into dopaminergic neuron-like cells were evaluated. Quantitative real-time polymerase chain reaction analysis 3 weeks after induction demonstrated that the mRNA expression of the dopaminergic activity markers tyrosine hydroxylase (TH), dopamine transporter (DAT), paired box gene 2 (PAX2), and PAX5 in the neuron-like cells derived from OB-NSCs was significantly higher than those derived from OE-MSCs. These findings were further supported by the immunocytochemistry staining showing that the expression of the tyrosine hydroxylase, DAT, PAX2, and paired like homeodomain 3 seemed to be slightly higher in OB-NSCs compared with OE-MSCs. Despite the lower differentiation capacity of OE-MSCs, other considerations such as a noninvasive and easier harvesting process, faster proliferation attributes, longer life span, autologous transplantability, and also the easier and inexpensive cultural process of the OE-MSCs, cumulatively make these cells the more appropriate alternative in the case of autologous transplantation during the treatment process of neurodegenerative disorders like Parkinson's disease.

Background Damage to olfactory sensory neurons (OSNs), situated within the neuroepithelium of the olfactory cleft, may be associated with anosmia. Although their direct contact with the nasal airspace make OSNs vulnerable to injury and death, multiple mechanisms maintain epithelium integrity and olfactory function. We hypothesized that BMI1, a polycomb protein found to be enriched in OSNs, may function in neuroprotection. Here, we explored BMI1 function in a mouse model. Methods Utilizing a mouse genetic approach to delete Bmi1 selectively in mature OSNs, we investigated changes in OE homeostasis by performing immunohistochemical, biochemical, and functional assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunostaining, and electro-olfactograms were used to compare gene expression, cell composition, and olfactory function in OSN-specific BMI1 knockout mice (n = 3 to 5) and controls. Chromatin studies were also performed to identify protein-DNA interactions between BMI1 and its target genes (n = 3). Results OSN-specific BMI1 knockout led to increased neuron death and basal cell activation. Chromatin studies suggested a mechanism of increased neurodegeneration due to de-repression of a pro-apoptosis gene, p19ARF. Despite the increased turnover, we found that olfactory neuroepithelium thickness and olfactory function remained intact. Our studies also revealed the presence of additional polycomb group proteins that may compensate for the loss of BMI1 in mature OSNs. Conclusion The olfactory neuroepithelium employs multiple mechanisms to maintain epithelial homeostasis. Our findings provide evidence that in a mouse model of BMI1 deletion, the overall integrity and function of the olfactory neuroepithelium are not compromised, despite increased neuronal turnover, reflecting a remarkable reparative capacity to sustain a critical sensory system.

Olfactory ectomesenchymal stem cells (OE-MSCs) possess the immunosuppressive activity and regeneration capacity and hold a lot of promises for neurodegenerative disorders treatment. This study aimed to determine OE-MSCs which are able to augment and differentiate into functional neurons and regenerate the CNS and also examine whether the implantation of OE-MSCs in the pars compacta of the substantia nigra (SNpc) can improve Parkinson's symptoms in a rat model-induced with 6-hydroxydopamine. We isolated OE-MSCs from lamina propria in olfactory mucosa and characterized them using flow cytometry and immunocytochemistry. The therapeutic potential of OE-MSCs was evaluated by the transplantation of isolated cells using a rat model of acute SN injury as a Parkinson's disease. Significant behavioral improvement in Parkinsonian rats was elicited by the OE-MSCs. The results demonstrate that the expression of PAX2, PAX5, PITX3, dopamine transporter, and tyrosine hydroxylase was increased by OE-MSCs compared to the control group which is analyzed with real-time polymerase chain reaction technique and immunohistochemical staining. In the outcome, the transplantation of 1,1'-dioctadecyl-3,3,3'3'-tetramethyl indocarbocyanine perchlorate labeled OE-MSCs that were fully differentiated to dopaminergic neurons contribute to a substantial improvement in patients with Parkinson's. Together, our results provide that using OE-MSCs in neurodegenerative disorders might lead to better neural regeneration.

The effects of caloric restriction (CR) on cell dynamics and gene expression in the mouse olfactory neuroepithelium are evaluated. Eight-week-old male C57BL/6 mice were fed either control pellets (104 kcal/week) or CR pellets (67 kcal/week). The cytoarchitecture of the olfactory neuroepithelium in the uninjured condition and its regeneration after injury by an olfactotoxic chemical, methimazole, were compared between mice fed with the control and CR diets. In the uninjured condition, there were significantly fewer olfactory marker protein (OMP)-positive olfactory receptor neurons and Ki67-positive proliferating basal cells at 3 months in the CR group than in the control group. The number of Ki67-positive basal cells increased after methimazole-induced mucosal injury in both the control and the CR groups, but the increase was less robust in the CR group. The recovery of the neuroepithelium at 2 months after methimazole administration was less complete in the CR group than in the control group. These histological changes were region-specific. The decrease in the OMP-positive neurons was prominent in the anterior region of the olfactory mucosa. Gene expression analysis using a DNA microarray and quantitative real-time polymerase chain reaction demonstrated that the expression levels of two inflammatory cytokines, interleukin-6 and chemokine ligand 1, were elevated in the olfactory mucosa of the CR group compared with the control group. These findings suggest that CR may be disadvantageous to the maintenance of the olfactory neuroepithelium, especially when it is injured.

Summary The olfactory epithelium (OE) regenerates after injury via two types of tissue stem cells: active globose cells (GBCs) and dormant horizontal basal cells (HBCs). HBCs are roused to activated status by OE injury when P63 levels fall. However, an in-depth understanding of activation requires a system for culturing them that maintains both their self-renewal and multipotency while preventing spontaneous differentiation. Here, we demonstrate that mouse, rat, and human HBCs can be cultured and passaged as P63+ multipotent cells. HBCs in vitro closely resemble HBCs in vivo based on immunocytochemical and transcriptomic comparisons. Genetic lineage analysis demonstrates that HBCs in culture arise from both tissue-derived HBCs and multipotent GBCs. Treatment with retinoic acid induces neuronal and non-neuronal differentiation and primes cultured HBCs for transplantation into the lesioned OE. Engrafted HBCs generate all OE cell types, including olfactory sensory neurons, confirming that HBC multipotency and neurocompetency are maintained in culture., Highlights • Horizontal basal cells (HBCs) expand in cultures from mouse and human OE • Globose basal cells, which are active stem cells of the OE, form HBCs in vitro • Retinoic acid (RA) activates HBCs in vitro to form neurons and non-neuronal cells • After RA, cultured HBCs transplant into OE in vivo and generate all OE cell types, In this article, Peterson and colleagues show that the reserve stem cells of the mammalian olfactory epithelium, the horizontal basal cells (HBCs), are maintained and expanded in vitro and closely resemble HBCs in vivo. Many HBCs are generated from transdifferentiation of globose basal cells during culturing. Retinoic acid induces differentiation and permits their engraftment back into the olfactory epithelium.

Benzo[a]pyrene (BaP) is a widespread pollutant that can act as an endocrine disrupting compound (EDC) and interferes with reproductive function. The central regulatory network of the reproductive system is mediated by gonadotropin-releasing hormone (GnRH) neurons, which originate in the olfactory placode and, during ontogenesis, migrate into the hypothalamus. Given the importance of the migratory process for GnRH neuron maturation, we investigated the effect of BaP (10 µM for 24 h) on GnRH neuroblasts isolated from the human fetal olfactory epithelium (FNCB4). BaP exposure significantly reduced the mRNA level of genes implicated in FNCB4 cell migration and affected their migratory ability. Our findings demonstrate that BaP may interfere with the central neuronal network controlling human reproduction affecting GnRH neuron maturation.

Human olfactory neurosphere-derived (ONS) cells are derived from the olfactory mucosa and display some progenitor- and neuronal cell-like properties, making them useful models of neurological disorders. However, they lack several important characteristics of true neurons, which can be overcome using induced pluripotent stem cell (iPSC) -derived neurons. Here we describe, for the first time, the generation and validation of an iPSC line from an olfactory biopsy from a control cohort member. This data lays the groundwork for future reprogramming of ONS cells, which can be used to generate neuronal models and compliment current ONS cell-based investigations into numerous neurological disorders.