From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.

Protocol for the derivation, culturing, and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro.

Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection. For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012).

Scribble mutation disrupts convergent extension and apical constriction during mammalian neural tube closure.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia.

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

Mechanical Brain Injury Increases Cells' Production of Cystathionine ß-Synthase and Glutamine Synthetase, but Reduces Pax2 Expression in the Telencephalon of Juvenile Chum Salmon, Oncorhynchus keta.

The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine ß-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.

Frontline Science: Conversion of neutrophils into atypical Ly6G+ SiglecF+ immune cells with neurosupportive potential in olfactory neuroepithelium.

Neutrophils are generally considered as short-lived, homogenous, and terminally differentiated phagocytes that play crucial roles in conquering infection, although they occasionally cause severe collateral tissue damage or chronic inflammation. Recent reports have indicated that neutrophils also play a protective role in inflammation resolution and tissue repair. However, how terminally differentiated neutrophils have diverse functions remains unclear. Here, we show that neutrophils undergo conversion into Ly6G+ SiglecF+ double-positive cells expressing neurosupportive genes in the olfactory neuroepithelium (OE) under an inflammatory state. Through comprehensive flow cytometric analysis of murine nose, we identified Ly6G+ SiglecF+ double-positive cells that reside only in the OE under steady-state conditions. Double-positive cells were neutrophil-derived cells and increased by more than 10-fold during inflammation or tissue injury. We found that neutrophils infiltrate into the nose to express proinflammatory genes in the acute phase of inflammatory state, and they gradually change their surface markers and gene expression, expressing some neurogenesis-related genes in addition to inflammation related genes in the later phase. As the OE is known to have exceptionally high regeneration capacity as a nervous system, these findings suggest that neutrophils have the potential to contribute neurogenesis after conversion in peripheral nervous tissues, providing a challenge on a classic view of neutrophils as terminally differentiated leukocytes.

Calcium Fluorescence Recordings from Neuroepithelial Stem Cells.

Neuroepithelial cells act as neural stem cells by renewing themselves during embryonic development. These cells are tightly interconnected and make contact with the basement membrane of the neuroepithelium. Under such circumstances, Ca2+ fluorescence recording is a successful method to study physiological properties of the neuroepithelial stem cell. This chapter describes detailed techniques of Ca2+ fluorescence recording from neuroepithelial stem cells.

The role of TASK-2 channels in CO2 sensing in zebrafish (Danio rerio).

Peripheral chemosensitivity in fishes is thought to be mediated by serotonin-enriched neuroepithelial cells (NECs) that are localized to the gills of adults and the integument of larvae. In adult zebrafish (Danio rerio), branchial NECs are presumed to mediate the cardiorespiratory reflexes associated with hypoxia or hypercapnia, whereas in larvae, there is indirect evidence linking cutaneous NECs to hypoxic hyperventilation and hypercapnic tachycardia. No study yet has examined the ventilatory response of larval zebrafish to hypercapnia, and regardless of developmental stage, the signaling pathways involved in CO2 sensing remain unclear. In the mouse, a background potassium channel (TASK-2) contributes to the sensitivity of chemoreceptor cells to CO2. Zebrafish possess two TASK-2 channel paralogs, TASK-2 and TASK-2b, encoded by kcnk5a and kcnk5b, respectively. The present study aimed to determine whether TASK-2 channels are expressed in NECs of larval zebrafish and whether they are involved in CO2 sensing. Using immunohistochemical approaches, TASK-2 protein was observed on the surface of NECs in larvae. Exposure of larvae to hypercapnia caused cardiac and breathing frequencies to increase, and these responses were blunted in fish experiencing TASK-2 and/or TASK-2b knockdown. The results of these experiments suggest that TASK-2 channels are involved in CO2 sensing by NECs and contribute to the initiation of reflex cardiorespiratory responses during exposure of larvae to hypercapnia.

Calbindin expression in adult vestibular epithelia.

The mammalian vestibular epithelia exhibit a remarkably stereotyped organization featuring cellular characteristics under planar cell polarity (PCP) control. PCP mechanisms are responsible for the organization of hair cell morphologic polarization vectors, and are thought to be responsible for the postsynaptic expression of the calcium-binding protein calretinin that defines the utricular striola and cristae central zone. However, recent analyses revealed that subtle differences in the topographic expression of oncomodulin, another calcium-binding protein, reflects heterogeneous factors driving the subtle variations in expression. Calbindin represents a third calcium-binding protein that has been previously described to be expressed in both hair cells and afferent calyces in proximity to the utricular striola and crista central zone. The objective of the present investigation was to determine calbindin's topographic pattern of expression to further elucidate the extent to which PCP mechanisms might exert control over the organization of vestibular neuroepithelia. The findings revealed that calbindin exhibited an expression pattern strikingly similar to oncomodulin. However, within calyces of the central zone calbindin was colocalized with calretinin. These results indicate that organizational features of vestibular epithelia are governed by a suite of factors that include PCP mechanisms as well others yet to be defined.

Reciprocal integrin/integrin antagonism through kindlin-2 and Rho GTPases regulates cell cohesion and collective migration.

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.

Stem-like cells of various origins showed therapeutic effect to improve the recovery of spinal cord injury.

We aimed to evaluate the therapeutic effects of exosomes, which were collected from human neuroepithelial stem cells (HNESCs) treated by miR-29b mimics, on the treatment of spinal cord injury (SCI). Computational analysis, real-time PCR, Western blot analysis and TUNEL assay, a BBB score system, the Nissl staining and IHC assay were conducted to explore the molecular signalling pathway underlying the function of exosomes in SCI. Exosomes isolated from cells treated with HNESC exhibited the strongest inhibitory effect on cell apoptosis while exhibiting the highest level of miR-29b expression and the lowest levels of PTEN and caspase-3 expression. Moreover, PTEN and caspase-3 were identified as the direct target genes of miR-29b. The exosomes isolated from the groups of HNESC and HNESC + miR-29b mimics exhibited in vivo therapeutic effects by restoring the BBB score and apoptosis index of post-SCI neuron cells to those of normal neuron cells, with the exosomes collected from the group of HNESC + miR-29b mimics showing the strongest effect. We suggested that the exosomes derived from the group of HNESC + miR-29b mimics exerted therapeutic effects on SCI by down-regulating the expression of PTEN/caspase-3 and subsequently suppressing the apoptosis of neuron cells.

Why is folate effective in preventing neural tube closure defects?

Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Interkinetic nuclear movements promote apical expansion in pseudostratified epithelia at the expense of apicobasal elongation.

Pseudostratified epithelia (PSE) are a common type of columnar epithelia found in a wealth of embryonic and adult tissues such as ectodermal placodes, the trachea, the ureter, the gut and the neuroepithelium. PSE are characterized by the choreographed displacement of cells' nuclei along the apicobasal axis according to phases of their cell cycle. Such movements, called interkinetic movements (INM), have been proposed to influence tissue expansion and shape and suggested as culprit in several congenital diseases such as CAKUT (Congenital anomalies of kidney and urinary tract) and esophageal atresia. INM rely on cytoskeleton dynamics just as adhesion, contractility and mitosis do. Therefore, long term impairment of INM without affecting proliferation and adhesion is currently technically unachievable. Here we bypassed this hurdle by generating a 2D agent-based model of a proliferating PSE and compared its output to the growth of the chick neuroepithelium to assess the interplay between INM and these other important cell processes during growth of a PSE. We found that INM directly generates apical expansion and apical nuclear crowding. In addition, our data strongly suggest that apicobasal elongation of cells is not an emerging property of a proliferative PSE but rather requires a specific elongation program. We then discuss how such program might functionally link INM, tissue growth and differentiation.

A concerted metabolic shift in early forebrain alters the CSF proteome and depends on MYC downregulation for mitochondrial maturation.

Massive, coordinated cellular changes accompany the transition of central nervous system (CNS) progenitors from forebrain neurectodermal cells to specified neuroepithelial cells. We have previously found that MYC regulates the changing ribosomal and proteostatic landscapes in mouse forebrain precursors at embryonic days E8.5 and E10.5 (before and after neural tube closure; NTC) (Chau et al., 2018). Here, we demonstrate parallel coordinated transcriptional changes in metabolic machinery during this same stage of forebrain specification. Progenitors showed striking mitochondrial structural changes transitioning from glycolytic cristae at E8.5, to more traditional mitochondria at E10.5. Accordingly, glucose use shifted in progenitors such that E8.5 progenitors relied on glycolysis, and after NTC increasingly used oxidative phosphorylation. This metabolic shift was matched by changes in surrounding amniotic and cerebrospinal fluid proteomes. Importantly, these mitochondrial morphological shifts depend on MYC downregulation. Together, our findings demonstrate that metabolic shifting accompanies dynamic organelle and proteostatic remodeling of progenitor cells during the earliest stages of forebrain development.

The Hedgehog receptor Patched1 regulates proliferation, neurogenesis, and axon guidance in the embryonic spinal cord.

The formation of the vertebrate nervous system depends on the complex interplay of morphogen signaling pathways and cell cycle progression to establish distinct cell fates. The Sonic hedgehog (Shh) signaling pathway is well understood to promote ventral cell fates in the developing spinal cord. A key regulator of Shh signaling is its receptor Patched1 (Ptch1). However, because the Ptch1 null mutation is lethal early in mouse embryogenesis, its role in controlling cell cycle progression, neurogenesis, and axon guidance in the developing spinal cord is not fully understood. An allele of Ptch1 called Wiggable (Ptch1Wig), which was previously shown to enhance Shh signaling, was used to test its ability to regulate neurogenesis and proliferation in the developing spinal cord. Ptch1Wig/Wig mutants displayed enhanced ventral proneural gene activation, and aberrant proliferation of the neural tube and floor plate cells, the latter normally being a quiescent population. The expression of the cell cycle regulators p27Kip1 and p57Kip2 were expanded in Ptch1Wig/Wig mutant spinal cords, as was the number of mitotic and S-phase nuclei, suggesting enhanced cell cycle progression. However, Ptch1Wig/Wig mutants also showed enhanced apoptosis in the ventral embryonic spinal cord, which resulted in thinner spinal cords at later embryonic stages. Commissural axons largely failed to cross the floor plate of Ptch1Wig/Wig mutant embryos, suggesting enhanced Shh signaling in these mutants led to a dorsal expansion of the chemoattraction front. These findings are consistent with a role of Ptch1 in regulating neurogenesis and proliferation of neural progenitors, and in restricting the influence of Shh signaling in commissural axon guidance to the floor plate.

Broad Promotes Neuroepithelial Stem Cell Differentiation in the Drosophila Optic Lobe.

Brain development requires the generation of the right number, and type, of neurons and glial cells at the right time. The Drosophila optic lobe, like mammalian brains, develops from simple neuroepithelia; they first divide symmetrically to expand the progenitor pool and then differentiate into neuroblasts, which divide asymmetrically to generate neurons and glial cells. Here, we investigate the mechanisms that control neuroepithelial growth and differentiation in the optic lobe. We find that the Broad/Tramtrack/Bric a brac-zinc finger protein Broad, which is dynamically expressed in the optic lobe neuroepithelia, promotes the transition of neuroepithelial cells to medulla neuroblasts. Loss of Broad function causes neuroepithelial cells to remain highly proliferative and delays neuroepithelial cell differentiation into neuroblasts, which leads to defective lamina and medulla. Conversely, Broad overexpression induces neuroepithelial cells to prematurely transform into medulla neuroblasts. We find that the ecdysone receptor is required for neuroepithelial maintenance and growth, and that Broad expression in neuroepithelial cells is repressed by the ecdysone receptor. Our studies identify Broad as an important cell-intrinsic transcription factor that promotes the neuroepithelial-cell-to-neuroblast transition.

Rgma-Induced Neo1 Proteolysis Promotes Neural Tube Morphogenesis.

Neuroepithelial cell (NEC) elongation is one of several key cell behaviors that mediate the tissue-level morphogenetic movements that shape the neural tube (NT), the precursor of the brain and spinal cord. However, the upstream signals that promote NEC elongation have been difficult to tease apart from those regulating apico-basal polarity and hingepoint formation, due to their confounding interdependence. The Repulsive Guidance Molecule a (Rgma)/Neogenin 1 (Neo1) signaling pathway plays a conserved role in NT formation (neurulation) and is reported to regulate both NEC elongation and apico-basal polarity, through signal transduction events that have not been identified. We examine here the role of Rgma/Neo1 signaling in zebrafish (sex unknown), an organism that does not use hingepoints to shape its hindbrain, thereby enabling a direct assessment of the role of this pathway in NEC elongation. We confirm that Rgma/Neo1 signaling is required for microtubule-mediated NEC elongation, and demonstrate via cell transplantation that Neo1 functions cell autonomously to promote elongation. However, in contrast to previous findings, our data do not support a role for this pathway in establishing apical junctional complexes. Last, we provide evidence that Rgma promotes Neo1 glycosylation and intramembrane proteolysis, resulting in the production of a transient, nuclear intracellular fragment (NeoICD). Partial rescue of Neo1a and Rgma knockdown embryos by overexpressing neoICD suggests that this proteolytic cleavage is essential for neurulation. Based on these observations, we propose that RGMA-induced NEO1 proteolysis orchestrates NT morphogenesis by promoting NEC elongation independently of the establishment of apical junctional complexes.SIGNIFICANCE STATEMENT The neural tube, the CNS precursor, is shaped during neurulation. Neural tube defects occur frequently, yet underlying genetic risk factors are poorly understood. Neuroepithelial cell (NEC) elongation is essential for proper completion of neurulation. Thus, connecting NEC elongation with the molecular pathways that control this process is expected to reveal novel neural tube defect risk factors and increase our understanding of NT development. Effectors of cell elongation include microtubules and microtubule-associated proteins; however, upstream regulators remain controversial due to the confounding interdependence of cell elongation and establishment of apico-basal polarity. Here, we reveal that Rgma-Neo1 signaling controls NEC elongation independently of the establishment of apical junctional complexes and identify Rgma-induced Neo1 proteolytic cleavage as a key upstream signaling event.

Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

Cell and tissue morphology determine actin-dependent nuclear migration mechanisms in neuroepithelia.

Correct nuclear position is crucial for cellular function and tissue development. Depending on cell context, however, the cytoskeletal elements responsible for nuclear positioning vary. While these cytoskeletal mechanisms have been intensely studied in single cells, how nuclear positioning is linked to tissue morphology is less clear. Here, we compare apical nuclear positioning in zebrafish neuroepithelia. We find that kinetics and actin-dependent mechanisms of nuclear positioning vary in tissues of different morphology. In straight neuroepithelia, nuclear positioning is controlled by Rho-ROCK-dependent myosin contractility. In contrast, in basally constricted neuroepithelia, a novel formin-dependent pushing mechanism is found for which we propose a proof-of-principle force generation theory. Overall, our data suggest that correct nuclear positioning is ensured by the adaptability of the cytoskeleton to cell and tissue shape. This in turn leads to robust epithelial maturation across geometries. The conclusion that different nuclear positioning mechanisms are favored in tissues of different morphology highlights the importance of developmental context for the execution of intracellular processes.

Regulation of chitosan-mediated differentiation of human olfactory receptor neurons by insulin-like growth factor binding protein-2.

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 ßIII tubulin, but decrease mature ORNs. Instead, down-regulation of phosphorylated IGF1R lifts the OMP expression, and lowers the ßIII 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.