Wnt-responsive Lgr5⁺ globose basal cells function as multipotent olfactory epithelium progenitor cells.

Persistent neurogenesis in the olfactory epithelium provides a unique model to study neural stem cell self-renewal and fate determination. In the olfactory neuroepithelium, globose basal cells (GBCs) are considered to be the direct progenitors of olfactory neurons. However, the study of neurogenesis from GBCs has been impeded by the paucity of GBC-specific markers. Here we report that Lgr5, a recently discovered adult stem cell marker, is exclusively expressed in GBCs in neonatal and adult mice. Lgr5(+) cells display characteristics of cycling stem cells, including Ki67 expression and EdU incorporation. Lineage tracing analysis demonstrates that Lgr5(+) GBCs regenerate multiple cell types under normal turnover condition or after olfactory lesion. Furthermore, upregulation or downregulation of Wnt signaling in vivo indicates a key role of Wnt signaling not only in maintaining Lgr5(+) cell proliferation and promoting neuroregeneration, but also in delaying sensory neuron maturation. Together, our observations provided new insights into the dynamics of neurogenesis in the olfactory epithelium.

Sox2 regulates globose basal cell regeneration in the olfactory epithelium.

BACKGROUND: In the olfactory epithelium, mitotically active globose basal cells (GBCs) continuously replenish olfactory sensory neurons (OSNs) 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+ OSNs. 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.

Integrated age-related immunohistological changes occur in human olfactory epithelium and olfactory bulb.

Olfactory epithelium (OE) is capable of lifelong regeneration due to presence of basal progenitor cells that respond to injury or neuronal loss with increased activity. However, this capability diminishes with advancing age and a decrease in odor perception in older individuals is well established. To characterize changes associated with age in the peripheral olfactory system, an in-depth analysis of the OE and its neuronal projections onto the olfactory bulb (OB) as a function of age was performed. Human olfactory tissue autopsy samples from 36 subjects with an average age of 74.1 years were analyzed. Established cell type-specific antibodies were used to identify OE component cells in whole mucosal sheets and epithelial sections as well as glomeruli and periglomerular structures in OB sections. With age, a reduction in OE area occurs across the mucosa progressing in a posterior-dorsal direction. Deterioration of the olfactory system is accompanied with diminution of neuron-containing OE, mature olfactory sensory neurons (OSNs) and OB innervation. On an individual level, the neuronal density within the epithelium appears to predict synapse density within the OB. The innervation of the OB is uneven with higher density at the ventral half that decreases with age as opposed to stable innervation at the dorsal half. Respiratory metaplasia, submucosal cysts, and neuromata, were commonly identified in aged OE. The finding of respiratory metaplasia and aneuronal epithelium with reduction in global basal cells suggests a progression of stem cell quiescence as an underlying pathophysiology of age-related smell loss in humans. KEY POINTS: A gradual loss of olfactory sensory neurons with age in human olfactory epithelium is also reflected in a reduction in glomeruli within the olfactory bulb. This gradual loss of neurons and synaptic connections with age occurs in a specific, spatially inhomogeneous manner. Decreasing mitotically active olfactory epithelium basal cells may contribute to age-related neuronal decline and smell loss in humans.

Globose basal cells for spinal cord regeneration.

Spinal cord injury (SCI) is a devastating condition with loss of motor and sensory functions below the injury level. Cell based therapies are experimented in pre-clinical studies around the world. Neural stem cells are located intra-cranially in subventricular zone and hippocampus which are highly invasive sources. The olfactory epithelium is a neurogenic tissue where neurogenesis takes place throughout the adult life by a population of stem/progenitor cells. Easily accessible olfactory neuroepithelial stem/progenitor cells are an attractive cell source for transplantation in SCI. Globose basal cells (GBCs) were isolated from rat olfactory epithelium, characterized by flow cytometry and immunohistochemically. These cells were further studied for neurosphere formation and neuronal induction. T10 laminectomy was done to create drop-weight SCI in rats. On the 9th day following SCI, 5 × 105 cells were transplanted into injured rat spinal cord. The outcome of transplantation was assessed by the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, motor evoked potential and histological observation. GBCs expressed neural stem cell markers nestin, SOX2, NCAM and also mesenchymal stem cell markers (CD29, CD54, CD90, CD73, CD105). These cells formed neurosphere, a culture characteristics of NSCs and on induction, differentiated cells expressed neuronal markers ßIII tubulin, microtubule-associated protein 2, neuronal nuclei, and neurofilament. GBCs transplanted rats exhibited hindlimb motor recovery as confirmed by BBB score and gastrocnemius muscle electromyography amplitude was increased compared to controls. Green fluorescent protein labelled GBCs survived around the injury epicenter and differentiated into ßIII tubulin-immunoreactive neuron-like cells. GBCs could be an alternative to NSCs from an accessible source for autologous neurotransplantation after SCI without ethical issues.

Immunohistological and electrophysiological characterization of Globose basal stem cells.

OBJECTIVES: In the past few decades, variety of foetal, embryonic and adult stem and progenitor cells have been tried with conflicting outcome for cell therapy of central nervous system injury and diseases. Cellular characteristics and functional plasticity of Globose basal stem cells (GBCs) residing in the olfactory epithelium of rat olfactory mucosa have not been studied in the past by the neuroscientists due to unavailability of specific markers for GBCs. In the present research, we standardized some techniques to isolate GBCs from rat olfactory epithelium in pure form using a highly selective GBC-III antibody passaged through fluorescence activated cell sorter (FACS). We also characterized these cells immunohistologically using various pluripotent stem cell markers. This work also throws some light on ionic channels present on these stem cells which are responsible for their neuron induction potential. MATERIALS AND METHODS: Globose basal stem cells were isolated from rat olfactory epithelium using GBC-III antibody and were characterized as multipotent stem cells using various neural progenitor markers. Ionic channels on GBCs were studied with voltage clamping. RESULTS: GBCs could be isolated in pure (99% purity) form and were found to be stained positive for all neural progenitor cell markers. Voltage gated Na(+) channels were completely absent, which proves the unexcitable nature of GBCs. Leaky K(+) channels were found to be present on the GBC which was of no significance. CONCLUSION: This research work can be helpful in understanding the nature of these stem cells and utilising them in future as potent candidates for neuro-regenerative therapies.

Isolating globose Basal stem cells from albino wistar rats using a highly specific monoclonal antibody.

INTRODUCTION: Olfactory mucosa which is situated in the roof of the nasal cavity possesses an extremely peculiar and exceptional type of pluripotent stem cells called Globose Basal Cells (GBCs) which help in lifelong regeneration of the olfactory mucosa. Previous literature doesn't provide much knowledge on the cytological, histochemical and electrophysiological properties of these cells, as they have never been isolated in pure form. MATERIAL AND METHODS: Olfactory mucosa was obtained from six Albino Wistar rats by using standardized surgical and chemical separation procedures. GBCs were isolated by using different chemical, surgical and fluorescent techniques. RESULTS: In this research work, we standardized the techniques for isolating these stem cells in pure form from rat olfactory mucosa by tagging them with GBC-III antibody and separating them from other epithelial cells by using Fluorescence Activated Cell Sorter (FACS). GBC-III antibody is a mouse monoclonal IgM antibody which recognizes a 40 kDa surface antigen, which is a laminin receptor surface protein present on the GBCs. It is a highly specific marker for GBCs, unlike the earlier antibodies used, like GBC-I, which were nonspecific markers for GBCs and showed positive reactions, even with Horizontal Basal Cells (HBCs), sustentacular cells (Sus) and duct cells. This study also standardized the techniques for surgically excising the olfactory mucosa from the nasal septum and chemically separating the olfactory epithelium from the lamina propria. CONCLUSION: GBCs are an important group of cells which can be exploited in future to study and treat neuro-degenerative disorders like multiple sclerosis, brain ischaemia, etc. and spinal cord trauma, as they reside in a niche similar to the microenvironment in the central nervous system and have the similar ectodermal development as the neuronal and non-neuronal cells of the CNS. Moreover, olfactory epithelium is easily accessible for autologous transplantation of GBCs for different CNS disorders.