Activating a Reserve Neural Stem Cell Population In Vitro Enables Engraftment and Multipotency after Transplantation.
ABSTRACT: 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.
Project description:The vertebrate olfactory epithelium (OE) is known for its ability to renew itself throughout life as well as to reconstitute after injury. Although this remarkable capacity demonstrates the persistence of stem cells and multipotent progenitor cells, their nature in the OE remains undefined and controversial, as both horizontal basal cells (HBCs) and globose basal cells (GBCs) have features in common with each other and with stem cells in other tissues. Here, we investigate whether some among the population of GBCs satisfy a key feature of stem cells, i.e., mitotic quiescence with retention of thymidine analogue label and activation by injury. Accordingly, we demonstrate that some GBCs express p27(Kip1) , a member of the Kip/Cip family of cyclin-dependent kinase inhibitors. In addition, some GBCs retain bromodeoxyuridine or ethynyldeoxyuridine for an extended period when the pulse is administered in neonates followed by a 1-month chase. Their identity as GBCs was confirmed by electron microscopy. All spared GBCs express Ki-67 in the methyl bromide (MeBr)-lesioned OE initially after lesion, indicating that the label-retaining (LR) GBCs are activated in response to injury. LR-GBCs reappear during the acute recovery period following MeBr exposure, as demonstrated with 2- or 4-week chase periods after labeling. Taken together, our data demonstrate the existence of LR-GBCs that are seemingly activated in response to epithelial injury and then re-established after the initial phase of recovery is completed. In this regard, some among the GBCs satisfy a common criterion for functioning like stem cells.
Project description:The biochemical identification and immunocytochemical characterization of a cell surface antigen, expressed on globose basal cells (GBCs) of the rodent olfactory epithelium (OE), are described. The monoclonal antibody (MAb) GBC-3 recognizes a surface protein, confirmed by both live cell staining and fluorescence-activated cell sorting. Two-dimensional SDS-PAGE/Western blot followed by tandem mass spectrometry demonstrates that the cell surface GBC-3 antigen is the 40 kDa laminin receptor precursor protein. The MAb GBC-3 labels the vast majority of cells among the GBC population and does not stain either sustentacular cells or horizontal basal cells (HBCs) in the normal rat OE. After epithelial lesion by exposure to methyl bromide, the remaining cells, which are mostly GBCs, are heavily stained by GBC-3, and colabeled with GBC-3 and sustentacular cell or HBC markers. GBC-3 will be a potentially useful tool for identifying and characterizing GBCs.
Project description:In the adult olfactory epithelium, the transcription factors Pax6 and Sox2 are co-expressed in sustentacular cells, horizontal basal cells (HBCs), and less-differentiated globose basal cells (GBCs)-both multipotent and transit amplifying categories-but are absent from immediate neuronal precursor GBCs and olfactory sensory neurons (OSNs). We used retroviral-vector transduction to over-express Pax6 and Sox2 individually and together during post-lesion recovery to determine how they regulate neuronal differentiation. Both Pax6 and Sox2, separately and together, can suppress the production of OSNs, as fewer clones contain neurons than with empty vector (EV), although this effect is not absolute. In this regard, Pax6 has the strongest effect when acting alone. In clones where neurons form, Pax6 reduces neuron numbers by comparison with EV, while Sox2 expands their numbers. Co-transduction with Pax6 and Sox2 produces an intermediate result. The increased production of OSNs driven by Sox2 is due to the expansion of neuronal progenitors, since proliferation and the numbers of Ascl1, Neurog1, and NeuroD1-expressing GBCs are increased. Conversely, Pax6 seems to accelerate neuronal differentiation, since Ascl1 labeling is reduced, while Neurog1- and NeuroD1-labeled GBCs are enriched. As a complement to the over-expression experiments, elimination of Sox2 in spared cells of floxed Sox2 mice, by retroviral Cre or by K5-driven CreERT2, reduces the production of OSNs and non-neuronal cells during OE regeneration. These data suggest that Pax6 and Sox2 have counteracting roles in regulating neurogenesis, in which Pax6 accelerates neuronal production, while Sox2 retards it and expands the pool of neuronal progenitors.
Project description:Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of enhanced green fluorescent protein (eGFP) and cell surface markers to FACS-isolate ?Sox2-eGFP(+) GBCs, Neurog1-eGFP(+) GBCs and immature neurons, and ?OMP-eGFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2,100 genes that are upregulated compared to sustentacular cells. A further cohort of >1,200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-eGFP(+) population. In contrast, the abbreviated lifespan of ?OMP-eGFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream ?Sox2-eGFP(+) GBCs and Neurog1-eGFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion.
Project description:Adult neurogenesis in the olfactory epithelium is often depicted as a unidirectional pathway during homeostasis and repair. We challenge the unidirectionality of this model by showing that epithelial injury unlocks the potential for Ascl1+ progenitors and Neurog1+ specified neuronal precursors to dedifferentiate into multipotent stem/progenitor cells that contribute significantly to tissue regeneration in the murine olfactory epithelium (OE). We characterize these dedifferentiating cells using several lineage-tracing strains and single-cell mRNA-seq, and we show that Sox2 is required for initiating dedifferentiation and that inhibition of Ezh2 promotes multipotent progenitor expansion. These results suggest that the apparent hierarchy of neuronal differentiation is not irreversible and that lineage commitment can be overridden following severe tissue injury. We elucidate a previously unappreciated pathway for endogenous tissue repair by a highly regenerative neuroepithelium and introduce a system to study the mechanisms underlying plasticity in the OE that can be adapted for other tissues.
Project description:Although olfactory mucosa possesses long-lived horizontal basal stem cells (HBCs) and remarkable regenerative capacity, the function of human olfactory neuroepithelium is significantly impaired in chronic inflammatory rhinosinusitis. Here, we show that, while inflammation initially damages olfactory neurons and activates HBC-mediated regeneration, continued inflammation locks HBCs in an undifferentiated state. Global gene expression in mouse HBCs reveals broad upregulation of NF-?B-regulated cytokines and chemokines including CCL19, CCL20, and CXCL10, accompanied by enhancement of "stemness"-related transcription factors. Loss-of-function studies identify an NF-?B-dependent role of HBCs in amplifying inflammatory signaling, contributing to macrophage and T cell local proliferation. Chronically activated HBCs signal macrophages to maintain immune defense and prevent Treg development. In diseased human olfactory tissue, activated HBCs in a P63+ undifferentiated state similarly contribute to inflammation through chemokine production. These observations establish a mechanism of chronic rhinosinusitis-associated olfactory loss, caused by a functional switch of neuroepithelial stem cells from regeneration to immune defense.
Project description:The basic helix-loop-helix transcription factor NeuroD1 is expressed in embryonic and adult mouse olfactory epithelium (OE), as well as during epithelial regeneration, suggesting that it plays an important role in olfactory neurogenesis. We characterized NEUROD1-expressing progenitors, determined their progeny in the adult OE, and identified a subtle phenotype in ?NeuroD1-knockout mice. All olfactory sensory neurons (OSNs) derive from a NeuroD1-expressing progenitor as shown by recombination-mediated lineage tracing, as do other sensory receptors of the nose, including vomeronasal, nasal septal, and Grunenberg ganglion neurons. NEUROD1-expressing cells are found among the globose basal cell population: they are actively proliferating and frequently coexpress Neurog1, but not the transit amplifying cell marker MASH1, nor the neuronal marker NCAM. As a consequence, NEUROD1-expressing globose basal cells are best classified as immediate neuronal precursors. In adolescent ?NeuroD1-LacZ knock-in null mice the OE displays subtle abnormalities, as compared to wildtype and heterozygous littermates. In some areas of the OE, mature neurons are absent, or sparse, although those same areas retain immature OSNs and LacZ-expressing progenitors, albeit both of these populations are smaller than expected. Our results support the conclusion that most, if not all, nasal chemosensory neurons derive from NeuroD1-expressing globose basal cells of the immediate neuronal precursor variety. Moreover, elimination of NeuroD1 by gene knockout, while it does not disrupt initial OSN differentiation, does compromise the integrity of parts of the olfactory epithelium by altering proliferation, neuronal differentiation, or neuronal survival there.
Project description:The olfactory epithelium is remarkable for the persistence of multipotent, neurocompetent progenitor and stem cells throughout life that can replace all of the various cell types of the epithelium following injury. The therapeutic exploitation of the neurocompetent stem cells of the adult olfactory epithelium would be facilitated by the development of a culture system that maintains the in vivo potency of the progenitors while they are expanded and/or manipulated. We have used an air-liquid interface culture protocol, in which a feeder cell layer of 3T3 cells is established on the underside of a culture insert and Facs-isolated or unsorted progenitor cells from the methyl bromide-lesioned adult rodent epithelium are seeded on upper side. Under these conditions, epithelial cells other than HBCs are capable of organizing themselves into complex three-dimensional, epithelium-lined spheres, which can be passaged. The spheres contain cells with the molecular phenotype of globose basal cells, horizontal basal cells, sustentacular cells and neurons. Spheres derived from mice that express the green fluorescent protein constitutively can be dissociated after 6 days in vitro and directly transplanted into the epithelium of wild-type, methyl bromide-lesioned mice via nasal infusion. The resulting clones contain the various cell types observed in aggregate when globose basal cells are transplanted acutely. In contrast, the same cells cultured as two-dimensional, submerged cultures undergo fibroblastic transition after transplantation and do not integrate into the epithelium. In conclusion, the culture system described here maintains the potency of progenitors, which can then participate in epitheliopoiesis in vivo.
Project description:The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ?Np63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ?Np63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ?Np63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ?Np63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ?Np63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.
Project description:The olfactory epithelium (OE) is composed of olfactory sensory neurons (OSNs), sustentacular supporting cells, and several types of non-neuronal cells. Stem and progenitor cells are located basally, and are the source of all cell types needed to maintain OE homeostasis. Here, we report that Ascl3, a basic helix-loop-helix transcription factor, is expressed in the developing OE. Lineage tracing experiments demonstrate that the non-neuronal microvillar cells and Bowman's glands are exclusively derived from Ascl3+ progenitor cells in the OE during development. Following chemically-induced injury, Ascl3 expression is activated in a subset of horizontal basal cells (HBCs), which repopulate all microvillar cells and Bowman's glands during OE regeneration. After ablation of Ascl3-expressing cells, the OE can regenerate, but lacks the non-neuronal microvillar and Bowman's gland support cells. These results demonstrate that Ascl3 marks progenitors that are lineage-committed strictly to microvillar cells and Bowman's glands, and highlight the requirement for these cell types to support OE homeostasis.