RESUMO
Olfactory epithelium (OE) has a lifelong capacity for neurogenesis due to the presence of basal stem cells. Despite the ability to generate short-term cultures, the successful in vitro expansion of purified stem cells from adult OE has not been reported. We sought to establish expansion-competent OE stem cell cultures to facilitate further study of the mechanisms and cell populations important in OE renewal. Successful cultures were prepared using adult mouse basal cells selected for expression of c-KIT. We show that c-KIT signaling regulates self-renewal capacity and prevents neurodifferentiation in culture. Inhibition of TGFß family signaling, a known negative regulator of embryonic basal cells, is also necessary for maintenance of the proliferative, undifferentiated state in vitro Characterizing successful cultures, we identified expression of BMI1 and other Polycomb proteins not previously identified in olfactory basal cells but known to be essential for self-renewal in other stem cell populations. Inducible fate mapping demonstrates that BMI1 is expressed in vivo by multipotent OE progenitors, validating our culture model. These findings provide mechanistic insights into the renewal and potency of olfactory stem cells.
Assuntos
Autorrenovação Celular/fisiologia , Neurogênese/fisiologia , Mucosa Olfatória/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 µm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.
Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração/efeitos dos fármacos , Hidralazina/farmacologia , Miócitos Cardíacos/metabolismo , Nitroglicerina/farmacologia , Vasodilatadores/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Acoplamento Excitação-Contração/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Oxirredução/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologiaRESUMO
The presence of active neurogenic niches in adult humans is controversial. We focused attention to the human olfactory neuroepithelium, an extracranial site supplying input to the olfactory bulbs of the brain. Using single-cell RNA sequencing analyzing 28,726 cells, we identified neural stem cell and neural progenitor cell pools and neurons. Additionally, we detailed the expression of 140 olfactory receptors. These data from the olfactory neuroepithelium niche provide evidence that neuron production may continue for decades in humans.
Assuntos
Neurogênese/fisiologia , Mucosa Olfatória/inervação , Mucosa Olfatória/fisiologia , Análise de Célula Única , Adulto , Envelhecimento/fisiologia , Humanos , Células-Tronco Neurais/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Análise de Sequência de RNA , OlfatoRESUMO
Stem cell-based therapies have been proposed as a strategy to replace damaged tissues, especially in the nervous system. A primary sensory modality, olfaction, is impaired in 12% of the US population, but lacks treatment options. We report here the development of a novel mouse model of inducible hyposmia and demonstrate that purified tissue-specific stem cells delivered intranasally engraft to produce olfactory neurons, achieving recovery of function. Adult mice were rendered hyposmic by conditional deletion of the ciliopathy-related IFT88 gene in the olfactory sensory neuron lineage and following experimentally induced olfactory injury, received either vehicle or stem cell infusion intranasally. Engraftment-derived olfactory neurons were identified histologically, and functional improvements were measured via electrophysiology and behavioral assay. We further explored mechanisms in culture that promote expansion of engraftment-competent adult olfactory basal progenitor cells. These findings provide a basis for translational research on propagating adult tissue-specific sensory progenitor cells and testing their therapeutic potential.
Assuntos
Ciliopatias , Células-Tronco Neurais , Transtornos do Olfato , Neurônios Receptores Olfatórios , Olfato , Transplante de Células-Tronco , Animais , Benzilatos , Ciliopatias/genética , Ciliopatias/metabolismo , Ciliopatias/patologia , Ciliopatias/terapia , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Células-Tronco Neurais/transplante , Transtornos do Olfato/genética , Transtornos do Olfato/metabolismo , Transtornos do Olfato/patologia , Transtornos do Olfato/terapia , Neurônios Receptores Olfatórios/metabolismo , Neurônios Receptores Olfatórios/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVES: To investigate epigenetic mechanisms contributing to regulation of cellular renewal and neurogenesis in adult olfactory epithelium (OE). STUDY DESIGN: Prospective basic science study. METHODS: Olfactory basal cell cultures were prepared from adult mice per established protocols. in vivo studies were performed using the mouse methimazole lesion-regeneration paradigm. Nasal tissue sections were prepared from adult mice 7 days following lesion, or from unlesioned controls. Polycomb proteins were assessed by Western blot from culture or nasal tissue lysates, and by gene expression studies from cultures. In addition, in vivo expression patterns of Polycomb proteins were examined using immunohistochemistry. Chromosome immunoprecipitation (ChIP) was performed to investigate epigenetic modifications and specific chromatin interactions for Polycomb proteins in olfactory basal cells. RESULTS: Subunits of Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) were identified in basal cell cultures and in vivo. In regenerating OE, basal progenitor cells identified by expression of the c-KIT receptor were found to coexpress the PRC2 protein EZH2. Because multiple variants of PRC1 subunits give rise to diverse PRC1 complexes serving different functions, expression of specific PRC1 variants was further examined. We identified PRC1 components including MEL18 (PCGF2) in immature neurons, and confirm BMI1 (PCGF4) expression in mature neurons. Moreover, we identified CBX8 as a neuron-specific PRC1 subunit. ChIP assays from OE cells demonstrated binding of PRC proteins to regulatory regions of specific transcription factors, consistent with PRC-mediated epigenetic silencing mechanisms active in adult OE. CONCLUSIONS: Multiple Polycomb proteins have cell type-specific expression patterns in the adult OE. Findings presented here, together with evidence from prior studies, suggest that PRC-mediated epigenetic silencing contributes to regulation of cellular renewal and tissue homeostasis in the OE. Efforts to define the mechanisms that regulate repair in the OE are essential for development of new therapeutic strategies for olfactory disorders. LEVEL OF EVIDENCE: N/A.
RESUMO
Despite a robust capacity for adult neurogenesis in the olfactory epithelium (OE), olfactory sensory losses are common. Identification of mechanisms regulating adult OE neurogenesis is, therefore, of interest. MicroRNAs (miRNAs) are broadly important in regulating vertebrate neurodevelopment, and are required in embryonic olfactory differentiation. We report here that a panel of miRNAs is differentially expressed by either progenitor or progeny cells in the regenerating mouse OE. Progenitor cells were purified from lesioned OE based on c-Kit expression, and miRNA expression was assayed in c-Kit (+) and c-Kit (-) cell populations. 28 miRNAs were significantly downregulated by at least 4 fold in the c-Kit (+) fraction, which marks the globose basal progenitor cell population. In addition, 10 miRNAs were upregulated in these basal cells. MiR-486, the most strongly downregulated miRNA identified, was further characterized to verify results. MiR-486 expression was confirmed in the c-Kit (-) OE layers using in situ hybridization. As a functional assay, over-expression of miR-486 in purified c-Kit (+) basal cell cultures resulted in a reduction in neurogenesis, consistent with a possible negative feedback regulatory model. Our data provide new insights regarding miRNA expression and function during adult OE neurogenesis, and identify candidate miRNAs warranting further study.
Assuntos
MicroRNAs/genética , Mucosa Olfatória/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese , Mucosa Olfatória/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Olfactory tissue undergoes lifelong renewal, due to the presence of basal neural stem cells. Multiple categories of globose basal stem cells have been identified, expressing markers such as Lgr5, Ascl1, GBC-2, and c-Kit. The differentiation potential of individual globose cells has remained unclear. Here, we utilized Cre/loxP lineage tracing with a multicolor reporter system to define c-Kit+ cell contributions at clonal resolution. We determined that reporter expression permitted identification of c-Kit derived progeny with fine cellular detail, and that clones were found to be comprised by neurons only, microvillar cells only, microvillar cells and neurons, or gland/duct cells. Quantification of reporter-labeled cells indicated that c-Kit+ cells behave as transit amplifying or immediate precursors, although we also found evidence for longer-term c-Kit+ cell contributions. Our results from the application of multicolor fate mapping delineate the clonal contributions of c-Kit+ cells to olfactory epithelial renewal, and provide novel insight into tissue maintenance of an adult neuroepithelium.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Neurais/citologia , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-kit/biossínteseRESUMO
The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.
Assuntos
Regeneração Nervosa/fisiologia , Células-Tronco Neurais/fisiologia , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Western Blotting , Contagem de Células , Dermoscopia , Imuno-Histoquímica , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Neurogênese/fisiologia , Mucosa Olfatória/crescimento & desenvolvimento , Mucosa Olfatória/lesões , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes deafness, affecting millions of people. Currently, there is no effective therapy to repair the inner ear sensory structures in humans. Cochlear implantation can restore input, but only if auditory neurons remain intact. Efforts to develop stem cell-based treatments for deafness have demonstrated progress, most notably utilizing embryonic-derived cells. In an effort to bypass limitations of embryonic or induced pluripotent stem cells that may impede the translation to clinical applications, we sought to utilize an alternative cell source. Here, we show that adult human mesenchymal-like stem cells (MSCs) obtained from nasal tissue can repair spiral ganglion loss in experimentally lesioned cochlear cultures from neonatal rats. Stem cells engraft into gentamicin-lesioned organotypic cultures and orchestrate the restoration of the spiral ganglion neuronal population, involving both direct neuronal differentiation and secondary effects on endogenous cells. As a physiologic assay, nasal MSC-derived cells engrafted into lesioned spiral ganglia demonstrate responses to infrared laser stimulus that are consistent with those typical of excitable cells. The addition of a pharmacologic activator of the canonical Wnt/ß-catenin pathway concurrent with stem cell treatment promoted robust neuronal differentiation. The availability of an effective adult autologous cell source for inner ear tissue repair should contribute to efforts to translate cell-based strategies to the clinic.