RESUMO
Irradiation (IR)-induced xerostomia is the most common side effect of radiation therapy in patients with head and neck cancer (HNC). Xerostomia diagnosis is mainly based on the patient's medical history and symptoms. Currently, no direct biomarkers are available for the early prediction of IR-induced xerostomia. Here, we identified PIEZO1 as a novel predictive tissue biomarker for xerostomia. Our data demonstrate that PIEZO1 is significantly upregulated at the gene and protein levels during IR-induced salivary gland (SG) hypofunction. Notably, PIEZO1 upregulation coincided with that of inflammatory (F4/80) and fibrotic markers (fibronectin and collagen fibers accumulation). These findings suggest that PIEZO1 upregulation in SG tissue may serve as a novel predictive marker for IR-induced xerostomia.
Assuntos
Biomarcadores , Canais Iônicos , Glândulas Salivares , Canais Iônicos/metabolismo , Canais Iônicos/genética , Biomarcadores/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/efeitos da radiação , Animais , Xerostomia/etiologia , Xerostomia/metabolismo , Camundongos , Masculino , Regulação para Cima/efeitos da radiação , Humanos , Camundongos Endogâmicos C57BLRESUMO
Error in Figure [...].
RESUMO
Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.
Assuntos
Receptores Odorantes , Ressonância de Plasmônio de Superfície , Antraquinonas , Descoberta de Drogas , Humanos , Ligantes , Células MCF-7 , Receptores Odorantes/genética , RutinaRESUMO
In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Doenças do Sistema Nervoso/terapia , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Transplante de Células-TroncoRESUMO
Soy isoflavones are popular ingredients with anti-adipogenic and anti-lipogenic properties. The anti-adipogenic and anti-lipogenic properties of genistein are well-known, but those of genistin and glycitein remain unknown, and those of daidzein are characterized by contrasting data. Therefore, the purpose of our study was to investigate the effects of daidzein, glycitein, genistein, and genistin on adipogenesis and lipogenesis in 3T3-L1 cells. Proliferation of 3T3-L1 preadipocytes was unaffected by genistin and glycitein, but it was affected by 50 and 100 µM genistein and 100 µM daidzein for 48 h. Among the four isoflavones, only 50 and 100 µM genistin and genistein markedly suppressed lipid accumulation during adipogenesis in 3T3-L1 cells through a similar signaling pathway in a dose-dependent manner. Genistin and genistein suppress adipocyte-specific proteins and genes, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and adipocyte binding protein 2 (aP2)/fatty acid-binding protein 4 (FABP4), and lipogenic enzymes such as ATP citrate lyase (ACL), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS). Both isoflavones also activate AMP-activated protein kinase α (AMPKα), an essential factor in adipocyte differentiation, and inhibited sterol regulatory element-binding transcription factor 1c (SREBP-1c). These results indicate that genistin is a potent anti-adipogenic and anti-lipogenic agent.
Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Isoflavonas/farmacologia , Lipogênese/efeitos dos fármacos , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetil-CoA Carboxilase/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Isoflavonas/química , Lipogênese/genética , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Glycine max/química , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Age-related macular degeneration (AMD) is a major cause of irreversible loss of vision with 80-90% of patients demonstrating dry type AMD. Dry AMD could possibly be prevented by polyphenol-rich medicinal foods by the inhibition of N-retinylidene-N-retinylethanolamine (A2E)-induced oxidative stress and cell damage. Arctium lappa L. (AL) leaves are medicinal and have antioxidant activity. The purpose of this study was to elucidate the protective effects of the extract of AL leaves (ALE) on dry AMD models, including in vitro A2E-induced damage in ARPE-19 cells, a human retinal pigment epithelial cell line, and in vivo light-induced retinal damage in BALB/c mice. According to the total phenolic contents (TPCs), total flavonoid contents (TFCs) and antioxidant activities, ALE was rich in polyphenols and had antioxidant efficacies on 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2',7'-dichlorofluorescin diacetate (DCFDA) assays. The effects of ALE on A2E accumulation and A2E-induced cell death were also monitored. Despite continued exposure to A2E (10 µM), ALE attenuated A2E accumulation in APRE-19 cells with levels similar to lutein. A2E-induced cell death at high concentration (25 µM) was also suppressed by ALE by inhibiting the apoptotic signaling pathway. Furthermore, ALE could protect the outer nuclear layer (ONL) in the retina from light-induced AMD in BALB/c mice. In conclusion, ALE could be considered a potentially valuable medicinal food for dry AMD.
Assuntos
Arctium/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Retina/efeitos dos fármacos , Retina/patologia , Retinoides/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Imuno-Histoquímica , Degeneração Macular/tratamento farmacológico , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system. Intestinal microbiota residing in the GI tract engages in a mutualistic relationship with the host. Different sections of the GI tract contain distinct proportions of the intestinal microbiota, resulting in the presence of unique bacterial products in each GI section. The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells. Metabolites act as messengers of information between the intestinal microbiota and host cells. The intestinal microbiota composition and resulting metabolites thus impact host development, health, and pathogenesis. Many recent studies have focused on modulation of the gut microbiota and their metabolites to improve host health and prevent or treat diseases. In this review, we focus on the production of microbial metabolites, their biological impact on the intestinal microbiota composition and host cells, and the effect of microbial metabolites that contribute to improvements in inflammatory bowel diseases and metabolic diseases. Understanding the role of microbial metabolites in protection against disease might offer an intriguing approach to regulate disease.
Assuntos
Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais/patologia , Doenças Metabólicas/patologia , Bactérias/química , Bactérias/metabolismo , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/toxicidade , Ácidos Graxos Voláteis/metabolismo , Ácidos Graxos Voláteis/toxicidade , Flavonas/metabolismo , Flavonas/toxicidade , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/microbiologia , Poliaminas/metabolismo , Poliaminas/toxicidadeRESUMO
Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.
Assuntos
Transplante de Tecido Encefálico , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/citologia , Doença de Parkinson/terapia , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Macaca mulatta , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos Sprague-DawleyRESUMO
Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.
Assuntos
Cílios/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas do Olho/genética , Audição/genética , Som , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Mecanotransdução Celular/genética , Neurônios/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Neural stem/progenitor cell (NSC/NPC) cultures can be a source of dopamine (DA) neurons for experimental and transplantation purposes. Nurr1, a steroid receptor transcription factor, can overcome the limitations associated with differentiation of cultured NPCs into DA neurons. However, forced Nurr1 expression in NPC cultures generates non-neuronal and/or immature DA cells. We show here that the Nurr1 level and period of expression crucially affect the differentiation and maturation of Nurr1-induced DA neurons. Mature DA neurons were generated by manipulating Nurr1 expression patterns to resemble those in the developing midbrain.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/crescimento & desenvolvimento , Neurogênese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Animais , Células Cultivadas , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Dopamina/biossíntese , Desenvolvimento Embrionário , Mesencéfalo/metabolismo , RatosRESUMO
The drug-eluting stent still has limitations such as thrombosis and inflammation. These limitations often occur in the absence of endothelialization. This study investigated the effects of WKYMVm- and sirolimus-coated stents on re-endothelialization and anti-restenosis. The WKYMVm peptide, specially synthesized for homing endothelial colony-forming cells, was coated onto a bare-metal stent with hyaluronic acid through a simple dip-coating method (designated HA-Pep). Thereafter, sirolimus was consecutively coated to onto the HA-Pep (designated Pep/SRL). The cellular response to stents by human umbilical-vein endothelial cells and vascular smooth-muscle cells was examined by XTT assay. Stents were implanted into rabbit iliac arteries, isolated 6 weeks post-implantation, and then subjected to histological analysis. The peptide was well attached to the surface of the stents and the sirolimus coating made the surface smooth. The release pattern for sirolimus was similar to that of commercial sirolimus-coated stents (57.2% within 7 days, with further release for up to 28 days). Endothelial-cell proliferation was enhanced in the HA-Pep group after 7 days of culture (38.2 ± 7.62%, compared with controls). On the other hand, the proliferation of smooth-muscle cells was inhibited in the Pep/SRL group after 7 days of culture (40.7 ± 6.71%, compared with controls). In an animal study, the restenosis rates for the Pep/SRL group (13.5 ± 4.50%) and commercial drug-eluting stents (Xience Prime™; 9.2 ± 7.20%) were lower than those for bare-metal stents (25.2 ± 4.52%) and HA-Pep stents (26.9 ± 3.88%). CD31 staining was incomplete for the bare-metal and Xience Prime™ groups. On the other hand, CD31 staining showed a consecutive linear pattern in the HA-Pep and Pep/SRL groups, suggesting that WKYMVm promotes endothelialization. These results indicate that the WKYMVm coating could promote endothelial healing, and consecutive coatings of WKYMVm and sirolimus onto bare-metal stents have a potential role in re-endothelialization and neointimal suppression.
Assuntos
Reestenose Coronária/prevenção & controle , Stents Farmacológicos , Oligopeptídeos/administração & dosagem , Sirolimo/administração & dosagem , Animais , Materiais Biocompatíveis/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Reestenose Coronária/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Artéria Ilíaca/efeitos dos fármacos , Artéria Ilíaca/patologia , Artéria Ilíaca/cirurgia , Masculino , Teste de Materiais , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Neointima/patologia , Neointima/prevenção & controle , Coelhos , RatosRESUMO
Current chemical treatments for cerebrovascular disease and neurological disorders have limited efficacy in tissue repair and functional restoration. Induced pluripotent stem cells (iPSCs) present a promising avenue in regenerative medicine for addressing neurological conditions. iPSCs, which are capable of reprogramming adult cells to regain pluripotency, offer the potential for patient-specific, personalized therapies. The modulation of molecular mechanisms through specific growth factor inhibition and signaling pathways can direct iPSCs' differentiation into neural stem cells (NSCs). These include employing bone morphogenetic protein-4 (BMP-4), transforming growth factor-beta (TGFß), and Sma-and Mad-related protein (SMAD) signaling. iPSC-derived NSCs can subsequently differentiate into various neuron types, each performing distinct functions. Cell transplantation underscores the potential of iPSC-derived NSCs to treat neurodegenerative diseases such as Parkinson's disease and points to future research directions for optimizing differentiation protocols and enhancing clinical applications.
RESUMO
Taste buds, the neuroepithelial organs responsible for the detection of gustatory stimuli in the oral cavity, arise from stem/progenitor cells among nearby basal keratinocytes. Using genetic lineage tracing, Lgr5 and Lgr6 were suggested as the specific markers for the stem/progenitor cells of taste buds, but recent evidence implied that taste buds may arise even in the absence of these markers. Thus, we wanted to verify the genetic lineage tracing of lingual Lgr5- and Lgr6-expressing cells. Unexpectedly, we found that antibody staining revealed more diverse Lgr5-expressing cells inside and outside the taste buds of circumvallate papillae than was previously suggested. We also found that, while tamoxifen-induced genetic recombination occurred only in cells expressing the Lgr5 reporter GFP, we did not see any increase in the number of recombined daughter cells induced by consecutive injections of tamoxifen. Similarly, we found that cells expressing Lgr6, another stem/progenitor cell marker candidate and an analog of Lgr5, also do not generate recombined clones. In contrast, Lgr5-expressing cells in fungiform papillae can transform into Lgr5-negative progeny. Together, our data indicate that lingual Lgr5- and Lgr6-expressing cells exhibit diversity in their capacity to transform into Lgr5- and Lgr6-negative cells, depending on their location. Our results complement previous findings that did not distinguish this diversity.
RESUMO
Insect survival depends on contact chemosensation to sense and avoid consuming plant-derived insecticides, such as L-canavanine. Members of a family of â¼60 gustatory receptors (GRs) comprise the main peripheral receptors responsible for taste sensation in Drosophila. However, the roles of most Drosophila GRs are unknown. In addition to GRs, a G protein-coupled receptor, DmXR, has been reported to be required for detecting L-canavanine. Here, we showed that GRs are essential for responding to L-canavanine and that flies missing DmXR displayed normal L-canavanine avoidance and L-canavanine-evoked action potentials. Mutations disrupting either Gr8a or Gr66a resulted in an inability to detect L-canavanine. We found that L-canavanine stimulated action potentials in S-type sensilla, which were where Gr8a and Gr66a were both expressed, but not in Gr66a-expressing sensilla that did not express Gr8a. L-canavanine-induced action potentials were also abolished in the Gr8a and Gr66a mutant animals. Gr8a was narrowly required for responding to L-canavanine, in contrast to Gr66a, which was broadly required for responding to other noxious tastants. Our data suggest that GR8a and GR66a are subunits of an L-canavanine receptor and that GR8a contributes to the specificity for L-canavanine.
Assuntos
Aprendizagem da Esquiva/fisiologia , Canavanina/farmacologia , Proteínas de Drosophila/fisiologia , Inseticidas/farmacologia , Receptores de Superfície Celular/fisiologia , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Animais Geneticamente Modificados , Aprendizagem da Esquiva/efeitos dos fármacos , Proteínas de Drosophila/genética , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Receptores de Superfície Celular/genéticaRESUMO
Aging is a complex series of multi-organ processes that occur in various organisms. As such, an in vivo study using an animal model of aging is necessary to define its exact mechanisms and identify anti-aging substances. Using Drosophila as an in vivo model system, we identified Crataegus pinnatifida extract (CPE) as a novel anti-aging substance. Regardless of sex, Drosophila treated with CPE showed a significantly increased lifespan compared to those without CPE. In this study, we also evaluated the involvement of CPE in aging-related biochemical pathways, including TOR, stem cell generation, and antioxidative effects, and found that the representative genes of each pathway were induced by CPE administration. CPE administration did not result in significant differences in fecundity, locomotion, feeding amount, or TAG level. These conclusions suggest that CPE is a good candidate as an anti-aging food substance capable of promoting a healthy lifespan.
Assuntos
Crataegus , Longevidade , Animais , Crataegus/química , Envelhecimento , Modelos Animais , DrosophilaRESUMO
Endometriosis is a gynecological disease prevalent in women of reproductive age, and it is characterized by the ectopic presence and growth of the eutopic endometrium. The pathophysiology and diagnostic biomarkers of endometriosis have not yet been comprehensively determined. To discover molecular markers and pathways underlying the pathogenesis of endometriosis, we identified differentially expressed genes (DEGs) in three Gene Expression Omnibus microarray datasets (GSE11691, GSE23339, and GSE7305) and performed gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network analyses. We also validated the identified genes via immunohistochemical analysis of tissues obtained from patients with endometriosis or healthy volunteers. A total of 118 DEGs (79 upregulated and 39 downregulated) were detected in each dataset with a lower (fold change) FC cutoff (log2|FC| > 1), and 17 DEGs (11 upregulated and six downregulated) with a higher FC cutoff (log2|FC| > 2). KEGG and GO functional analyses revealed enrichment of signaling pathways associated with inflammation, complement activation, cell adhesion, and extracellular matrix in endometriotic tissues. Upregulation of seven genes (C7, CFH, FZD7, LY96, PDLIM3, PTGIS, and WISP2) out of 17 was validated via comparison with external gene sets, and protein expression of four genes (LY96, PDLIM3, PTGIS, and WISP2) was further analyzed by immunohistochemistry and western blot analysis. Based on these results, we suggest that TLR4/NF-κB and Wnt/frizzled signaling pathways, as well as estrogen receptors, regulate the progression of endometriosis. These pathways may be therapeutic and diagnostic targets for endometriosis.
Assuntos
Endometriose , Humanos , Feminino , Endometriose/diagnóstico , Endometriose/genética , Endometriose/metabolismo , Biologia Computacional/métodos , Mapas de Interação de Proteínas/genética , Biomarcadores/metabolismo , Via de Sinalização WntRESUMO
Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/stem cell-based therapy of Parkinson's disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non-dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)-type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain-type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell-based therapy for PD.
Assuntos
Diferenciação Celular/genética , Fator 3-beta Nuclear de Hepatócito/genética , Neurônios/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Dopamina/metabolismo , Humanos , Camundongos , Neurogênese/genética , Neurônios/citologia , Neurônios/transplante , Doença de Parkinson/cirurgia , Fenótipo , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Substância Negra/citologia , Substância Negra/metabolismo , Transfecção/métodos , Resultado do TratamentoRESUMO
Recently, biocompatibility report revealed that the TiO2 nano-network (TiO2 NT) structure has much higher cells colonization than the native TiO2 on Ti surface. In this study, we prepared the hyaluronic acid (HA) immobilized TiO2 NT layer by plasma surface modification and then evaluated biological behavior of MC3T3-E1 on the Ti, TiO2 NT and TiO2 NT/NH2/HA surface. The cell viability tests revealed slightly enhanced viability on the TiO2 NT/NH2/HA surfaces than on the untreated Ti surfaces.
Assuntos
Ácido Hialurônico/química , Nanoestruturas , Poliaminas/química , Titânio/química , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Proliferação de Células , Camundongos , Microscopia Eletrônica de Varredura , Propriedades de SuperfícieRESUMO
Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.
Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Rombencéfalo/citologia , Serotonina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Humanos , Imuno-Histoquímica/métodos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rombencéfalo/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Tretinoína/farmacologiaRESUMO
Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.