RESUMEN
Cellular agriculture, an alternative and innovative approach to sustainable food production, has gained momentum in recent years. However, there is limited research into the production of cultivated seafood. Here, we investigated the ability of fish mackerel cells (Scomber scombrus) to adhere to plant, algal and fungal-based biomaterial scaffolds, aiming to optimize the cultivation of fish cells for use in cellular agriculture. A mackerel cell line was utilized, and metabolic assays and confocal imaging were utilized to track cell adhesion, growth, and differentiation on the different biomaterials. The mackerel cells adhered and grew on gelatin (positive control), zein, and soy proteins, as well as on alginate, chitosan, and cellulose polysaccharides. The highest adhesion and growth were on the zein and chitosan substrates, apart from the gelatin control. These findings provide a blueprint to enhance scaffold selection and design, contributing to the broader field of cellular agriculture through the development of scalable and eco-conscious solutions for meeting the growing global demand for seafood.
Asunto(s)
Materiales Biocompatibles , Adhesión Celular , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Gelatina/química , Quitosano/química , Hongos/metabolismo , Hongos/química , Plantas/química , Plantas/metabolismo , Plantas/microbiología , Línea Celular , Alginatos/química , Peces , Andamios del Tejido/químicaRESUMEN
Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.
Asunto(s)
NAD , Mononucleótido de Nicotinamida , Humanos , NAD/metabolismo , Mononucleótido de Nicotinamida/metabolismo , Neuronas/metabolismo , Mitocondrias/metabolismo , Autofagia , Niacinamida/metabolismoRESUMEN
Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.
Asunto(s)
Adrenérgicos , Neuroblastoma , Humanos , Linaje de la Célula/genética , Inhibidores de Puntos de Control Inmunológico , Neuroblastoma/genética , Citocinas/genética , FenotipoRESUMEN
Autophagy is an evolutionarily conserved catabolic pathway for the degradation of cytoplasmic constituents in eukaryotic cells. It is the primary disposal route for selective removal of undesirable cellular materials like aggregation-prone proteins and damaged organelles for maintaining cellular homeostasis, and for bulk degradation of intracellular macromolecules and recycling the breakdown products for providing energy homeostasis during starvation. These functions of autophagy are attributed to cellular survival and thus pertinent for human health; however, malfunction of this process is detrimental to the cells, particularly for post-mitotic neurons. Thus, basal autophagy is vital for maintaining neuronal homeostasis, whereas autophagy dysfunction contributes to neurodegeneration. Defective autophagy has been demonstrated in several neurodegenerative diseases wherein pharmacological induction of autophagy is beneficial in many of these disease models. Elucidating the mechanisms underlying defective autophagy is imperative for the development of therapies targeting this process. Disease-affected human neuronal cells can be established from patient-derived human induced pluripotent stem cells (hiPSCs) that provide a clinically relevant platform for studying disease mechanisms and drug discovery. Thus, modeling autophagy dysfunction as a phenotypic readout in patient-derived neurons provides a more direct platform for investigating the mechanisms underlying defective autophagy and evaluating the therapeutic efficacy of autophagy inducers. Toward this, several hiPSC-derived neuronal cell models of neurodegenerative diseases have been employed. In this review, we highlight the key methodologies pertaining to hiPSC maintenance and neuronal differentiation, and studying autophagy at an endogenous level in hiPSC-derived neuronal cells.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Autofagia , Homeostasis , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismoRESUMEN
Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.
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Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario , Microscopía Intravital/métodos , Cresta Neural , Animales , Encéfalo/embriología , Encéfalo/fisiología , División Celular , Movimiento Celular , Quimera/embriología , Quimera/fisiología , Electroporación , Femenino , Técnicas de Transferencia de Gen , Ratones , Ratones Transgénicos , Neovascularización Fisiológica , Cresta Neural/irrigación sanguínea , Cresta Neural/citología , Cresta Neural/embriología , Placenta/fisiología , Embarazo , Retina/embriología , Retina/fisiología , Transmisión Sináptica , ÚteroRESUMEN
Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.
Asunto(s)
Cresta Neural , Neuroblastoma , Animales , Niño , Quimera , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis, and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases, including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers, as well as the nature of the autophagy defects, may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell types, such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modeling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation. It outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models.
Asunto(s)
Autofagia , Diferenciación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Animales , HumanosRESUMEN
Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.
Asunto(s)
Diferenciación Celular/fisiología , Metilación de ADN/fisiología , Elementos de Facilitación Genéticos/fisiología , Células Madre Embrionarias de Ratones/metabolismo , Transcripción Genética/fisiología , Animales , Línea Celular , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Embrionarias de Ratones/citología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation.
Asunto(s)
Encéfalo/patología , Cinetocoros/patología , Microcefalia/genética , Microcefalia/patología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Empalme del ARN , Encéfalo/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Humanos , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , FenotipoRESUMEN
Chimeric mice have been generated by injecting pluripotent stem cells into morula-to-blastocyst stage mouse embryo or by introducing more mature cells into later stage embryos that correspond to the differentiation stage of the donor cells. It has not been rigorously tested, however, whether successful chimera formation requires the developmental stage of host embryo and donor cell to be matched. Here, we compared the success of chimera formation following injection of primary neural crest cells (NCCs) into blastocysts or of embryonic stem cells (ESCs) into E8.5 embryos (heterochronic injection) with that of injecting ESCs cells into the blastocyst or NCCs into the E8.5 embryos (isochronic injection). Chimera formation was efficient when donor and host were matched, but no functional chimeric contribution was found in heterochronic injections. This suggests that matching the developmental stage of donor cells with the host embryo is crucial for functional engraftment of donor cells into the developing embryo.
Asunto(s)
Quimera/metabolismo , Cresta Neural/citología , Animales , Apoptosis , Blastocisto/citología , Células Cultivadas , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Gastrulación , Inyecciones , Ratones Endogámicos C57BL , Cresta Neural/trasplante , Trasplante de Células Madre , Factores de TiempoRESUMEN
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Factor de Transcripción YY1/metabolismo , Animales , Factor de Unión a CCCTC/metabolismo , Células Madre Embrionarias/metabolismo , Humanos , RatonesRESUMEN
Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETß, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histona Acetiltransferasas/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Proliferación Celular , Supervivencia Celular/genética , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Placa Neural/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Isoformas de ProteínasRESUMEN
Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Movimiento Celular , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Recurrencia Local de Neoplasia/patología , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Recurrencia Local de Neoplasia/metabolismo , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.
Asunto(s)
Células Madre Pluripotentes/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular/genética , Línea Celular , Quimera/metabolismo , Cromosomas Humanos X/genética , Fase de Segmentación del Huevo/metabolismo , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , ADN Mitocondrial/metabolismo , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Impresión Genómica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Mórula/citología , Mórula/metabolismo , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W(sh)/W(sh)), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases.
Asunto(s)
Quimera/metabolismo , Embrión de Mamíferos/citología , Cresta Neural/citología , Pigmentación de la Piel , Negro o Afroamericano , Animales , Animales Recién Nacidos , Supervivencia Celular , Células Cultivadas , ADN/metabolismo , Fibroblastos/citología , Gastrulación , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones Endogámicos C57BL , Microinyecciones , Reacción en Cadena de la Polimerasa , Ratas , Especificidad de la Especie , Donantes de TejidosRESUMEN
Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.
Asunto(s)
Reprogramación Celular , Células Epiteliales/citología , Fibroblastos/citología , Epitelio Pigmentado de la Retina/citología , Factores de Transcripción/genética , Línea Celular , Simulación por Computador , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Disautonomía Familiar/genética , Células Madre Embrionarias Humanas/patología , Neuronas/metabolismo , Sistema Nervioso Periférico/patología , Vesículas Sinápticas/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Proteínas Portadoras/genética , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Disautonomía Familiar/metabolismo , Disautonomía Familiar/patología , Feto , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Cinetina/farmacología , Masculino , Mutación , Cresta Neural/efectos de los fármacos , Cresta Neural/patología , Neuronas/efectos de los fármacos , Sistema Nervioso Periférico/efectos de los fármacos , Fenotipo , Vesículas Sinápticas/efectos de los fármacos , Factores de Elongación TranscripcionalRESUMEN
Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-ß-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-ß-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.
Asunto(s)
Autofagia , Glicoproteínas de Membrana/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Animales , Células Cultivadas , Colesterol/deficiencia , Colesterol/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Ratas , Proteínas SNARE/metabolismo , beta-Ciclodextrinas/farmacologíaRESUMEN
The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3-4 x 2 x 1 mm(3). A total of 40-60% of these cells was positive for Oct4, Sox2, and Nanog and 60-80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis-cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue.
Asunto(s)
Células Madre Adultas/citología , Células Madre Mesenquimatosas/citología , Mucosa Bucal/citología , Adulto , Células Madre Adultas/clasificación , Células Madre Adultas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/clasificación , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones SCID , Persona de Mediana Edad , Mucosa Bucal/fisiología , Membrana Mucosa/citología , Membrana Mucosa/fisiología , Adulto JovenRESUMEN
Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.