RESUMEN
Recent advances in cell reprogramming showed that OSKM induction is able to improve cell physiology in vitro and in vivo. Here, we show that a single short reprogramming induction is sufficient to prevent musculoskeletal functions deterioration of mice, when applied in early life. In addition, in old age, treated mice have improved tissue structures in kidney, spleen, skin, and lung, with an increased lifespan of 15% associated with organ-specific differential age-related DNA methylation signatures rejuvenated by the treatment. Altogether, our results indicate that a single short reprogramming early in life might initiate and propagate an epigenetically related mechanism to promote a healthy lifespan.
Asunto(s)
Reprogramación Celular , Longevidad , Ratones , Animales , Longevidad/genética , Reprogramación Celular/genética , Estado de SaludRESUMEN
Cell therapy approaches to treat a wide range of pathologies have greatly benefited from cell reprogramming techniques that allow the conversion of a somatic cell into a pluripotent cell. Many technological developments have been made since the initial major discovery of this biological process. Recently reprogramming methods based on the use of RNA have emerged and seem very promising. Thus, in this review we will focus on presenting the interest of such methods for cell reprogramming but also how these RNA-based strategies can be extended to eventually lead to medical applications to improve healthspan and longevity.
RESUMEN
Aging is associated with a progressive and functional decline of all tissues and a striking increase in many "age-related diseases". Although aging has long been considered an inevitable process, strategies to delay and potentially even reverse the aging process have recently been developed. Here, we review emerging rejuvenation strategies that are based on reprogramming toward pluripotency. Some of these approaches may eventually lead to medical applications to improve healthspan and longevity.
Asunto(s)
Reprogramación Celular , Senescencia Celular , Rejuvenecimiento , Envejecimiento/fisiología , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Medicina RegenerativaRESUMEN
In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquito-borne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.
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Astrocitos/virología , Virus de la Encefalitis Japonesa (Subgrupo)/fisiología , Células-Madre Neurales/virología , Neuroglía/virología , Neuronas/virología , Tropismo Viral , Animales , Astrocitos/fisiología , Encéfalo/virología , Células Cultivadas , Virus de la Encefalitis Japonesa (Subgrupo)/crecimiento & desarrollo , Ratones , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Virus Zika/crecimiento & desarrollo , Virus Zika/fisiologíaRESUMEN
Cellular senescence plays an important role in the process of aging and is often associated with age-related diseases. Senescence was originally considered as a barrier to cell reprogramming, however we developed a strategy to overcome this hurdle and derive induced pluripotent stem cells (iPSCs) from senescent cells and cells from centenarians. Furthermore we showed that the newly generated iPSCs could be re-differentiated into fully rejuvenated cells. That has increased the known beneficial properties of iPSCs to include them as a tool to model age-related diseases or even to cure them through cell therapy. In this review, we describe the hallmarks of cellular senescence before presenting how we reprogrammed aged and senescent cells into iPSCs and obtained rejuvenated re-differentiated cells. Finally, we take an interest in the way iPSCs can be used to understand and cure age-related diseases and we present their advantages for patient-specific therapy.
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Envejecimiento/metabolismo , Senescencia Celular , Células Madre Pluripotentes Inducidas/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/patología , Animales , Desdiferenciación Celular , Proliferación Celular , Reprogramación Celular , Senescencia Celular/genética , Susceptibilidad a Enfermedades , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/trasplante , Esperanza de Vida , Persona de Mediana Edad , Fenotipo , RejuvenecimientoRESUMEN
DNA replication is highly regulated, ensuring faithful inheritance of genetic information through each cell cycle. In metazoans, this process is initiated at many thousands of DNA replication origins whose cell type-specific distribution and usage are poorly understood. We exhaustively mapped the genome-wide location of replication origins in human cells using deep sequencing of short nascent strands and identified ten times more origin positions than we expected; most of these positions were conserved in four different human cell lines. Furthermore, we identified a consensus G-quadruplex-forming DNA motif that can predict the position of DNA replication origins in human cells, accounting for their distribution, usage efficiency and timing. Finally, we discovered a cell type-specific reprogrammable signature of cell identity that was revealed by specific efficiencies of conserved origin positions and not by the selection of cell type-specific subsets of origins.
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G-Cuádruplex , Origen de Réplica/genética , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Secuencia de Consenso , Cartilla de ADN/genética , Replicación del ADN/genética , Genoma Humano , Células HeLa , Humanos , Motivos de NucleótidosRESUMEN
Understanding the complex mechanisms by which infectious agents can disrupt behavior represents a major challenge. The Borna disease virus (BDV), a potential human pathogen, provides a unique model to study such mechanisms. Because BDV induces neurodegeneration in brain areas that are still undergoing maturation at the time of infection, we tested the hypothesis that BDV interferes with neurogenesis. We showed that human neural stem/progenitor cells are highly permissive to BDV, although infection does not alter their survival or undifferentiated phenotype. In contrast, upon the induction of differentiation, BDV is capable of severely impairing neurogenesis by interfering with the survival of newly generated neurons. Such impairment was specific to neurogenesis, since astrogliogenesis was unaltered. In conclusion, we demonstrate a new mechanism by which BDV might impair neural function and brain plasticity in infected individuals. These results may contribute to a better understanding of behavioral disorders associated with BDV infection.
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Enfermedad de Borna/fisiopatología , Virus de la Enfermedad de Borna/fisiología , Neurogénesis , Neuronas/citología , Células Madre/citología , Enfermedad de Borna/virología , Encéfalo/citología , Encéfalo/virología , Células Cultivadas , HumanosRESUMEN
Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.
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Diferenciación Celular , Reprogramación Celular , Senescencia Celular/genética , Células Madre Pluripotentes Inducidas/citología , Rejuvenecimiento , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/metabolismo , Telómero/genética , Telómero/metabolismo , Factores de Transcripción/farmacologíaRESUMEN
Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.
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Estratos Germinativos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Humanos , Células Madre Pluripotentes/fisiologíaRESUMEN
HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.
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Medios de Cultivo/química , HEPES/metabolismo , Priones/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Inhibidores Enzimáticos/metabolismo , Pliegue de Proteína/efectos de los fármacosRESUMEN
Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as α-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human α-synuclein, C57BL/6S Δsnca mice presenting a natural deletion of the α-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of α-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human α-synuclein in C57BL/6S Δsnca mice. This monoclonal antibody, recognizing an N-terminal α-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of α-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human α-synuclein, and cellular models expressing human α-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of α-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.
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Anticuerpos Monoclonales/biosíntesis , alfa-Sinucleína/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Línea Celular , Células Cultivadas , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , alfa-Sinucleína/genéticaRESUMEN
Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.
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Células Madre Embrionarias/química , Células Madre Mesenquimatosas/química , Proteínas/análisis , Proteómica/métodos , Tejido Adiposo/citología , Análisis de Varianza , Antígenos CD/metabolismo , Médula Ósea/química , Células Cultivadas , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Células Madre Embrionarias/metabolismo , Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Proteínas/aislamiento & purificación , Membrana Sinovial/citología , Venas Umbilicales/citologíaRESUMEN
BACKGROUND: ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed. RESULTS: Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of approximately 42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites. CONCLUSION: The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.
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Evolución Molecular , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Xenopus/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , ADN Complementario/metabolismo , Expresión Génica , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Células 3T3 NIH , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Testículo/metabolismo , Xenopus , Proteínas de Xenopus/análisis , Proteínas de Xenopus/metabolismoRESUMEN
The study of prion transmission and targeting is a major scientific issue with important consequences for public health. Only a few cell culture systems that are able to convert the cellular isoform of the prion protein into the pathologic scrapie isoform of the prion protein (PrP(Sc)) have been described. We hypothesized that central nervous system neural stem cells (NSCs) could be the basis of a new cell culture model permissive to prion infection. Here, we report that monolayers of differentiated fetal NSCs and adult multipotent progenitor cells isolated from mice were able to propagate prions. We also demonstrated the large influence of neural cell fate on the production of PrP(Sc), allowing the molecular study of prion neuronal targeting in relation with strain differences. This new stem cell-based model, which is applicable to different species and to transgenic mice, will allow thoughtful investigations of the molecular basis of prion diseases, and will open new avenues for diagnostic and therapeutic research.
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Neuronas/citología , Priones/metabolismo , Células Madre/citología , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Cultivadas , Corteza Cerebral , Modelos Animales de Enfermedad , Femenino , Feto , Ratones , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Embarazo , Enfermedades por Prión/metabolismo , Células Madre/metabolismoRESUMEN
The activation of the transcription factor nuclear factor-kappaB (NF-kappaB) by growth factors, cytokines, and cellular stress can prevent apoptosis, but the underlying mechanism is unknown. Here we provide evidence for an action of NF-kappaB on calcium signaling that accounts for its anti-apoptotic function. Embryonic fibroblasts lacking the transactivating subunit of NF-kappaB RelA (p65) exhibit enhanced inositol 1,4,5-trisphosphate (IP(3)) receptor-mediated calcium release and increased sensitivity to apoptosis, which are restored upon re-expression of RelA. The size of the endoplasmic reticulum (ER) calcium pool and the number of IP(3) receptors per cell are decreased in response to stimuli that activate NF-kappaB and are increased when NF-kappaB activity is suppressed. The selective antagonism of IP(3) receptors blocks apoptosis in RelA-deficient cells, whereas activation of NF-kappaB in normal cells leads to decreased levels of the type 1 IP(3) receptor and decreased calcium release. Overexpression of Bcl-2 normalizes ER calcium homeostasis and prevents calcium-mediated apoptosis in RelA-deficient cells. These findings establish an ER calcium channel as a pivotal target for NF-kappaB-mediated cell survival signaling.
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Apoptosis , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , FN-kappa B/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Supervivencia Celular , Ceramidas/farmacología , Citosol/metabolismo , ADN/metabolismo , Retículo Endoplásmico/metabolismo , Immunoblotting , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato , Metabolismo de los Lípidos , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Microsomas/metabolismo , FN-kappa B/química , Oligonucleótidos Antisentido/química , Estrés Oxidativo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transducción de Señal , Factores de Tiempo , Factor de Transcripción ReIA , Activación TranscripcionalRESUMEN
First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complex that degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins--RNAi technology may eventually make that dream a reality.
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Quimioterapia , Farmacología/tendencias , Interferencia de ARN/fisiología , Tecnología Farmacéutica/métodos , Animales , Muerte Celular , Humanos , Neoplasias/terapia , Interferencia de ARN/efectos de los fármacos , Transducción de Señal , Tecnología Farmacéutica/tendenciasRESUMEN
Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.