RESUMEN
Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.
Asunto(s)
Blastocisto/citología , Línea Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Pluripotentes/citología , Animales , Proteína Morfogenética Ósea 4/metabolismo , Cadherinas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Embrión de Mamíferos/citología , Estratos Germinativos/citología , RatonesRESUMEN
Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.
Asunto(s)
Degeneración Macular , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Proteínas del Sistema Complemento/metabolismo , Coroides/metabolismo , Proteínas/genética , Genómica , Polimorfismo de Nucleótido Simple , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/genéticaRESUMEN
Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.
Asunto(s)
Corteza Cerebral/metabolismo , Perfilación de la Expresión Génica/métodos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Retina/metabolismo , Transcriptoma , Animales , Linaje de la Célula/genética , Corteza Cerebral/citología , Criopreservación/métodos , Femenino , Congelación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas del Tejido Nervioso/clasificación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/clasificación , Neuronas/citología , Retina/citología , Análisis de la Célula Individual/métodos , Bancos de TejidosRESUMEN
Tau is an axonal microtubule-binding protein. Tau pathology in brain and increased tau concentration in the cerebrospinal fluid (CSF) are hallmarks of Alzheimer's disease (AD). Most of tau in CSF is present as fragments. We immunoprecipitated tau from CSF and identified several endogenous peptides ending at amino acid (aa) 123 or 224 using high-resolution mass spectrometry. We raised neo-epitope-specific antibodies against tau fragments specifically ending at aa 123 and 224, respectively. With these antibodies, we performed immunohistochemistry on brain tissue and designed immunoassays measuring N-123, N-224, and x-224 tau. Immunoassays were applied to soluble brain fractions from pathologically confirmed subjects (81 AD patients, 33 controls), CSF from three cross-sectional and two longitudinal cohorts (a total of 133 AD, 38 MCI, 20 MCI-AD, 31 PSP, 15 CBS patients, and 91 controls), and neuronally- and peripherally-derived extracellular vesicles (NDEVs and PDEVs, respectively) in serum from four AD patients and four controls. Anti-tau 224 antibody stained neurofibrillary tangles and neuropil threads, while anti-tau 123 only showed weak cytoplasmic staining in AD. N-224 tau was lower in the AD soluble brain fraction compared to controls, while N-123 tau showed similar levels. N-224 tau was higher in AD compared to controls in all CSF cohorts (p < 0.001), but not N-123 tau. Decrease in cognitive performance and conversion from MCI to AD were associated with increased baseline CSF levels of N-224 tau (p < 0.0001). N-224 tau concentrations in PSP and CBS were significantly lower than in AD (p < 0.0001) and did not correlate to t-tau and p-tau. In a longitudinal cohort, CSF N-224 tau levels were stable over 6 months, with no significant effect of treatment with AChE inhibitors. N-224 tau was present in NDEVs, while N-123 tau showed comparable concentrations in both vesicle types. We suggest that N-123 tau is produced both in CNS and PNS and represents a general marker of tau metabolism, while N-224 tau is neuron-specific, present in the tangles, secreted in CSF, and upregulated in AD, suggesting a link between tau cleavage and propagation, tangle pathology, and cognitive decline.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Disfunción Cognitiva/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Biomarcadores/líquido cefalorraquídeo , Encéfalo/metabolismo , Encéfalo/patología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Estudios de Cohortes , Femenino , Humanos , Masculino , Proteínas tau/metabolismoRESUMEN
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3ß and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/fisiología , Células Madre Pluripotentes/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Reprogramación Celular , Citosina/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Estratos Germinativos/fisiología , Ratones , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , TranscriptomaRESUMEN
Transcriptome profiling at single-cell resolution allows us to identify and assess functional cell types and cellular states, including those within degenerating ocular tissues in retinitis pigmentosa. The technology is particularly valuable when studying tissues with high cellular heterogeneity, or when specific cell types are of interest. In this chapter, we introduce a detailed protocol of a medium-throughput single-nucleus RNA sequencing technique that utilizes frozen tissue as input sample. This protocol can be executed by any researcher with basic training in molecular biology techniques. With this protocol, a single experimenter can easily process two samples per day up to cDNA amplification, and library preparations can be done in batches of 8. Routinely we can obtain ~20 K nuclei per eye from 3 to 4 library preparations.
Asunto(s)
ARN , Análisis de Secuencia de ARNRESUMEN
Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n = 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223. Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2, as mechanistic players in disease.
RESUMEN
Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases.
Asunto(s)
Susceptibilidad a Enfermedades/metabolismo , Regulación de la Expresión Génica/genética , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Anciano , Anciano de 80 o más Años , Alelos , Coroides/metabolismo , Bases de Datos Genéticas , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Degeneración Macular/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , RNA-Seq , Factores de Riesgo , Análisis de la Célula Individual , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismo , Transcriptoma/genéticaRESUMEN
The aggregation of intracellular tau protein is a major hallmark of Alzheimer's disease (AD). The extent and the stereotypical spread of tau pathology in the AD brain are correlated with cognitive decline during disease progression. Here we present an in-depth analysis of endogenous tau fragmentation in a well-characterized cohort of AD and age-matched control subjects. Using protein mass spectrometry and Edman degradation to interrogate endogenous tau fragments in the human brain, we identified two novel proteolytic sites, G323 and G326, as major tau cleavage events in both normal and AD cortex. These sites are located within the sequence recently identified as the structural core of tau protofilaments, suggesting an inhibitory mechanism of fibril formation. In contrast, a different set of novel cleavages showed a distinct increase in late stage AD. These disease-associated sites are located outside of the protofilament core sequence. We demonstrate that calpain 1 specifically cleaves at both the normal and diseased sites in vitro, and the site selection is conformation-dependent. Monomeric tau is predominantly cleaved at G323/G326 (normal sites), whereas oligomerization increases cleavages at the late-AD-associated sites. The fragmentation patterns specific to disease and healthy states suggest novel regulatory mechanisms of tau aggregation in the human brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Calpaína/metabolismo , Progresión de la Enfermedad , Proteínas tau/química , Proteínas tau/metabolismo , Anciano de 80 o más Años , Encéfalo/metabolismo , Femenino , Humanos , Masculino , ProteolisisRESUMEN
Neuronal development requires a complex choreography of transcriptional decisions to obtain specific cellular identities. Realizing the ultimate goal of identifying genome-wide signatures that define and drive specific neuronal fates has been hampered by enormous complexity in both time and space during development. Here, we have paired high-throughput purification of pyramidal neuron subclasses with deep profiling of spatiotemporal transcriptional dynamics during corticogenesis to resolve lineage choice decisions. We identified numerous features ranging from spatial and temporal usage of alternative mRNA isoforms and promoters to a host of mRNA genes modulated during fate specification. Notably, we uncovered numerous long noncoding RNAs with restricted temporal and cell-type-specific expression. To facilitate future exploration, we provide an interactive online database to enable multidimensional data mining and dissemination. This multifaceted study generates a powerful resource and informs understanding of the transcriptional regulation underlying pyramidal neuron diversity in the neocortex.
Asunto(s)
Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Neocórtex/metabolismo , Células Piramidales/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , Transcriptoma , Animales , Secuencia de Bases , Cuerpo Calloso/citología , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas Motoras , Neurogénesis/genética , Neuronas/metabolismo , Tractos Piramidales/citología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Myelin is a defining feature of the vertebrate nervous system. Variability in the thickness of the myelin envelope is a structural feature affecting the conduction of neuronal signals. Conversely, the distribution of myelinated tracts along the length of axons has been assumed to be uniform. Here, we traced high-throughput electron microscopy reconstructions of single axons of pyramidal neurons in the mouse neocortex and built high-resolution maps of myelination. We find that individual neurons have distinct longitudinal distribution of myelin. Neurons in the superficial layers displayed the most diversified profiles, including a new pattern where myelinated segments are interspersed with long, unmyelinated tracts. Our data indicate that the profile of longitudinal distribution of myelin is an integral feature of neuronal identity and may have evolved as a strategy to modulate long-distance communication in the neocortex.
Asunto(s)
Vaina de Mielina/fisiología , Neocórtex/citología , Células Piramidales/fisiología , Corteza Somatosensorial/citología , Corteza Visual/citología , Animales , Axones/fisiología , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Neocórtex/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Células Piramidales/citología , Corteza Somatosensorial/fisiología , Corteza Visual/fisiologíaRESUMEN
The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype-specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neurotransmisores/genética , Tractos Piramidales/metabolismo , Transducción de Señal/genética , Animales , Proteínas de Unión al ADN/genética , Genes Supresores , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas Motoras/clasificación , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/genética , Neurotransmisores/biosíntesis , Neurotransmisores/metabolismo , Regiones Promotoras Genéticas/genética , Tractos Piramidales/citología , Tractos Piramidales/fisiologíaRESUMEN
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
Asunto(s)
Apoptosis/genética , Diferenciación Celular/genética , Embrión de Mamíferos/metabolismo , Células Germinativas/metabolismo , Proteínas de Unión al ARN/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
L3mbtl2 has been implicated in transcriptional repression and chromatin compaction but its biological function has not been defined. Here we show that disruption of L3mbtl2 results in embryonic lethality with failure of gastrulation. This correlates with compromised proliferation and abnormal differentiation of L3mbtl2(-/-) embryonic stem (ES) cells. L3mbtl2 regulates genes by recruiting a Polycomb Repressive Complex1 (PRC1)-related complex, resembling the previously described E2F6-complex, and including G9A, Hdac1, and Ring1b. The presence of L3mbtl2 at target genes is associated with H3K9 dimethylation, low histone acetylation, and H2AK119 ubiquitination, but the latter is neither dependent on L3mbtl2 nor sufficient for repression. Genome-wide studies revealed that the L3mbtl2-dependent complex predominantly regulates genes not bound by canonical PRC1 and PRC2. However, some developmental regulators are repressed by the combined activity of all three complexes. Together, we have uncovered a highly selective, essential role for an atypical PRC1-family complex in ES cells and early development.
Asunto(s)
Desarrollo Embrionario , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Ensamble y Desensamble de Cromatina/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Genoma/genética , Ratones , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 2 , Unión Proteica/genética , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Transcripción Genética , Proteínas Supresoras de Tumor/química , Dedos de ZincRESUMEN
Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine ESCs. We termed these hLR5 iPSCs because they require the expression of five ectopic reprogramming factors, Oct4, Sox2, Klf4, cMyc, and Nanog, to maintain this more naive state. The cells are "metastable" and upon ectopic factor withdrawal they revert to standard human iPSCs. Finally, we demonstrate that the hLR5 state facilitates gene targeting, and as such provides a powerful tool for the generation of recombinant human pluripotent stem cell lines.
Asunto(s)
Células Madre Embrionarias/metabolismo , Técnicas de Transferencia de Gen , Células Madre Pluripotentes Inducidas/metabolismo , Factor Inhibidor de Leucemia/farmacología , Factores de Transcripción/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Desdiferenciación Celular/efectos de los fármacos , Desdiferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/patología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Terapia Genética/métodos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Ratones , Recombinación Genética/genética , Homología de Secuencia , Factores de Transcripción/genéticaRESUMEN
Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.
Asunto(s)
Técnicas de Cultivo de Embriones , Células Madre Embrionarias/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Animales , Técnicas de Cultivo de Célula , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Transgénicos , Retroviridae/genética , Especificidad de la Especie , Teratoma/genética , Cromosoma XRESUMEN
Dual specificity phosphatases (DSPs) are members of the protein-tyrosine phosphatase superfamily that dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Many DSPs have been found to play important roles in various aspects of cellular function and to be involved in human disease. We have identified a gene located on human chromosome 10q22.2, which utilizes alternative open reading frames (ORFs) to encode the following two distinct DSPs: the previously described testis and skeletal muscle-specific dual specificity phosphatase (TMDP) and a novel DSP, muscle-restricted dual specificity phosphatase (MDSP). Use of alternative ORFs encoding distinct proteins from a single gene is extremely rare in eukaryotes, and in all previously reported cases the two proteins produced from one gene are unrelated. To our knowledge this is the first example of a gene from which two distinct proteins of the same family are expressed using alternative ORFs. Here we provide evidence that both MDSP and TMDP proteins are expressed in vivo and are restricted to specific tissues, skeletal muscle and testis, respectively. Most interestingly, the protein expression profiles of both MDSP and TMDP during mouse postnatal development are strikingly similar. MDSP is expressed at very low levels in myotubes and early postnatal muscle. TMDP is not detectable in testis lysate in the first 3 weeks of life. The expression of both MDSP and TMDP proteins was markedly increased at approximately the 3rd week after birth and continued to increase gradually into adulthood, implying that the physiological functions of both DSPs are specific to the mature/late-developing organs. The conserved gene structure and the similarity in postnatal expression profile of these two proteins suggest biological significance of the unusual gene arrangement.
Asunto(s)
Cromosomas Humanos Par 10 , Genes , Sistemas de Lectura Abierta , Fosfoproteínas Fosfatasas/genética , Proteínas Tirosina Fosfatasas/genética , Animales , Secuencia de Bases , Fosfatasas de Especificidad Dual , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Especificidad de Órganos , Fosfoproteínas Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Testículo/enzimología , Distribución TisularRESUMEN
Free cholesterol (FC) has been reported to efflux from cells through caveolae, which are 50-100 nm plasma membrane pits. The 22 kDa protein caveolin-1 is concentrated in caveolae and is required for their formation. The HDL scavenger receptor BI (SR-BI), which stimulates both FC efflux and selective uptake of HDL-derived cholesteryl ester (CE), has been reported to be concentrated in caveolae, suggesting that this localization facilitates flux of FC and CE across the membrane. However, we found that overexpression of caveolin-1 in Fischer rat thyroid (FRT) cells, which lack caveolin-1 and caveolae, or HEK 293 cells, which normally express very low levels of caveolin-1, did not affect FC efflux to HDL or liposomes. Transient expression of SR-B1 did not affect this result. Similarly, caveolin-1 expression did not affect selective uptake of CE from labeled HDL particles in FRT or HEK 293 cells transfected with SR-BI. We conclude that basal and SR-BI-stimulated FC efflux to HDL and liposomes and SR-BI-mediated selective uptake of HDL CE are not affected by caveolin-1 expression in HEK 293 or FRT cells.