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1.
Nature ; 545(7653): 229-233, 2017 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-28445466

RESUMEN

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.


Asunto(s)
Genes Dominantes/genética , Genes p53 , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Selección Genética , Proteína p53 Supresora de Tumor/genética , Alelos , Recuento de Células , Diferenciación Celular/genética , División Celular/genética , Línea Celular , ADN/metabolismo , Análisis Mutacional de ADN , Exoma/genética , Células Madre Embrionarias Humanas/citología , Humanos , Pérdida de Heterocigocidad/genética , Mosaicismo , Neoplasias/genética , Dominios Proteicos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo
2.
Proc Natl Acad Sci U S A ; 109(41): 16498-503, 2012 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-23019579

RESUMEN

The coatomer module of the nuclear pore complex borders the cylinder-like nuclear pore-membrane domain of the nuclear envelope. In evolution, a single coatomer module increases in size from hetero-heptamer (Saccharomyces cerevisiae) to hetero-octamer (Schizosaccharomyces pombe) to hetero-nonamer (Metazoa). Notably, the heptamer-octamer transition proceeds through the acquisition of the nucleoporin Nup37. How Nup37 contacts the heptamer remained unknown. Using recombinant nucleoporins, we show that Sp-Nup37 specifically binds the Sp-Nup120 member of the hetero-heptamer but does not bind an Sc-Nup120 homolog. To elucidate the Nup37-Nup120 interaction at the atomic level, we carried out crystallographic analyses of Sp-Nup37 alone and in a complex with an N-terminal, ~110-kDa fragment of Sp-Nup120 comprising residues 1-950. Corroborating structural predictions, we determined that Nup37 folds into a seven-bladed ß-propeller. Several disordered surface regions of the Nup37 ß-propeller assume structure when bound to Sp-Nup120. The N-terminal domain of Sp-Nup120(1-950) also folds into a seven-bladed propeller with a markedly protruding 6D-7A insert and is followed by a contorted helical domain. Conspicuously, this 6D-7A insert contains an extension of 50 residues which also is highly conserved in Metazoa but is absent in Sc-Nup120. Strikingly, numerous contacts with the Nup37 ß-propeller are located on this extension of the 6D-7A insert. Another contact region is situated toward the end of the helical region of Sp-Nup120(1-950). Our findings provide information about the evolution and the assembly of the coatomer module of the nuclear pore complex.


Asunto(s)
Membrana Nuclear/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Schizosaccharomyces pombe/química , Secuencia de Aminoácidos , Evolución Biológica , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Aminoácido
3.
Nat Aging ; 4(7): 984-997, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38907103

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by a progressive loss of motor function linked to degenerating extratelencephalic neurons/Betz cells (ETNs). The reasons why these neurons are selectively affected remain unclear. Here, to understand the unique molecular properties that may sensitize ETNs to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In both patients and unaffected individuals, we found significantly higher expression of ALS risk genes in THY1+ ETNs, regardless of diagnosis. In patients, this was accompanied by the induction of genes involved in protein homeostasis and stress responses that were significantly induced in a wide collection of ETNs. Examination of oligodendroglial and microglial nuclei revealed patient-specific downregulation of myelinating genes in oligodendrocytes and upregulation of an endolysosomal reactive state in microglia. Our findings suggest that selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitizing them to genetics and mechanisms of degeneration.


Asunto(s)
Esclerosis Amiotrófica Lateral , Neuronas , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Neuronas/metabolismo , Neuronas/patología , Factores de Riesgo , Microglía/metabolismo , Microglía/patología , Núcleo Celular/metabolismo , Núcleo Celular/genética , Oligodendroglía/metabolismo , Oligodendroglía/patología , Masculino , Análisis de la Célula Individual , Análisis de Secuencia de ARN , Femenino , Persona de Mediana Edad , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Degeneración Nerviosa/metabolismo
4.
Cell Rep ; 42(1): 111988, 2023 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-36640364

RESUMEN

The maturation of neurons and the development of synapses, although emblematic of neurons, also relies on interactions with astrocytes and other glia. Here, to study the role of glia-neuron interactions, we analyze the transcriptomes of human pluripotent stem cell (hPSC)-derived neurons, from 80 human donors, that were cultured with or without contact with glial cells. We find that the presence of astrocytes enhances synaptic gene-expression programs in neurons when in physical contact with astrocytes. These changes in neurons correlate with increased expression, in the cocultured glia, of genes that encode synaptic cell adhesion molecules. Both the neuronal and astrocyte gene-expression programs are enriched for genes associated with schizophrenia risk. Our results suggest that astrocyte-expressed genes with synaptic functions are associated with stronger expression of synaptic genetic programs in neurons, and they suggest a potential role for astrocyte-neuron interactions in schizophrenia.


Asunto(s)
Astrocitos , Esquizofrenia , Humanos , Astrocitos/metabolismo , Adhesión Celular/genética , Esquizofrenia/genética , Esquizofrenia/metabolismo , Neuronas/metabolismo , Neuroglía , Sinapsis/fisiología
5.
Cell Rep ; 42(1): 111896, 2023 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-36596304

RESUMEN

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.


Asunto(s)
Señales (Psicología) , Células Madre Pluripotentes Inducidas , Humanos , Diferenciación Celular , Neuronas Motoras/metabolismo , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Homeodominio/metabolismo
6.
Cell Stem Cell ; 30(3): 312-332.e13, 2023 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-36796362

RESUMEN

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes.


Asunto(s)
Células-Madre Neurales , Infección por el Virus Zika , Virus Zika , Humanos , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Encéfalo/metabolismo , Virus Zika/metabolismo , Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo
7.
J Cell Biol ; 191(3): 505-21, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20974814

RESUMEN

Nuclear pore complexes (NPCs) control the movement of molecules across the nuclear envelope (NE). We investigated the molecular interactions that exist at the interface between the NPC scaffold and the pore membrane. We show that key players mediating these interactions in mammalian cells are the nucleoporins Nup155 and Nup160. Nup155 depletion massively alters NE structure, causing a dramatic decrease in NPC numbers and the improper targeting of membrane proteins to the inner nuclear membrane. The role of Nup155 in assembly is likely closely linked to events at the membrane as we show that Nup155 interacts with pore membrane proteins Pom121 and NDC1. Furthermore, we demonstrate that the N terminus of Pom121 directly binds the ß-propeller regions of Nup155 and Nup160. We propose a model in which the interactions of Pom121 with Nup155 and Nup160 are predicted to assist in the formation of the nuclear pore and the anchoring of the NPC to the pore membrane.


Asunto(s)
Glicoproteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Humanos , Modelos Biológicos , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo
8.
Am J Physiol Endocrinol Metab ; 291(5): E922-8, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16757545

RESUMEN

Oxytocin (OT) is a potent uterine agonist. Its receptor (OTR) is a G protein-coupled receptor that is downregulated by prolonged exposure to OT. We hypothesized that activation of PKC mediated this OT-induced decrease in OTR expression. Diminished PKC activity in late pregnancy could underlie the increased expression of uterine OTR preceding labor onset. Using cell cultures of transformed human uterine myocytes, we determined the effects of PKC agonists and antagonists on the expression of OTR. We also explored the effects of overexpression of activator protein-1 (AP-1, a mediator of many PKC- and phorbol ester-induced effects) using adenoviral expression vectors for the AP-1 subunits c-Jun and c-Fos. Stimulation of PKC using the phorbol ester 12-O-tetradecanoylphorbol 13-acetate caused a rapid, significant (P < or = 0.05) increase in c-Jun and c-Fos concentrations but a significant decrease in mRNA for OTR within 6 h followed by a significant decrease in OT binding by 24 h. Adenoviral infection of the cells with expression vectors for c-Jun and c-Fos increased the AP-1 subunits but had no effect on OTR expression. Furthermore, there were no changes in c-Fos or c-Jun levels in human intrauterine tissues around the time of labor onset, as measured by Western analyses. We conclude that phorbol ester treatment decreases OTR expression, likely through a mechanism that does not involve AP-1.


Asunto(s)
Carcinógenos/farmacología , Miometrio/efectos de los fármacos , Miometrio/fisiología , Receptores de Oxitocina/genética , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo , Proteínas Sanguíneas/farmacología , Línea Celular Transformada , Femenino , Humanos , Miometrio/citología , Parto/fisiología , Embarazo , Tercer Trimestre del Embarazo/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Transcripción AP-1/genética
9.
Am J Physiol Endocrinol Metab ; 289(4): E658-64, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15870103

RESUMEN

Increased concentrations of IL-1beta and TNF-alpha have been associated with parturition. However, the role of these cytokines is unknown. Before parturition, the uterus undergoes a process of activation, during which there are significant changes in expression of genes associated with increased uterine contractility, including the receptors for oxytocin (OT) and prostaglandin (PG)F(2alpha) (FP), PGH(2) synthase isoform 2 (PGHS2), the gap junction protein connexin-43 (Cx-43), and the inducible isoform of nitric oxide synthase (iNOS). To determine whether IL-1beta or TNF-alpha was part of the causal mechanism for increased uterine contractions, we placed osmotic pumps infusing IL-1beta or TNF-alpha into the peritoneal cavity of late pregnant rats (gestation day 19) and measured the effects on uterine contractility and on the uterine concentrations of mRNA for the contraction-associated genes 24 h later. Maternal serum concentrations of IL-1beta and TNF-alpha were increased significantly. By day 21, the control animals had significant increases (P < or = 0.05) in mRNA for OT, FP, PGHS2, and Cx-43, a decrease (P < or = 0.05) in iNOS, and an increase (P < or = 0.05) in uterine sensitivity and responsiveness to OT. Infusion of IL-1beta or TNF-alpha had no effect on uterine contractility or on expression of the activation-associated genes. We conclude that intraperitoneal infusion of IL-1beta or TNF-alpha resulting in significantly increased maternal serum cytokine levels does not cause uterine activation. The role of proinflammatory cytokines in the mechanism of parturition remains unclear.


Asunto(s)
Interleucina-1/sangre , Parto/fisiología , Preñez/fisiología , Embarazo/fisiología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismo , Contracción Uterina/fisiología , Animales , Citocinas/sangre , Femenino , Infusiones Intravenosas , Interleucina-1/administración & dosificación , Preñez/sangre , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/administración & dosificación
10.
J Physiol ; 565(Pt 1): 125-35, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15774515

RESUMEN

Compromised fetal growth impairs vascular function; however, it is unclear whether chronic hypoxia in utero affects adult endothelial function. We hypothesized that maternal hypoxia (H, 12% O2, n= 9) or nutrient restriction (NR, 40% of control, n= 7) imposed from day 15-21 pregnancy in rats would impair endothelial function in adult male offspring (relative to control, C, n= 10). Using a wire myograph, endothelium-dependent relaxation in response to methacholine was assessed in small mesenteric arteries from 4- and 7-month-old (mo) male offspring. Nitric oxide (NO) mediation of endothelium-dependent relaxation was evaluated using N(omega)-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor). Observed differences in the NO pathway at 7 months were investigated using exogenous superoxide dismutase (SOD) to reduce NO scavenging, and sodium nitroprusside (SNP; NO donor) to assess smooth muscle sensitivity to NO. Sensitivity to methacholine-induced endothelium-dependent relaxation was reduced in H offspring at 4 months (P < 0.05), but was not different among groups at 7 months. L-NAME reduced methacholine sensitivity in C (P < 0.01), H (P < 0.01) and NR (P < 0.05) offspring at 4 months, but at 7 months L-NAME reduced sensitivity in C (P < 0.05), tended to in NR (P= 0.055) but had no effect in H offspring. SOD did not alter sensitivity to methacholine in C, but increased sensitivity in H offspring (P < 0.01). SNP responses did not differ among groups. In summary, prenatal hypoxia, but not nutrient restriction impaired endothelium-dependent relaxation at 4 months, and reduced NO mediation of endothelial function at 7 months, in part through reduced NO bio-availability. Distinct effects following reduced maternal oxygen versus nutrition suggest that decreased oxygen supply during fetal life may specifically impact adult vascular function.


Asunto(s)
Envejecimiento/metabolismo , Endotelio Vascular/metabolismo , Privación de Alimentos , Hipoxia/fisiopatología , Efectos Tardíos de la Exposición Prenatal , Animales , Peso Corporal/fisiología , Femenino , Hipoxia/embriología , Masculino , Intercambio Materno-Fetal , Óxido Nítrico Sintasa/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley
11.
Am J Obstet Gynecol ; 192(4): 1304-13; discussion 1313-5, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15846226

RESUMEN

OBJECTIVE: The purpose of this study was to determine the role of 5beta-dihydroprogesterone (5beta-DHP), acting through the nuclear receptor pregnane X receptor (PXR), in regulating uterine contractility. STUDY DESIGN: Uterine contractility was studied in tissues from women, rats, and mice. Messenger RNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR), and protein was measured using enzyme assays, immunofluorescence microscopy, and Western analyses. RESULTS: Human and rat uterine tissues contain mRNA and protein for 5beta-reductase and for PXR. Acute in vitro treatment with 5beta-DHP causes rapid uterine relaxation that is not mediated by PXR. Chronic in vivo administration of 5beta-DHP to mice with intact PXR, but not in mice with disrupted PXR, causes an increased effect of 1400W, a specific inhibitor of inducible nitric oxide synthase (iNOS). This suggests that 5beta-DHP increased iNOS-modulated uterine tone, as occurs during pregnancy. CONCLUSION: These data support the hypothesis that metabolites of progesterone may act chronically through a PXR-mediated mechanism to regulate uterine contractility.


Asunto(s)
Progesterona/metabolismo , Progesterona/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de Esteroides/efectos de los fármacos , Contracción Uterina/efectos de los fármacos , Análisis de Varianza , Animales , Secuencia de Bases , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Ratones Noqueados , Microscopía Fluorescente , Modelos Animales , Datos de Secuencia Molecular , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Técnicas de Cultivo de Órganos , Embarazo , Preñez , Receptor X de Pregnano , Probabilidad , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Regulación hacia Arriba , Contracción Uterina/fisiología
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