Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 41
Filtrar
Más filtros

Banco de datos
Tipo del documento
País de afiliación
Intervalo de año de publicación
1.
Mol Cell ; 58(6): 1001-14, 2015 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-26004228

RESUMEN

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.


Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Secuencia de Aminoácidos , Animales , Factor Inductor de la Apoptosis/genética , Línea Celular Tumoral , Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Humanos , Immunoblotting , Ratones Noqueados , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas/genética , Interferencia de ARN , Factores de Tiempo
2.
Dev Biol ; 461(1): 86-95, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-31982375

RESUMEN

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".


Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Corteza Motora/embriología , Neurogénesis/fisiología , Células Piramidales/trasplante , Animales , Encéfalo/embriología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Corteza Motora/citología , Células Piramidales/citología , Trasplante Heterólogo
3.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-30965622

RESUMEN

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.


Asunto(s)
MicroARNs/metabolismo , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes/metabolismo
4.
Int J Mol Sci ; 20(19)2019 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-31575031

RESUMEN

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.


Asunto(s)
Carcinoma Papilar/etiología , Carcinoma de Células Renales/etiología , Cuerpos Embrioides/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-met/genética , Alelos , Carcinoma Papilar/diagnóstico , Carcinoma de Células Renales/diagnóstico , Cuerpos Embrioides/metabolismo , Cuerpos Embrioides/ultraestructura , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genotipo , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética/métodos , Reproducibilidad de los Resultados
5.
Biochem Biophys Res Commun ; 503(3): 1861-1867, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30057314

RESUMEN

Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the "gatekeeper" T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.


Asunto(s)
Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Espectroscopía Infrarroja por Transformada de Fourier , Animales , Línea Celular , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Mutación
6.
Am J Pathol ; 180(5): 2084-96, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22425713

RESUMEN

Because many of the genes used to produce induced pluripotent stem cells (iPSCs) from somatic cells are either outright established oncogenes, such as c-myc and Klf4, or potentially related to tumorigenesis in various cancers, both the safety and the risks of tumorigenesis linked to iPSC generation require evaluation. In this work, we generated, by lentivirus-mediated gene transfer of Oct4, Sox2, Nanog, and Lin28, two types of iPSCs from human mesenchymal stem cells and human amniotic fluid-derived cells: fully reprogrammed iPSCs with silencing of the four transgenes and partially reprogrammed iPSCs that still express one or several transgenes. We assessed the behavior of these cells during both their differentiation and proliferation using in vivo teratoma assays in nonobese diabetic mice with severe combined immunodeficiency. In contrast to fully reprogrammed iPSCs, 43% of partially reprogrammed iPSC cases (6 of 14 teratomas) generated major dysplasia and malignant tumors, with yolk sac tumors and embryonal carcinomas positive for α-fetoprotein, cytokeratin AE1/AE3, and CD30. This correlated with the expression of one or several transgenes used for the reprogramming, down-regulation of CDK 1A mRNA (p21/CDKN1A), and up-regulation of antiapoptotic Bcl-2 mRNA. Therefore, the oncogenicity of therapeutically valuable patient-specific iPSC-derived cells should be scrupulously evaluated before they are used for any clinical applications.


Asunto(s)
Transformación Celular Neoplásica/patología , Células Madre Pluripotentes Inducidas/patología , Antígeno Ki-1/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Reprogramación Celular/fisiología , Células Madre Embrionarias/citología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Cariotipo , Factor 4 Similar a Kruppel , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Teratoma/metabolismo , Teratoma/patología , Transgenes/genética
7.
Blood ; 114(8): 1506-17, 2009 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-19478046

RESUMEN

The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA(+)CD41(-)) and MK (GPA(-)CD41(+)) lineages. In contrast to erythropoietin (EPO)-dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.


Asunto(s)
Células Madre Embrionarias/fisiología , Células Eritroides , Hematopoyesis/fisiología , Células Progenitoras de Megacariocitos y Eritrocitos/fisiología , Megacariocitos/fisiología , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias/metabolismo , Células Eritroides/citología , Células Eritroides/metabolismo , Glicoforinas/metabolismo , Humanos , Leucosialina/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Ratones , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo
8.
Apoptosis ; 15(12): 1529-39, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20640889

RESUMEN

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.


Asunto(s)
Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Fragmentos de Péptidos/fisiología , Proteínas Proto-Oncogénicas/fisiología , Serina Proteasas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Clorometilcetona de Tosilfenilalanila , Factor de Crecimiento Transformador beta/farmacología , Animales , Anisomicina/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Silenciador del Gen/fisiología , Humanos , Ratones , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Serina Proteasas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Clorometilcetona de Tosilfenilalanila/farmacología , Factor de Crecimiento Transformador beta/genética
9.
Stem Cells ; 27(8): 1750-9, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19544443

RESUMEN

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.


Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Antígenos CD/biosíntesis , Cadherinas/biosíntesis , Proteínas Portadoras/farmacología , Diferenciación Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Células Cultivadas , Citocinas/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Citometría de Flujo , Humanos , Cinética , Factores de Transcripción/biosíntesis
10.
Oncotarget ; 10(28): 2693-2708, 2019 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-31105870

RESUMEN

Recent development of cell reprogramming technologies brought a major hope for future cell therapy applications by the use of these cells or their derivatives. For this purpose, one of the major requirements is the absence of genomic alterations generating a risk of cell transformation. Here we analyzed by microarray-based comparative genomic hybridization human iPSC generated by two non-integrative and one integrative method at pluripotent stage as well as in corresponding teratomas. We show that all iPSC lines exhibit copy number variations (CNV) of several genes deregulated in oncogenesis. These cancer-associated genomic alterations were more pronounced in virally programmed hiPSCs and their derivative teratoma as compared to those found in iPSC generated by mRNA-mediated reprogramming. Bioinformatics analysis showed the involvement of these genes in human leukemia and carcinoma. We conclude that genetic screening should become a standard procedure to ensure that hiPSCs are free from cancer-associated genomic alterations before clinical use.

11.
J Clin Invest ; 129(5): 2145-2162, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30985297

RESUMEN

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.


Asunto(s)
Sistema Nervioso Central/fisiopatología , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/genética , Células Madre Pluripotentes/metabolismo , Receptores de Superficie Celular/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adolescente , Empalme Alternativo , Animales , Apoptosis , Encéfalo/diagnóstico por imagen , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Preescolar , Eliminación de Gen , Variación Genética , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/fisiología , Receptores de Superficie Celular/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiología
12.
Stem Cell Res ; 29: 56-59, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29597128

RESUMEN

Heterozygous non-synonymous (p.S142F) mutation in HNF1A leads to maturity-onset diabetes of the young (MODY) type 3, which is a subtype of dominant inherited young-onset non-autoimmune diabetes due to the defect of insulin secretion from pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs) from a patient with HNF1A p.S142F mutation. Cells from this patient, which were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the HNF1A p.S142F mutation, expressed pluripotency hallmarks.


Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Masculino , Mutación
13.
Stem Cell Res ; 26: 8-16, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29197744

RESUMEN

MEN2A is a hereditary cancer-predisposing syndrome that affects patients with germline RET mutations. The effects of this oncogenic tyrosine kinase in the context of primitive stem cells are not known. In order to study these events, we generated a MEN2A induced Pluripotent Stem Cell (iPSC) line from a patient with RET mutation and an isogenic counterpart by CRISPR-Cas9 correction of the mutation. Whole exome sequencing of iPSC before and after CRISPR-Cas9 genome edition revealed no major exonic off target effect of the CRISPR correction. However, an integrative differential gene expression analysis of iPSC with oncogenic RETC634Y and its gene-corrected iPSC with RETY634C as well as RETwt iPSCs revealed activation of the Early Growth Response 1 (EGR1) transcriptional program in RET-mutated iPSC, a pathway shown to be involved in RET-induced oncogenesis. These data constitute the first proof of concept of the feasibility of the use of an iPSC and its genome-corrected counterpart to unravel the molecular mechanisms underlying the development of the hereditary MEN2A cancer predisposing syndrome.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células Madre Pluripotentes Inducidas/patología , Neoplasia Endocrina Múltiple Tipo 2a/genética , Mutación , Proteínas Proto-Oncogénicas c-ret/genética , Transcriptoma , Adulto , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Neoplasia Endocrina Múltiple Tipo 2a/patología , Neoplasia Endocrina Múltiple Tipo 2a/prevención & control , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Células Tumorales Cultivadas
15.
Stem Cell Res ; 23: 178-181, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28925365

RESUMEN

Heterozygous activating mutation (p.Glu227Lys) in KCNJ11 leads to maturity-onset diabetes of the young (MODY) type 13, that is a subtype of dominant inherited young-onset non-autoimmune diabetes due to a primary defect in pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs) from a patient with KCNJ11p.Glu227Lys mutation who developed MODY at 13years old. KCNJ11p.Glu227Lys-mutated cells that were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the KCNJ11p.Glu227Lys mutation, expressed pluripotency hallmarks and had the differentiation capacity into the three germ layers.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Canales de Potasio de Rectificación Interna/genética , Animales , Secuencia de Bases , Forma de la Célula , Humanos , Masculino , Ratones , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Fenotipo
16.
Stem Cell Res ; 24: 135-138, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-29034880

RESUMEN

BRCA1 germline mutation confers hereditary predisposition for breast and ovarian cancer. To understand the physiopathology of mammary and ovarian epithelial cancer transformation, and to identify early driver molecular events, we have generated an iPSC line from a patient carrying a germline exon 17 deletion in BRCA1 gene (BRAC1Ex17 iPSC) in a high-risk family context. Blood cells were reprogrammed used non-integrative virus of Sendaï. The BRCA1-deleted iPSC had normal karyotype, harboured a deletion in the exon 17 of the BRCA1 gene, expressed pluripotent hallmarks and had the differentiation capacity into the three germ layers.


Asunto(s)
Proteína BRCA1/genética , Exones/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Proteína BRCA1/metabolismo , Línea Celular , Femenino , Humanos , Persona de Mediana Edad , Eliminación de Secuencia , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología
17.
Exp Hematol ; 53: 48-58, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28602946

RESUMEN

We report here the first use of whole-genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML), who presented in chronic phase (CP) with doubly marked BCR-ABL1+/JAK2V617F-mutant cells and, over a 9-year period, progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS revealed that the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1, and RREB1 as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only six additional coding somatic mutations, despite retention by the hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells revealed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2 of 101 cases of myeloproliferative neoplasms, but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.


Asunto(s)
Janus Quinasa 2/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Factores de Transcripción/genética
18.
Sci Rep ; 6: 27059, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27245508

RESUMEN

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.


Asunto(s)
Reprogramación Celular , Inestabilidad Cromosómica , Cromosomas de los Mamíferos/química , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Biomarcadores/metabolismo , Ciclo Celular/genética , Diferenciación Celular , Línea Celular , Fibroblastos/citología , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Cariotipificación , Lentivirus/genética , Lentivirus/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Porcinos
19.
PLoS One ; 11(3): e0149291, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26938212

RESUMEN

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.


Asunto(s)
Reprogramación Celular/genética , Células Madre Embrionarias/citología , Heterogeneidad Genética , Supervivencia de Injerto , Hematopoyesis/genética , Células Madre Pluripotentes Inducidas/citología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula/genética , Quimerismo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Expresión Génica , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Lentivirus/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Retroviridae/genética , Donantes de Tejidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante Heterólogo
20.
J Mol Endocrinol ; 34(1): 127-37, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15691883

RESUMEN

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion protein that is localised at cell-cell contacts. This molecule is an important determinant of vascular architecture and endothelial cell survival. In the adrenal cortex, steroidogenic and endothelial cells form a complex architecture. The adrenocorticotrophin hormone (ACTH) regulates gland homeostasis whose secretion is subjected to a negative feedback by adrenocorticosteroids. The aim of the present study was to determine whether VE-cadherin expression in the adrenal gland was regulated by hormonal challenge. We demonstrated that VE-cadherin protein levels were dramatically decreased (23.5+/-3.7%) by dexamethasone injections in the mouse and were restored by ACTH within 7 days (94.9+/-18.6%). Flow cytometry analysis of adrenal cells showed that the ratios of endothelial versus total adrenal cells were identical (35%) in dexamethasone- or ACTH-treated or untreated mice, suggesting that VE-cadherin expression could be regulated by ACTH. We demonstrate the existence of a transcriptional regulation of the VE-cadherin gene using transgenic mice carrying the chloramphenicol acetyl transferase gene under the control of the VE-cadherin promoter. Indeed, the promoter activity in the adrenals, but not in the lung or liver, was decreased in response to dexamethasone treatment (40+/-1.3%) and was partially restored after gland regeneration by ACTH injection (82+/-3%). In conclusion, our results show that transcription of a specific endothelial gene is controlled by the hypothalamo-pituitary axis and the data expand the knowledge regarding the role of ACTH in the regulation of the adrenal vascular network.


Asunto(s)
Glándulas Suprarrenales/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Cadherinas/genética , Animales , Antígenos CD , Cadherinas/biosíntesis , Endotelio/metabolismo , Citometría de Flujo , Ratones , Regiones Promotoras Genéticas
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA