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1.
Nature ; 464(7286): 292-6, 2010 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-20164838

RESUMEN

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.


Asunto(s)
Disqueratosis Congénita/genética , Células Madre Pluripotentes , Telómero/genética , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Reprogramación Celular/genética , Disqueratosis Congénita/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Proteínas Nucleares/genética , Células Madre Pluripotentes/enzimología , ARN/genética , ARN/metabolismo , Eliminación de Secuencia/genética , Telomerasa/genética , Telomerasa/metabolismo , Regulación hacia Arriba
2.
Stem Cells ; 31(7): 1287-97, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23400930

RESUMEN

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here, we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.


Asunto(s)
ADN Mitocondrial/genética , Células Madre Pluripotentes Inducidas/fisiología , Enfermedades Mitocondriales/genética , Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , Diferenciación Celular/genética , Línea Celular , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , ADN Mitocondrial/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/patología , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Eliminación de Secuencia
3.
Blood ; 115(17): 3453-62, 2010 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-20089964

RESUMEN

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.


Asunto(s)
Células Madre Embrionarias/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Anemia de Fanconi/metabolismo , Técnicas de Silenciamiento del Gen , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular , Células Madre Embrionarias/patología , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Modelos Biológicos , Interferencia de ARN
4.
Blood ; 113(22): 5476-9, 2009 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-19299331

RESUMEN

Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.


Asunto(s)
Células Sanguíneas/citología , Desdiferenciación Celular , Proliferación Celular , Células Madre Pluripotentes/citología , Adulto , Antígenos CD34/metabolismo , Células Sanguíneas/metabolismo , Técnicas de Cultivo de Célula , Desdiferenciación Celular/fisiología , Células Cultivadas , Humanos , Factor 4 Similar a Kruppel , Masculino , Modelos Biológicos , Células Madre Pluripotentes/metabolismo
5.
J Vis Exp ; (126)2017 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-28829422

RESUMEN

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.


Asunto(s)
Trasplante de Células/métodos , Epitelio Pigmentado de la Retina/trasplante , Trasplante Heterólogo/métodos , Animales , Humanos , Inyecciones Intraoculares/métodos , Degeneración Macular/terapia , Ratas , Retina/trasplante , Epitelio Pigmentado de la Retina/citología
6.
Cell Stem Cell ; 20(5): 635-647.e7, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-28132833

RESUMEN

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.


Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Niacinamida/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genotipo , Humanos , Inmunohistoquímica , Retina/efectos de los fármacos , Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
7.
Cell Stem Cell ; 13(6): 691-705, 2013 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-24315443

RESUMEN

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.


Asunto(s)
Envejecimiento/patología , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Reprogramación Celular , Senescencia Celular , Niño , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/trasplante , Neuronas Dopaminérgicas/ultraestructura , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Mesencéfalo/patología , Ratones , Persona de Mediana Edad , Enfermedad de Parkinson/patología , Fenotipo , Donantes de Tejidos
8.
Methods Mol Biol ; 767: 55-65, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21822867

RESUMEN

This chapter describes a protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with a cocktail of retroviral vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC. The culture conditions are then switched to conditions that support human embryonic stem cell growth and emerging iPSC colonies that morphologically resemble human embryonic stem cell (hESC) colonies and have silenced the retroviral vectors (as evidenced by downregulation of retroviral GFP expression) that are mechanically isolated and subsequently cultured in identical fashion to hESCs. Putative iPSC lines are validated to be bona fide human iPSC lines by analyzing them for the expression of pluripotency markers and by differentiation in vitro and in vivo.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Fibroblastos/virología , Técnicas de Transferencia de Gen , Células Madre Pluripotentes Inducidas/citología , Retroviridae/genética , Línea Celular , Proliferación Celular , Reprogramación Celular/genética , Células Clonales , Ensayo de Unidades Formadoras de Colonias , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Reproducibilidad de los Resultados , Retroviridae/fisiología , Coloración y Etiquetado , Factores de Transcripción/metabolismo , Transfección , Replicación Viral/fisiología
9.
Nat Biotechnol ; 27(11): 1033-7, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19826408

RESUMEN

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.


Asunto(s)
Reprogramación Celular/genética , Imagenología Tridimensional/métodos , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular , Línea Celular , Forma de la Célula , Supervivencia Celular , Ensayo de Unidades Formadoras de Colonias , Células Madre Embrionarias/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Teratoma/patología , Factores de Tiempo
10.
Nat Protoc ; 3(5): 923-33, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18451800

RESUMEN

Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos that are discarded during the IVF procedure because of poor morphology and a low likelihood for generating viable pregnancies or surviving the cryopreservation process are also a viable source of hES cells. In this protocol, we describe how to derive novel hES cells from discarded poor-quality embryos and how to maintain the hES cell lines.


Asunto(s)
Masa Celular Interna del Blastocisto/citología , Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Fertilización In Vitro , Humanos , Bancos de Tejidos
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