RESUMEN
We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast-derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.
Asunto(s)
Células Madre Pluripotentes/citología , Seguridad , Animales , Línea Celular , Transformación Celular Neoplásica/patología , Ratones , Neuronas/citología , Teratoma/patologíaRESUMEN
Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Fibroblastos/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular/fisiología , Ingeniería Genética/métodos , Factor 4 Similar a Kruppel , Ratones , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Nuclear factor-kappaB (NF-kappaB) is a transcription factor important for various cellular events such as inflammation, immune response, proliferation, and apoptosis. In this study, we performed a yeast two-hybrid screening using the N-terminal domain of the p65 subunit (RelA) of NF-kappaB as bait and isolated centrosomal P4.1-associated protein (CPAP) as a candidate for a RelA-associating partner. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments followed by Western blotting also showed association of CPAP with RelA. When overexpressed, CPAP enhanced NF-kappaB-dependent transcription induced by tumor necrosis factor-alpha (TNFalpha). Reduction of the protein level of endogenous CPAP by RNA interference resulted in decreased activation of NF-kappaB by TNFalpha. After treatment with TNFalpha, a portion of CPAP was observed to accumulate in the nucleus, although CPAP was found primarily in the cytoplasm without any stimulation. Moreover, CPAP was observed in a complex recruited to the transcriptional promoter region containing the NF-kappaB-binding motif. One hybrid assay showed that CPAP has the potential to activate gene expression when tethered to the transcriptional promoter. These data suggest that CPAP functions as a coactivator of NF-kappaB-mediated transcription. Since a physiological interaction between CPAP and the coactivator p300/CREB-binding protein was also observed and synergistic activation of NF-kappaB-mediated transcription was achieved by these proteins, CPAP-dependent transcriptional activation is likely to include p300/CREB-binding protein.
Asunto(s)
Centrosoma/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Transcripción Genética , Secuencias de Aminoácidos , Sitios de Unión , Western Blotting , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica , Genes Reporteros , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Luciferasas/metabolismo , Modelos Genéticos , FN-kappa B/química , FN-kappa B/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factor de Transcripción ReIA , Activación Transcripcional , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Several cellular signaling systems exhibit cross talk. Cross talk seems to play an important role in modifying signal effects. In vertebrates, the nuclear factor kappa B (NF-kappaB) signaling pathway plays important roles in immune response, inflammation and apoptosis. Meanwhile, the Wnt/beta-catenin signaling pathway is involved in oncogenesis and development. We show here that RelA, a component of NF-kappaB, specifically suppressed beta-catenin/Tcf-dependent transcription. This suppression did not depend on the trans-acting transcriptional ability of RelA. Furthermore, RelA neither affected the nuclear import of beta-catenin nor the DNA binding ability of the beta-catenin/Tcf complex, suggesting that NF-kappaB modifies this signaling pathway after the binding of the beta-catenin/Tcf complex with target DNA.