RESUMEN
Nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax1) is regarded as an important component of the transcription factor network that governs pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Nr0b1 using the Cre-loxP system and analyzed its precise function. We succeeded in establishing the Nr0b1-null ES cells and confirmed their pluripotency by showing their contribution to chimeric embryos. However, they proliferated slowly with over-expression of 2-cell stage specific transcripts including Zscan4c, which is known to be involved in telomere elongation in ES cells. We revealed that over-expression of Zscan4c prevents normal self-renewal by inducing arrest at G2 phase followed by cell death and that Nr0b1 directly represses the Zscan4c promoter. These data indicated that Nr0b1 is not essential to maintain pluripotency but is involved in the proper activation of 2-cell specific transcripts for self-renewal.
Asunto(s)
Receptor Nuclear Huérfano DAX-1/metabolismo , Células Madre Embrionarias/metabolismo , Factores de Transcripción/metabolismo , Animales , Ciclo Celular/genética , Muerte Celular/genética , Línea Celular , Proliferación Celular , Autorrenovación de las Células , Receptor Nuclear Huérfano DAX-1/genética , Células Madre Embrionarias/citología , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Sitios Genéticos , Ratones , Fenotipo , Unión ProteicaRESUMEN
Tumor suppressor Trp53 works as a guardian of the genome in somatic cells. In mouse embryonic stem (ES) cells, it was reported that Trp53 represses pluripotency-associated transcription factor Nanog to induce differentiation. However, since Trp53-null mice develop to term, Trp53 is dispensable for both the maintenance and differentiation of the pluripotent stem cell population in vivo, suggesting the differential functions of Trp53 in ES cells and embryos. To reveal the basis of this discrepancy, here we established a new line of Trp53-null ES cells by sequential gene targeting and evaluated their ability to differentiate in vitro and in vivo. We found that Trp53-null ES cells had defects in differentiation in vitro as reported previously, whereas they were able to contribute to normal development in chimeric embryos. These data indicated that the requirement of Trp53 for maintaining and executing the ES pluripotency is not absolute.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Aneuploidia , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Quimera , Desarrollo Embrionario/genética , Células Madre Embrionarias/efectos de los fármacos , Expresión Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Genotipo , Cariotipificación , Factor Inhibidor de Leucemia/farmacología , Ratones , Transporte de Proteínas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.
Asunto(s)
Blastocisto/citología , Cadherinas/genética , Estratos Germinativos/citología , Células Madre Pluripotentes/citología , Quimera por Trasplante/genética , Animales , Blastocisto/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Elementos Transponibles de ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Expresión Génica , Vectores Genéticos , Estratos Germinativos/metabolismo , Masculino , Ratones , Microinyecciones , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Transgenes , Quimera por Trasplante/crecimiento & desarrolloRESUMEN
In mice, coat pigmentation requires a stem cell (SC) system in which the survival, proliferation, and differentiation of melanocytes (MCs) are regulated by microenvironments in hair follicles (HFs). In vitro systems are required to analyze the behavior of single melanocyte stem cells (MCSCs) and their potential to form SC systems in vivo. We describe here an experimental system for the isolation, self-renewal, and differentiation of MCSCs, as well as an in vivo reconstitution assay for assessing their potential. Using Dct(tm1(Cre)Bee)/CAG-CAT-GFP mice, we show that, in the presence of stem cell factor and basic fibroblast growth factor and the XB2 feeder cell line, purified MCSCs can undergo clonogenic proliferation, resulting in c-Kit(low) side scatter(low) cells. In culture, these cells maintain their capacity to differentiate and reconstitute an MCSC system in HFs. As these cells are present in the upper part of the HF near the bulge region, express only low levels of housekeeping genes, and are resistant to neonatal treatment with ACK2, it is likely that only MCSCs that are quiescent in vivo have clonogenic activity in vitro. We also found that MCSCs can be purified from wild-type mice by fluorescent cell sorting and can be characterized in vitro.