RESUMEN
Although many factors have been identified to be involved in the development of the neuroectoderm during embryogenesis, it is still important to identify novel factors that convert undifferentiated embryonic cells into neuroectoderm. RuvB-like protein 2 (Ruvbl2) is known to regulate gene expression via chromatin remodeling by participating in multi-protein complexes, but its role during embryonic development is not well known. In this study, we established Ruvbl2-overexpressing mouse embryonic stem cells and examined their capacity to specifically differentiate into neuroectoderm and confirmed the specific expression of RUVBL2 in early embryonic neuroectoderm. Our results suggest that Ruvbl2 has a role in the differentiation of neuroectoderm during early embryogenesis.
Asunto(s)
Diferenciación Celular/genética , ADN Helicasas/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Placa Neural/citología , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Biomarcadores/metabolismo , Proliferación Celular , ADN Helicasas/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Feto/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Organogénesis/genética , Regulación hacia Arriba/genéticaRESUMEN
Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes.
Asunto(s)
Reprogramación Celular/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Proteínas Nucleares/genética , Factores de Transcripción/genética , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Western Blotting , Células Cultivadas , Análisis por Conglomerados , Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Biología de Sistemas/métodos , Factores de Transcripción/metabolismoRESUMEN
In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; â¼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.
