RESUMO
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.
Assuntos
Desdiferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Retrovirus Endógenos/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Totipotentes/citologia , Células-Tronco Totipotentes/metabolismo , Animais , Desdiferenciação Celular/fisiologia , Linhagem da Célula/genética , Quimera/embriologia , Cromatina/genética , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Células-Tronco Embrionárias/virologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Histonas/química , Histonas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisina/química , Lisina/metabolismo , Metilação , Camundongos , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/virologia , Sequências Repetidas Terminais/genética , Células-Tronco Totipotentes/virologia , Transcriptoma/genéticaRESUMO
It has been reported recently that the cytomegalovirus (CMV) immediate-early promoter is transcriptionally inactive in undifferentiated mouse embryonic stem (ES) cells. This result is surprising, since the CMV promoter is used to express transgenes in a variety of cell lines. We studied the expression of a human CMV-driven enhanced green fluorescent protein (EGFP) reporter gene (pEGFP-N1) in five undifferentiated mouse ES cell lines (BL/6III, D3, E14TG2a, MESC20, and 129) and found EGFP expression in all of these cell lines. Under optimal conditions, between 50%-80% transfection efficiencies could be achieved, and EGFP expression levels were maintained for at least 72 hours. Therefore, the human CMV promoter remains a useful system for transgene expression in undifferentiated ES cells.