RESUMEN
Hybrid embryonic stem (ES)-like clones were generated by fusion of murine ES cells with somatic cells that carried a neo resistance gene under the transcriptional control of the Oct-4 promoter. The Oct-4 promoter was reactivated in hybrid ES cells formed by fusion with fetal fibroblasts, and all hybrid colonies were of ES rather than fibroblast phenotype, suggesting efficient reprogramming of fibroblast chromosomes. Like normal diploid murine ES cells, hybrid lines expressed alkaline phosphatase activity and formed differentiated cells derived from the three embryonic germ layers both in vitro and in vivo. Treatments thought to affect nuclear transfer efficiency (ES cell confluence and serum starvation of primary embryonic fibroblasts) were investigated to determine whether they had an effect on reprogramming in cell hybrids. Serum starvation of primary embryonic fibroblasts increased hybrid colony number 50-fold. ES cells were most effective at reprogramming when they contained a high proportion of cells in the S and G2/M phases of the cell cycle. These data suggest that nuclear reprogramming requires an initial round of somatic DNA replication of quiescent chromatin in the presence of ES-derived factors produced during S and G2/M phases.
Asunto(s)
Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Células Híbridas/citología , Células Madre Pluripotentes/citología , Fosfatasa Alcalina/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Núcleo Celular/genética , Medio de Cultivo Libre de Suero , Cartilla de ADN/genética , Replicación del ADN , Regulación de la Expresión Génica , Células Híbridas/metabolismo , Ratones , Fenotipo , Células Madre Pluripotentes/metabolismoRESUMEN
Cells of different types can be induced to fuse by electroshock. Cells of one type are typically dominant and are able to reprogram the nuclei derived from cells of the other type, in fusion hybrids derived from one cell of each type. Flow cytometry provides a quick and objective technique to assess cell fusion for nuclear reprogramming studies. Two cell types are each stained with a different fluorescent dye and then induced to fuse to form fusion products called heterokaryons. Heterokaryons can be identified and quantified by flow cytometry as double-stained events. Protocols are provided for the optimization of cell staining under conditions that minimize cell clumping and dye leakage. If spectral overlap occurs between emission spectra of the two stained cell types, the data will need to be electronically compensated.
Asunto(s)
Núcleo Celular/metabolismo , Citometría de Flujo/métodos , Células Híbridas/citología , Animales , Fusión Celular , Embrión de Mamíferos/citología , Femenino , Ratones , Ratones Endogámicos CBA , Modelos Biológicos , Células Madre/citología , Timo/metabolismoRESUMEN
The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.
Asunto(s)
Embrión de Mamíferos/citología , Células Germinativas/citología , Células Madre/citología , Animales , Western Blotting , Linaje de la Célula , Regulación hacia Abajo , MAP Quinasa Quinasa 1 , Ratones , Ratones Endogámicos CBA , Ratones Transgénicos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismoRESUMEN
Primary mouse brain cells were cultured with HPRT (hypoxanthine phosphoribosyl transferase)-deficient ES (embryonic stem) cells to see if the ES cells could provide cues sufficient to reprogram a pluripotential state. After 5 days of coculture, HPRT-deficient ES cells were killed by selection in HAT (hypoxanthine, aminopterin, thymidine) medium. We observed islands of HAT-resistant ES-like cells surrounded by differentiated cells. Cell lines generated from three such "islands" proved to be spontaneous, pluripotential ES-neural hybrids, and gave rise to a chimera following blastocyst injection. Re-expression of the ES-specific gene Foxd3 from somatic-derived chromosomes suggested that the somatic nucleus had been reprogrammed. Our results raise the intriguing possibility that ASCs shown to contribute to multiple tissues in blastocyst-injection studies may not contribute as a result of pluripotency. Instead contributions may arise from spontaneous fusion events in which phenotype is determined by either cytoplasmic dominance, nuclear reprogramming, or both.