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Steve Pells

Publications and source records attributed to Steve Pells.

4 recordsLinked to original sources
PubMed2006

Nuclear reprogramming of somatic cells by embryonic stem cells is affected by cell cycle stage.

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.

Alkaline Phosphatase↗
PubMed2006

Quantification of cell fusion by flow cytometry.

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.

Animals↗
PubMed2002

Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro.

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.

Animals↗