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Molecular & Cellular Proteomics 8:1401-1412, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Proteomics Analysis of Epithelial Cells Reprogrammed in Cell-free Extract^*,

Emma Pewsey, Christine Bruce, A. Stephen Georgiou, Mark Jones, Duncan Baker, Saw Yen Ow^¶, Phillip C. Wright^¶, Christel K. Freberg^||, Philippe Collas^|| and Alireza Fazeli^,**

From the Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, The Jessop Wing, Sheffield S10 2SF, United Kingdom,
The Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom,
¶Department of Chemical and Process Engineering, Chemical Engineering Life Science Interface (ChELSI), Mappin Street, Sheffield S1 3JD, United Kingdom, and
||Department of Biochemistry, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in regenerative medicine. We report here the proteomic profile of 293T epithelial cells reprogrammed to a pluripotent state using undifferentiated embryonal carcinoma (NCCIT) cellular extracts. 293T cells were reversibly permeabilized with streptolysin O, incubated in an extract of NCCIT cells or a control extract of 293T cells for 1 h, resealed with CaCl₂, and cultured. OCT4 and SOX2 gene expression were up-regulated in NCCIT extract-treated cells relative to control cells, whereas there was no alteration in DNMT3B gene expression. Thirty percent of NCCIT extract-treated cells were positive for SSEA-4, and karyotyping confirmed their 293T origin, excluding the possibility of contamination from NCCIT cells. Two-dimensional PAGE revealed 400 protein spots for each cell type studied. At least 10 protein spots in the proteome of NCCIT extract-treated cells had an expression profile similar to that of NCCIT and remained unaltered in control cells. Using tandem mass spectrometry, we identified these proteins, which include 78-kDa glucose-regulated protein precursor and tropomyosin -3 chain. This investigation provides the first evidence that proteins are altered in a specific manner in NCCIT extract-treated cells. This is the first report on the proteomic characterization of the nuclear reprogramming process.

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