title: Proteomics analysis of epithelial cells reprogrammed in cell-free extract.
abstract: 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 CaCl(2), 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 approximately 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 alpha-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.
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, The Jessop Wing, Sheffield S102SF, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Pewsey
authors.forename: Christine
authors.initials: C
authors.lastname: Bruce
authors.forename: A Stephen
authors.initials: AS
authors.lastname: Georgiou
authors.forename: Mark
authors.initials: M
authors.lastname: Jones
authors.forename: Duncan
authors.initials: D
authors.lastname: Baker
authors.forename: Saw Yen
authors.initials: SY
authors.lastname: Ow
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
authors.forename: Christel K
authors.initials: CK
authors.lastname: Freberg
authors.forename: Philippe
authors.initials: P
authors.lastname: Collas
authors.forename: Alireza
authors.initials: A
authors.lastname: Fazeli
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D002853:Chromatography, Liquid; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D007621:Karyotyping; D016133:Polymerase Chain Reaction; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D015854:Up-Regulation
chemical_list: D017931:DNA Primers