Proteomics analysis of epithelial cells reprogrammed in cell-free extract.
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.
Pewsey
Emma
E
Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, The Jessop Wing, Sheffield S102SF, United Kingdom.
Bruce
Christine
C
Georgiou
A Stephen
AS
Jones
Mark
M
Baker
Duncan
D
Ow
Saw Yen
SY
Wright
Phillip C
PC
Freberg
Christel K
CK
Collas
Philippe
P
Fazeli
Alireza
A
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
D017931:DNA Primers