Proteomics Analysis of Epithelial Cells Reprogrammed in Cell-free Extract

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

doi:10.1074/mcp.m800478-mcp200

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.

Highlights

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in regenerative medicine
The method involves the retroviral introduction of four defined transcription factors, Oct4/Sox2/c-myc/Klf4 [13, 15] or Oct-4/Sox2/Nanog/Lin28 [17], into somatic cells, which is sufficient to reprogram them into embryonic-like stem cells
The levels of OCT4 (p Ͻ 0.05) and SOX2 (p Ͻ 0.05) in Nex were elevated by ϳ20- and ϳ2-fold, respectively, relative to expression in 293T cells not subjected to the reprogramming procedure

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—293T cells (human embryonic kidney epithelial cells) were grown to ϳ70% confluency at 37 °C and 5% CO2 in complete culture medium containing Dulbecco’s modified Eagle’s medium (Sigma) with 10% FCS (Invitrogen), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Invitrogen), and nonessential amino acids (Sigma). 293T cells treated with NCCIT (Nex) or 293T extract (293Tex) were seeded at 100,000 cells/well in a 48-well plate and cultured in 250 ␮l of complete RPMI 1640 medium with antibiotics. The Nex sample group consisted of two biological replicates of 293T cells treated with an NCCIT cell extract. The free cysteine residues of proteins were alkylated to prevent reformation of disulfide bonds by rocking each strip for 15 min in 10 ml of solution containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl, pH 8.8, 0.25% (w/v) bromphenol blue, and 2.5% (w/v) iodoacetamide. These strips were affixed onto homogeneous 12.5% polyacrylamide slab gels (2550 ϫ 2100 ϫ 1 mm) for SDS-PAGE. For further insights into mechanisms, quantitative PCR analyses were conducted

RESULTS

DISCUSSION

26 S protease regulatory subunit 6B