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 NC-CIT 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 ϳ400 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.

Proteomic Analysis of Early Reprogramming Events in Murine Somatic Cells Incubated withXenopus laevisOocyte Extracts Demonstrates Network Associations with Induced Pluripotency Markers

“…Other proteomic studies have compared proteomic profiles of iPSCs with ESCs ( Jin et al, 2011;Kim et al, 2012;Munoz et al, 2011;Wang et al, 2012), or protein expression changes of iPSCs that have been cultured for days or weeks post-reprogramming compared to the somatic cells from which they were derived (Huang et al, 2012;Kim et al, 2012;Munoz et al, 2011;Pewsey et al, 2009). In the current study, we chose to concentrate on global nuclear protein expression changes during the early stages of reprogramming, before the cells express pluripotency markers.…”

Estimating the Quality of Reprogrammed Cells Using ES Cell Differentiation Expression Patterns

“…However, how the cell state alteration process happens from terminal differentiation to pluripotency is unclear. The similarities and differences in the transcriptomes of iPSCs and ES cells have been estimated [7] , [8] , while other properties of iPSCs are also different compared with ESCs, such as the genome methylation state [16] , [17] , microRNA profiling [18] , histone modification, proteomic profiles [19] , and so on. It is still a challenge to find an accurate and easy method to estimate the pluripotency of iPSC candidates based on these cellular properties.…”

A poor imitation of a natural process

“…of reprogramming efficiency. Additional assays for aspects such as the genomic methylation state, 80 miRNA profiling, 81 histone modification and proteomic profiling 82 have demonstrated the difference between iPSCs and ESCs.…”