Analysis of Differential Proteomes of Induced Pluripotent Stem Cells by Protein-Based Reprogramming of Fibroblasts

Abstract

The recent generation of induced pluripotent stem (iPS) cells represents a novel opportunity to complement embryonic stem (ES) cell-based approaches. iPS cells can be generated by viral transduction of specific transcription factors, but there is a potential risk of tumorigenicity by random retroviral integration. We have generated novel iPS (sFB-protein-iPS) cells from murine dermal fibroblasts (FVB-sFB) that have ES cell characteristics, using ES cell-derived cell extracts instead of performing viral transduction. Notably, only cell extracts from an ES cell line (C57-mES) on the C57/BL6 background generated iPS cells in our protocol-not an ES cell line (E14-mES) on the 129 background. Hypothesizing that determining the differences in these 2 mES cell lines will provide vital insight into the reprogramming machinery, we performed proteomic and global gene expression analysis by iTRAQ and mRNA microarray, respectively. We observed that pluripotent ES cells and ES cell extract-derived iPS cells had differential proteomes and global gene expression patterns. Notably, reprogramming-competent C57-mES cells highly expressed proteins that regulate protein synthesis and metabolism, compared with reprogramming-incompetent 129-mES cells, suggesting that there is a threshold that protein synthetic machinery must exceed to initiate reprogramming.

Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc

“…Interestingly, with a gap of four days (TF3-B) instead of three (TF3-A) between the second and third transfection, the levels of the introduced factors as well as Nanog and SSEA-1 were significantly lower, indicating that the level of the proteins should be maintained above a certain threshold for the induction of pluripotency. These results are in agreement with the findings of Jin et al [18].…”

Role of Zscan4 in secondary murine iPSC derivation mediated by protein extracts of ESC or iPSC

“…Interestingly, ribosomal protein levels were higher in C57 strain mES cells than in E14 mES cell lines. These results indicate that a large capacity of protein synthetic machinery may be required for the initiation of reprogramming [22].…”

“…To investigate the key proteins involved in this improvement of efficiency, we compared the proteomics of C57-mES cells to that of primary iPS cells. However, as we recently reported, these two cell types show very similar patterns of protein expression [22]. Due to the sensitivity limit of Q-TOF LCeMS/MS, iTRAQ™ may not be able to identify all the proteomic differences between mES and iPS cell lines.…”

Proteomic analysis revealed the altered kidney protein profile of a Cyld knockout mouse model

“…iTRAQ labeling enabled the comprehensive analysis of differential proteome expression, as described previously (Jin et al, 2009(Jin et al, , 2011. For iTRAQ LC-MS/MS analysis, 50 µg clarified supernatants was denatured for 1 h at 60°C, the disulfide bonds were reduced, and the cysteine residues were blocked, as described in the iTRAQ protocol (Applied Biosystems, Foster City, CA, USA).…”