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

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.

新一代诱导多能干细胞（induced pluripotent stem cells, iPS细胞）为基于胚胎干细胞（embryonic stem cells, ES细胞）的研究策略提供了全新的补充契机。iPS细胞可通过特定转录因子的病毒转导获得，但随机逆转录病毒整合存在潜在的致瘤风险。本研究采用胚胎干细胞来源的细胞提取物替代病毒转导手段，从FVB品系小鼠皮肤成纤维细胞（FVB-sFB）中成功构建了具备胚胎干细胞特性的新型iPS细胞（sFB-protein-iPS）。值得注意的是，本研究中仅C57/BL6背景的ES细胞系（C57-mES）的提取物可在本实验方案下诱导生成iPS细胞，而129背景的ES细胞系（E14-mES）则无此效果。我们推测，解析这两种小鼠ES细胞系之间的差异将为重编程机制研究提供关键见解，因此分别通过同位素标记相对和绝对定量（iTRAQ）技术与mRNA芯片开展了蛋白质组学与全基因表达分析。研究结果显示，多能ES细胞与胚胎干细胞提取物诱导获得的iPS细胞在蛋白质组与全基因表达模式上均存在显著差异。尤为关键的是，相较于不具备重编程能力的129-mES细胞，具备重编程能力的C57-mES细胞高表达调控蛋白质合成与代谢的相关蛋白，这表明蛋白质合成系统需达到一定阈值方可启动细胞重编程过程。