著名的四大轉(zhuǎn)錄因子：Oct4,、Sox2,、Klf4和c-Myc對(duì)iPSC的形成至關(guān)重要,，但轉(zhuǎn)錄因子激發(fā)iPSC形成的步驟及機(jī)制一直尚不明確,。近期Cell和Cell Report陸續(xù)發(fā)表了三項(xiàng)新研究成果，分別解析了iPS細(xì)胞如何一步步形成的步驟，以及在重編程的第一天和最后三天里的一個(gè)兩步復(fù)位方法,，這揭開了iPSCs形成的謎底,，也指出了相應(yīng)提高重編程效率的一些新基因。
iPSC具有和胚胎干細(xì)胞（ESC）類似的特征和功能,，卻極大程度避免了ESC研究和應(yīng)用中面臨的倫理和排斥等諸多障礙,，因此這一新技術(shù)給基于干細(xì)胞的個(gè)性化治療和再生醫(yī)學(xué)帶來了光明的前景。諾貝爾獎(jiǎng)得主Yamanaka教授及后來的大量研究都表明Oct4,、Sox2,、Klf4和c-Myc（OSKM）等轉(zhuǎn)錄因子對(duì)iPSC的形成具有至關(guān)重要的作用，但是對(duì)于上述轉(zhuǎn)錄因子激發(fā)iPSC形成的步驟和機(jī)制尚不明確,。
為了揭開這個(gè)謎底,，來自哈佛-麻省總醫(yī)院的兩位知名科學(xué)家：Sridhar Ramaswamy和Konrad Hochedlinger進(jìn)行了iPS細(xì)胞形成的轉(zhuǎn)錄分析，發(fā)現(xiàn)了一種雙相過程,。其中c-Myc/Klf4驅(qū)動(dòng)了第一波，Oct4/Sox2/Klf4驅(qū)動(dòng)了第二波,，這種雙相過程導(dǎo)致了一些細(xì)胞難以重編程,，如果能提高4個(gè)因子的表達(dá)，就可以解決這一問題,。
由此研究人員不僅解析出了iPS細(xì)胞由這些轉(zhuǎn)錄因子誘導(dǎo)恢復(fù)多能性的步驟,，而且也提出了令體細(xì)胞更易形成iPSCs的方法。
在另外一篇文章中,，研究人員采用了一種稱為深度定量蛋白質(zhì)組學(xué)（in-depth quantitative proteomics）的方法,，分析成纖維細(xì)胞重編程為iPS細(xì)胞過程中蛋白質(zhì)組的變化，從蛋白種類和數(shù)量上的變化來闡述重編程過程,。
研究人員收集了6個(gè)時(shí)間段的樣品進(jìn)行分析：蛋白收集,，在多肽上加上穩(wěn)定的同位素標(biāo)記，然后利用高通量納米液相色譜-串聯(lián)質(zhì)譜進(jìn)行分析,，由此發(fā)現(xiàn)重編程的第一天和倒數(shù)第三天出現(xiàn)了蛋白質(zhì)組的一個(gè)兩步復(fù)位過程,，這些蛋白以一個(gè)高度協(xié)調(diào)的方式發(fā)生著變化，出現(xiàn)了幾種生物學(xué)進(jìn)程,，比如電子傳遞鏈復(fù)合物的電化學(xué)變化,，中間階段囊泡的運(yùn)輸，還有最后階段中的EMT樣進(jìn)程等,。
這項(xiàng)研究以定量蛋白質(zhì)組學(xué)為基礎(chǔ),，進(jìn)行了大規(guī)模（8000種蛋白），大范圍（7個(gè)數(shù)量級(jí)）的分析,，明確的指出了重編程過程是一種多步驟進(jìn)程,，目前大部分研究集中在起始階段，而這項(xiàng)研究發(fā)現(xiàn)了前三天和后十二天的變化,，解析了其中微妙的中間階段,，將進(jìn)一步增強(qiáng)我們對(duì)細(xì)胞重編程機(jī)理的認(rèn)識(shí),。(生物谷Bioon.com)
doi:10.1016/j.cell.2012.08.023
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Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase
Yosef Buganim1, 7, Dina A. Faddah1, 2, 7, Albert W. Cheng1, 3, Elena Itskovich1, Styliani Markoulaki1, Kibibi Ganz1, Sandy L. Klemm5, Alexander van Oudenaarden2, 4, 6 and Rudolf Jaenisch1, 2, ,
During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
DOI:10.1016/j.celrep.2012.10.014
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Highly Coordinated Proteome Dynamics during Reprogramming of Somatic Cells to Pluripotency
Jenny Hansson, Mahmoud Reza Rafiee, Sonja Reiland, Jose M. Polo, Julian Gehring, Satoshi Okawa, Wolfgang Huber, Konrad Hochedlinger, Jeroen KrijgsveldSee Affiliations
Generation of induced pluripotent stem cells (iPSCs) is a process whose mechanistic underpinnings are only beginning to emerge. Here, we applied in-depth quantitative proteomics to monitor proteome changes during the course of reprogramming of fibroblasts to iPSCs. We uncover a two-step resetting of the proteome during the first and last 3 days of reprogramming, with multiple functionally related proteins changing in expression in a highly coordinated fashion. This comprised several biological processes, including changes in the stoichiometry of electron transport-chain complexes, repressed vesicle-mediated transport during the intermediate stage, and an EMT-like process in the late phase. In addition, we demonstrate that the nucleoporin Nup210 is essential for reprogramming by its permitting of rapid cellular proliferation and subsequent progression through MET. Along with the identification of proteins expressed in a stage-specific manner, this study provides a rich resource toward an enhanced mechanistic understanding of cellular reprogramming.
DOI:10.1016/j.cell.2012.11.039
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A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells
Jose M. Polo, Endre Anderssen, Ryan M. Walsh, Benjamin A. Schwarz, Christian M. Nefzger, Sue Mei Lim, Marti Borkent, Effie Apostolou, Sara Alaei, Jennifer Cloutier, Ori Bar-Nur, Sihem Cheloufi, Matthias Stadtfeld, Maria Eugenia Figueroa, Daisy Robinton, Sridaran Natesan, Ari Melnick, Jinfang Zhu, Sridhar Ramaswamy, Konrad HochedlingerSee Affiliations
Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
