著名的四大转录因子：Oct4、Sox2、Klf4和c-Myc对iPSC的形成至关重要，但转录因子激发iPSC形成的步骤及机制一直尚不明确。近期Cell和Cell Report陆续发表了三项新研究成果，分别解析了iPS细胞如何一步步形成的步骤，以及在重编程的第一天和最后三天里的一个两步复位方法，这揭开了iPSCs形成的谜底，也指出了相应提高重编程效率的一些新基因。
iPSC具有和胚胎干细胞（ESC）类似的特征和功能，却极大程度避免了ESC研究和应用中面临的伦理和排斥等诸多障碍，因此这一新技术给基于干细胞的个性化治疗和再生医学带来了光明的前景。诺贝尔奖得主Yamanaka教授及后来的大量研究都表明Oct4、Sox2、Klf4和c-Myc（OSKM）等转录因子对iPSC的形成具有至关重要的作用，但是对于上述转录因子激发iPSC形成的步骤和机制尚不明确。
为了揭开这个谜底，来自哈佛-麻省总医院的两位知名科学家：Sridhar Ramaswamy和Konrad Hochedlinger进行了iPS细胞形成的转录分析，发现了一种双相过程。其中c-Myc/Klf4驱动了第一波，Oct4/Sox2/Klf4驱动了第二波，这种双相过程导致了一些细胞难以重编程，如果能提高4个因子的表达，就可以解决这一问题。
由此研究人员不仅解析出了iPS细胞由这些转录因子诱导恢复多能性的步骤，而且也提出了令体细胞更易形成iPSCs的方法。
在另外一篇文章中，研究人员采用了一种称为深度定量蛋白质组学（in-depth quantitative proteomics）的方法，分析成纤维细胞重编程为iPS细胞过程中蛋白质组的变化，从蛋白种类和数量上的变化来阐述重编程过程。
研究人员收集了6个时间段的样品进行分析：蛋白收集，在多肽上加上稳定的同位素标记，然后利用高通量纳米液相色谱-串联质谱进行分析，由此发现重编程的第一天和倒数第三天出现了蛋白质组的一个两步复位过程，这些蛋白以一个高度协调的方式发生着变化，出现了几种生物学进程，比如电子传递链复合物的电化学变化，中间阶段囊泡的运输，还有最后阶段中的EMT样进程等。
这项研究以定量蛋白质组学为基础，进行了大规模（8000种蛋白），大范围（7个数量级）的分析，明确的指出了重编程过程是一种多步骤进程，目前大部分研究集中在起始阶段，而这项研究发现了前三天和后十二天的变化，解析了其中微妙的中间阶段，将进一步增强我们对细胞重编程机理的认识。(生物谷Bioon.com)
doi:10.1016/j.cell.2012.08.023
PMC:
PMID:
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
PMC:
PMID:
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
PMC:
PMID:
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.
