English

Abstract

Introduction It has been more than 6 years since the four Yamanaka transcription factors— Oct4, Klf4, Sox2 and c-Myc—boosted research on reprogramming somatic cells into embryonic-like cells. However, methods for increasing the reprogramming efficiency and identifying the reprogramming-resistant cells remain unknown. To summarize the updated knowledge and the progress of the molecular mechanism of reprogramming, this review has introduced and evaluated the work done by Polo et al., who established a method to define the intermediate state of prereprogramming and the two waves in which reprogramming is triggered. This article provides a comprehensive and critical overview of the study conducted by Polo et al. by discussing the first wave triggered by c-Myc/ Klf4 and the second one regulated by Oct4/Sox2/Klf4. First, we focused on the methods used for the definition of the two waves followed by a discussion of how to distinguish the reprogrammable and reprogrammingresistant cells from mouse embryonic fibroblasts with flow cytometry. It also placed significant emphasis on the necessity of the Yamanaka factors in the observed waves. Finally, the article asked whether or not the reprogramming model used by Polo et al. could be adapted to human cell reprogramming. Topics ranging from how to comprehensively understand the preand post-reprogramming processes to future complementary work in the field are also discussed in this article. Conclusion This review highly evaluates the work of Polo et al., which provided evidence of the two waves during the post-reprogramming process. Moreover, we raise some critical questions which would be more helpful to understand the molecular mechanism of reprograming. Introduction The four famous Yamanaka transcription factors Oct4, Klf4, Sox2 and c-Myc (OKSM) are critical to the formation of induced pluripotent stem cells (iPSCs)1,2, but the mechanism of reprogramming and the intermediate state during the reprogramming remain unclear. To uncover the black box of reprogramming, intriguingly, Polo et al. found a two-wave process during iPSC formation3. In this study, by way of gene-targeting doxycycline-inducible reprogramming system4, they generated iPSCs from mouse embryonic fibroblast cells (MEFCs) with OKSM overexpression. The results revealed two waves from pre-reprogrammed fibroblast cells to post-reprogrammed iPSCs. The first wave was triggered by c-Myc/Klf4 and the second was driven by Oct4/Sox2/Klf4. Somatic cells that failed to be reprogrammed could not start the second wave, even though they could activate the first wave. In addition, the researchers found that after the first wave, some bivalent genes gradually developed, but DNA methylation happens only after the second wave, during which a stable pluripotent state is acquired. The conclusions from this study provide us with detailed insights into the characteristics and molecular mechanisms of inherent molecular events in reprogramming. Since Yamanaka generated the iPSCs, we have entered a post-reprogramming era. However, the precise regulation of reprogramming factors and why the OKSM factors can reprogram MEFCs have remained unknown. Polo et al. have first defined the intermediate state of reprogramming into separate time points. By starting with defining the reprogramming process by FACSsorted Thy1-negative, SSEA1-positive and Oct4-GFP–positive cells, they demonstrated the different cell-surface markers in the bulk cells. Combined with this observation, the geneexpression assay also showed the reprogramming and embryonic stem cell (ESC)–related gene level in different points. Based on these observations, they found two waves before fully being reprogrammed: days 0–3 is prereprogramming wave, day 9 is the post-reprogramming wave and days 3–9 is the intermediate state. This report is critical for improving the efficiency of reprogramming and its future therapeutic applications, but the results are preliminary and some important limitations must be addressed. The aim of this review was to discuss cell reprogramming in the post-Yamanaka era.