Abstract
Induced pluripotent stem (iPS) cells were first generated by forced expression of transcription factors (TFs) in fibroblasts. Recently, iPS cells have been generated more rapidly and efficiently using miRNAs with or without other transcription factors. However, the specific and collaborative roles of miRNAs and transcription factors in pluripotency acquisition and maintenance remain to be further investigated. Here, based on the miRNA profiling in mouse embryonic fibroblasts (MEFs), MEFs infected with Oct3/4, Sox2, Klf4 and c-Myc (OSKM) for 1, 2, 4, or 8 day, two iPS cell lines and ES cells, representing iPS activation and maintenance steps, we found that two unique miRNA sets are responsible for different steps of iPS generation, and the miRNA expression profiles of iPS cells are very similar to that of ES cells. Furthermore, we searched for transcription factors binding sites at the promoter regions of up-regulated miRNAs, and found that up-regulated miRNAs such as the miR-429-200 and miR-17 clusters are directly activated by exogenous TFs. The GO and pathway enrichment for candidate target gene sets of miRNAs or OSKM provided a clear picture of division and collaboration between miRNAs and OSKM during completion of the iPS process. Compared with the pathways regulated by OSKM, we found that miRNAs play critical roles in regulating iPS-specific pathways, such as the adherens junction and Wnt signaling pathways. Furthermore, we blocked miRNA expression using Dicer knockdown, and found that the level of miRNAs was decreased following this treatment, and the efficiency of iPS generation was significantly repressed. By combining high-throughput analysis, biostatistical analysis and functional experiments, this study provides new ideas for investigating the important roles of miRNAs, the mechanisms of miRNAs and related signaling pathways, and the potential for many more applications of miRNAs in somatic cell reprogramming.
Highlights
Mouse embryonic fibroblasts (MEFs) can be successfully reprogrammed to a pluripotent state using four transcription factors (TFs): Oct3/4, Sox2, Klf4 and c-Myc (OSKM), which are identified as reprogramming factors [1]
Even though induced pluripotent stem cells (iPS) cells are considered more similar to embryonic stem cells (ES) cells than to MEFs, iPSC1 cells are more similar to iPSC2 cells than to ES cells (Figure 1B), which is consistent with previous reports [18,19]
Earlyinfected series cluster more closely with MEFs than with iPS and ES cells, which in agreement with Pearson correlation analysis (Figure 1B). These results indicate that the iPS process is long and continuous, in which exogenous transcription factors gradually regulate gene expression patterns to that of ES cells, resulting in pluripotency
Summary
Mouse embryonic fibroblasts (MEFs) can be successfully reprogrammed to a pluripotent state using four transcription factors (TFs): Oct3/4, Sox, Klf and c-Myc (OSKM), which are identified as reprogramming factors [1]. In 2007, the generation of human induced pluripotent stem cells (iPS) was reported [2,3]. As iPS cells exhibit pluripotency and an infinite capacity for selfrenewal like embryonic stem cells (ES), and can be established from somatic cells isolated from patients, they are expected to provide new opportunities for disease modeling, the screening of new drugs and personal clinic treatment. Recent reports have revealed that iPS cells can be generated more rapidly and efficiently by miR-302/367 without any transcription factors, than by OSKM factors [12], indicating a previously known and important role of miRNAs in iPS reprogramming
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