Human embryonic stem cells
(hESCs) have gained popularity as a potentially ideal cell candidate for regenerative medicine. First isolated by James Thomson and colleagues in 1998 [1
], hESCs are derived from the inner cell mass of the human blastocyte and can be kept in an undifferentiated, self-renewing state indefinitely. In contrast to adult stem cells, hESCs have the advantage of being pluripotent, which endows them with the ability to differentiate into virtually every cell type in the human body. However, the use of human embryos is controversial in the United States, and potential tissue rejection following transplantation in patients remains problematic [2
One way to circumvent these issues is to generate induced pluripotent stem cells (iPSCs). Mouse and human cells can be reprogrammed to pluripotency through ectopic expression of defined transcription factors [3
]. The first successful reprogramming of human fibroblast cells into iPSCs was reported independently by Shinya Yamanaka (using OCT4, SOX2, KLF4, c-MYC) [8
] and James Thomson (using OCT4, SOX2, NANOG, LIN28) [12
]. The main advantage of this approach is that it does not need human embryos or oocytes to generate patient-specific stem cells, and therefore can potentially bypass the ethical and political debates that have surrounded this field for the past decade. Another important benefit is that for the first time, disease-specific stem cells can be created, which will help scientists understand the molecular mechanisms of many common inherited diseases [13
For a number of reasons, these reprogramming methods have so far been gradual and slow, requiring weeks of cell culture with very low yield of iPSCs [4
]. Inefficient delivery of factors to the cells is certainly one obstacle, and this challenge is being actively addressed by many groups. Another obstacle is the general lack of understanding of the molecular changes that underlie reprogramming [16
]. Understanding the molecular circuitry of reprogramming will greatly benefit the field by providing new targets and pathways that could increase the yield of iPSCs. Efforts to better integrate the genomic and epigenomic networks that control reprogramming have been undertaken [18
], but overall the specific mechanisms required for more efficient reprogramming remain elusive.
One potential regulatory mechanism of reprogramming that has so far received little attention is microRNAs (miRNAs). These small, noncoding RNAs play important posttranscriptional regulatory roles by targeting messenger RNAs (mRNAs) for cleavage or translational repression [19
], and are key components of an evolutionarily conserved system of RNA-based gene regulation in eukaryotes [20
]. Interestingly, hESCs are known to express miRNAs that are often undetectable in adult organs such as miR-371, miR-372, miR-302a, miR-302b, miR-302c, and miR-302d [21
], whereas Dicer-deficient murine embryonic stem cells (ESCs), which cannot generate miRNAs, have been shown to be defective in differentiation [26
]. These and other studies suggest that miRNAs likely play key roles in human and murine ESC gene regulation [24
]. A recent study has attempted to incorporate miRNA gene regulation into a model of transcriptional regulatory circuitry of ESCs by generating genome-wide maps of binding sites for key ESC transcription factors such as Oct4, Sox2, and Nanog [33
]. These ESC transcription factors were found to bind at many start sites of miRNA transcripts that have been detected in ESCs, such as the miR-302 cluster. Clearly, at least a subset of miRNAs seems to be involved in pluripotency, and these studies have contributed greatly to the understanding of miRNA networks in ESCs.
While significant efforts are being applied to ESCs, no study has yet analyzed the miRNA profile of human iPSCs. Determining the miRNAs that are associated with reprogramming may yield significant insight into the specific miRNA expression patterns needed for pluripotency. In this report, we use miRNA microarrays to compare the “microRNA-omes” of human iPSCs, hESCs, and fetal fibroblasts. We confirm the presence of a signature group of miRNAs that are up-regulated in both iPSCs and hESCs, such as the miR-302 and 17–92 clusters, as well as some subtle differences between the two pluripotent cell types, including the miR-371/372/373 cluster. We also note the broad changes in miRNA patterns between pluripotent cells and differentiated fibroblasts. These miRNA profiles are an initial step toward a better understanding of the regulatory networks that govern pluripotency and reprogramming.