Abstract
Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.
Highlights
In addition to the previously reported iPS cell lines, mouse embryonic fibroblasts (MEF), neural stem cells, and adult tail tip fibroblasts were collected from two mouse strains (B6 ϫ D2 F1 and B6 ϫ 129S2 F1)
By examining the miRNA expression differences, we identified a group of miRNAs with significantly lower abundances in the partially pluripotent stem cells as compared with the fully pluripotent cells (Fig. 1, B–D)
We identified an imprinted genomic region in mouse that was actively expressed in fully pluripotent stem cell lines, such as ES and 4n-iPS
Summary
Generation of iPS Cell Lines and iPS Mice—Five iPS cell lines and their derived iPS mice used in this study were reported previously [3]. Microarray Hybridization and Identification of Differentially Expressed Genes—Microarray hybridization data of all cell lines were obtained using identical procedures as described before [3]. Expression heat maps of genes and small RNAs within the imprinted region were drawn using the heatmap. package of R. Deep Sequencing Data Analysis, miRNA Identification, and miRNA Target Prediction—After removing adaptor sequences, the resulting Solexa sequencing data were mapped back to the mouse genome using BLAST; only sequences with perfect genomic matches were kept for future analysis. The 3Ј-untranslated region sequences of mouse genes were downloaded from the Ensemble database (NCBIM37). Genes whose 3Ј-untranslated region sequences contained at least two perfect complementary sequences to the 5Ј 2–8-nucleotide seed region of a miRNA were selected as putative miRNA targets. Pathway analysis was performed using the DAVID Bioinformatics Resources with the BioCarta pathway database [15]
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