Profiling of MicroRNA in Human and Mouse ES and iPS Cells Reveals Overlapping but Distinct MicroRNA Expression Patterns

<div><p>Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.</p></div

Alteration of level of miRNAs during formation of human EB.

<p>Human EB was formed, and expression profile of miRNAs was examined at day 7, 14, and 21 after starting formation of EB. Levels of miRNAs were categorized by patterns of changing of expression (ΔCt values).</p

Cell lines used in this study.

<p>Abbreviation: OSKM; Oct3/4, Sox2, Klf4, c-Myc, retroV; retrovirus, sendai V; sendai virus, RI; Rock Inhibitor.</p

Comparison of miRNA expression levels of ES and iPS cells.

<p>A, B. Average ΔCt of human (A) or mouse (B) ES and human (A) or mouse (B) iPS are shown. miRNAs that have less than 0.05 p value between ES and iPS cells are listed by their miRNA number. Clustered miRNAs are indicated by violet under bar. p value: non marked bars <0.01. *(red asterisk) <0.05.</p

Comparison of expression level of miRNAs in human pluripotent stem cells and human somatic cells.

<p>Expression level of miRNAs in various cells was examined by qPCR based array, and ΔCt value was calculated as described. Average of ΔCt of immature ES/iPS (hES/hiPS-g, blue bar) and somatic cells and iPS derived NSCs (hSomatic-g, red bar) was calculated with standard deviation. A. Top 50 miRNAs from the highest expression level in ES/iPS are shown. miRNAs labeled with “H” are human specific miRNAs. B, C. Top 50 miRNAs from biggest difference of ΔCt between hES/hiPS-g and hSomatic-g are shown as well as values in hES/hiPS-g is bigger (B) and in hSomatic-g is bigger (C). In A–C. p value: non marked bars <0.01. * <0.05, ns not significant. Purple asterisk indicates miRNAs which have suppressive to p21 expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073532#pone.0073532-Jung1" target="_blank">[28]</a>.</p

Comparison of miRNAs between human and mouse pluripotent stem cells.

<p>A. Top 60 miRNAs from lowest average ΔCt value of human and mouse pluripotent stem cells are listed. Ranking number in human and mouse are compared, and additional information are as indicated in the panel below of the figure. B. Expression levels (ΔCt) of miRNAs which are differentially expressed in human and mouse ES/iPS. Listed miRNAs are labeled with pink color but not with green asterisk in the panel A, which means miRNAs with more than 50 difference in mouse and human ranking and at the same time, having more than 2ΔCt value difference with somatic cells.</p

Human miRNAs which have eigenvectors of the first component more than 2 in absolute value.

<p>Human miRNAs which have eigenvectors of the first component more than 2 in absolute value.</p

Clustering analyses of expression pattern of miRNA in mouse pluripotent cells, differentiated cells, and somatic cells.

<p>A. Comparison of relative expression levels of 201 miRNAs in mouse pluripotent cells, and somatic cells. Red indicates low expression and green indicates high expression. B. Consensus clustering of cells by NMF with two metaprofiles. The NMF-transformed miRNA profiles distinguished two clusters. C, D. PCA was done using the same set of miRNA above. The first three components clustered cells (the first and second in C, the second and third in D) are shown. The first component accounts for 41.99%, the second for 16.62% and the third for 11.08% of the system variance. E. Expression levels of newly identified miRNAs are shown.</p

Mouse miRNAs which have eigenvectors of the first component more than 2 in absolute value.

<p>Mouse miRNAs which have eigenvectors of the first component more than 2 in absolute value.</p

Angiographic Score after Implantation of MSCs.

<p>(A and B) Representative angiography at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia, (C) Angiographic score at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia. *P<0.05 vs. the control group.</p