Imprinted miRNA Signature in Male GS Cells culture conditions or physiological function of the miRNA [11,22,25,29,37]. The DNA methylation and expression of imprinted miRNA in maGS cells may also be modified by the level of reprogramming [22,25]. In particular, imprinting status of the imprinted miRNAs in the Dlk1-Dio3 locus was shown to correlate with the pluripotency levels of the mouse stem cells [22,25]. However, we found that, unlike partially reprogrammed iPS cells, mouse maGS cells did not show aberrant DNA methylation at Dlk1-Dio3 IG-DMR and had hemi-zygotic methylation pattern (59.2% vs. 46.3%) of ES cells. Ability to generate partial, incomplete and fully reprogrammed maGS cells in future may help determine, if imprinting status of Dlk1-Dio3 IG- DMR could be an indicator of reprogramming in maGS cells, as has been reported earlier for the ES cells [22,25]. The difference in DNA methylation and miRNA expression was not due to difference in their chromosomal composition as both GS, maGS and ES cells, used in this study, were of male origin and contained XY sex chromosomes. Figure 4. Expression of miR-296-3p (A), miR-296-5p (B), miR-127 (C), miR-127-5p (D) and miR-483 (E) imprinted miRNAs during in vitro differentiation of male germ-line (GS) and multipotent adult germ-line (maGS) stem cells. Embryonic stem (ES) cells were used as control for comparison. Undifferentiated colonies were induced to form embryoid bodies (EB) by hanging drop method for 4 days and further treated with all-trans retinoic acid (EBRA) for another 4 days. Values above the bars indicate relative abundance of miRNAs normalized to sno202 RNA as internal controls and calibrated on the expression of respective miRNA in undifferentiated colonies of respective group. Different alphabet (a, b, c) on the bars indicate statistical difference (P,0.01) in gene expression within GS, maGS or ES cell group. doi:10.1371/journal.pone.0022481.g004 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22481