We also evaluated whether expression of imprinted miRNAs in mouse GS and maGS cells alters during their in vitro differenti- ation. As expected, stem cell (Oct4, Nanog, Cd9, Rex1), and GS cell (Stra8, Cd9) marker genes were silenced during differentiation of GS and maGS cells but was not complete in ES cells (Supplementary Figure S1). During differentiation, expression of imprinted miRNAs also underwent changes but varied both with the differentiation stage and the miRNA (Figure 4). Given that the EBs contain a mixed population of cells from three germ layers [39] and that the expression of individual imprinted miRNA may vary with the cell/tissue types wherein they may have different functions [14,15,20,21,23,24,26], the differential expression pat- tern of imprinted miRNAs in differentiating GS, maGS and ES cells may reflect their differential ability to differentiate into various cell types [11,40,41,42,43]. Interestingly, although a clear pattern was not evident, changes in the expression pattern of imprinted miRNA during in vitro differentiation of maGS cells were apparently more similar to those of ES cells and clearly differed from differentiating GS cells (Figure 4). This observation is similar to those of Zovoilis et al., [28,44] who found that maGS cells resembled ES cells, but the expression of certain miRNAs such as miR-290 cluster was retained during their in vitro differentiation. We also observed that, similar to previous reports on several miRNAs [45,46], mature miRNA originating from both 39 (miR-296-3p) and 59 (miR-296-5p) arms of the miR-296 accumulated as sister pairs in undifferentiated testis-derived germ- line stem cells (Figure 1B). However, their expression pattern differed among the differentiating cells of the three groups (Figure 4A and 4B) and, the EBs generated from GS cells resembled those of ES cells for the expression pattern of miR-296- 3p. Since the phenomenon of miRNA strand selection for the functional stability (-3p vs. -5p vs. both) occurs in a tissue- dependent manner [45,46,47], the differences in the expression of miR-296-3p and miR-296-5p among the EBs of the three groups probably reflects the different proportion of cells of three germ- layers in them [11,39,40,41]. It was also observed that, differentiating EBs generated from GS cells had significantly high level of miR-127 and miR-127-5p (Figure 4C and 4D), which might suggest their possible role during in vitro differentiation of SSC. Our result is similar to a previous study which showed high expression of miR-127 in testicular samples [46]. The miR-127 was preferentially expressed in immature mouse testes that principally contained mitotically active spermatogonia (day 7), meiosis I spermatocyte (day 12) and round spermatid (day 21), but remained at medium level in purified pachytene spermatocyte and round spermatid and declined in adult testis (containing fully differentiated germ cells and spermatozoa) [46]. However, the mechanism by which miR- 27 affects the SSC biology remains unclear. It is likely that miR- 127 may act by down-regulating its target proteins such as Bcl6 [48] and Brd2 [49] which are important for GS cell self-renewal and for the inhibition of differentiation, respectively [50,51,52,53]. The Brd2 was also shown to be expressed at high levels in diplotene spermatocytes and round spermatids and at low levels in spermatogonia that negatively co-related with the expression pattern of miR-127 [49,53]. The functions of imprinted miRNAs in testis-derived male germ-line stem cells are not known. They acts in trans, generally outside the genomic region from where they arise [13,14], and may even cleave the mRNAs encoded by the same imprinted gene cluster in partner chromosome (for example, maternally expressed miRNAs can silence a paternally expressed gene or vice versa) in a RNAi-like manner [23,54,55]. Liu et al., [22] found that imprinted miRNAs encoded by Dlk1-Dio3 locus (e.g. miR-127 and Imprinted miRNA Signature in Male GS Cells miR-127-5p) had 717 putative targets that were related to multiple aspects of growth, differentiation, metabolism and other develop- mental processes in pluripotent cells. Furthermore, several miRNAs from this cluster potentially target the PRC2 silencing complex to form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this locus [22]. On the other hand, Gnas-Nespas cluster encode miR- 296 and miR-298 which are derived from non-coding Nespas gene transcript. The miR-296 regulates the expression of growth factor receptor in endothelial cells [56] and increases upon in vitro differentiation of ES cells to target the Nanog gene transcript [57]. The Igf2-H19 cluster encodes miR-675 and miR-483 but their precise role in stem cells is not known. An in silico bioinformatic analysis using web-based TargetScan (URL: http://www.targetscan. org/mmu_50/) and MicroCosm Targets Version 5 (URL: http:// www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) soft- wares showed that miR-483 has numerous putative targets (,975 in mouse and ,1072 in human) that included Jarid1b (data not shown). Jarid1b (Histone demethylase KDM5b) directly regulates genes that control cancer cell proliferation [58] and may be essential for the stem cell pluripotency [57,59]. Although Jarid1b is yet to be validated as a target of miR-483, we found that consistent with the high expression of miR-483 in GS cells, the expression of Jarid1b was significantly lower in GS cells than in maGS or ES cells (Figure 5). Moreover, consistent with the expression of Jarid1b, GS cells proliferated slower than maGS cells (,4–6 days for GS cells and ,2–3 days for maGS cells), as has also been reported earlier [11,60]. In conclusion, our data suggest that genomic imprinting and expression of imprinted miRNAs are androgenetic in mouse GS cells but changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus serve as epigenetic signature or molecular marker to distinguish GS cells from maGS or ES cells. Since maGS cells originate from GS cells during their extended in vitro culture [1,5,6,7,8,11], our data may have implications in clinical settings to distinguish GS cell colonies from maGS cell colonies and thereby minimize the likelihood of teratoma formation by contaminating maGS cells generated from the GS cells. Conversely, in experi- mental research settings and regenerative medicine, analysis of imprinted miRNA may help in discriminating maGS cells from GS Figure 5. Expression of Jarid1b, a putative target of miR-483 miRNA in male germ-line (GS) and multipotent adult germ-line (maGS) stem cells. Embryonic stem (ES) cells were used as control for comparison. Values above the bars indicate relative abundance of mRNAs normalized to the expression of respective mRNA in undiffer- entiated ES cells. Bars with different alphabet (a, b, c) differ significantly (P,0.01). doi:10.1371/journal.pone.0022481.g005 PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22481