Differential Genomic Imprinting and Expression of Imprinted microRNAs in Testes-Derived Male Germ-Line Stem Cells in Mouse Ji Young Shin, Mukesh Kumar Gupta*, Yoon Hee Jung, Sang Jun Uhm, Hoon Taek Lee* Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, South Korea Abstract Background: Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings. Methods and Findings: Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P,0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P,0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA. Conclusions: Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells. Citation: Shin JY, Gupta MK, Jung YH, Uhm SJ, Lee HT (2011) Differential Genomic Imprinting and Expression of Imprinted microRNAs in Testes-Derived Male Germ-Line Stem Cells in Mouse. PLoS ONE 6(7): e22481. doi:10.1371/journal.pone.0022481 Editor: Joseph Najbauer, City of Hope National Medical Center and Beckman Research Institute, United States of America Received March 6, 2011; Accepted June 22, 2011; Published July 22, 2011 Copyright: 2011 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from BioGreen 21 Program (#PJ007188), Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: htl3675@konkuk.ac.kr (HTL); goops@konkuk.ac.kr (MKG) Introduction Germ-line stem (GS) cells, the in vitro counterpart of spermato- gonial stem cells (SSC) in the testis, can self-renew in vitro for more than two years and, when transplanted into the seminiferous tubules of an infertile male mouse, can establish donor-derived spermatogenesis to transmit the donor haplotype to progeny [1,2,3,4]. Upon extended in vitro culture, a second cell type, called multipotent GS (mGS; from neonatal testes) or multipotent adult GS (maGS; from adult testes) cells, also appears from GS cells that can be expanded selectively under culture conditions used for embryonic stem (ES) cells. Unlike GS cells, mGS or maGS cells show multipotency and produce teratoma upon transplantation into the seminiferous tubules of the recipient testis [5,6]. The mGS and maGS cells originate from the cultured GS cells themselves at a low frequency and are not some leftover of earlier type of germ cells [1,6,7,8]. During this conversion, the androgenetic genomic imprinting in GS cells also changes to ES cell-like pattern in mGS or maGS cells [6,9]. Recently, we showed that mouse maGS cells are epigenetically stable for DNA methylation at imprinted Igf2- H19 gene cluster during in vitro culture and differentiation [10] but re-acquire GS cell-like growth and differentiation characteristics with altered DNA methylation pattern when they are re-cultured in the GS-like conditions [11]. Thus, at any particular time point, in vitro cultured GS cells may contain some contaminating mGS or maGS cells which may produce teratoma instead of initiating spermatogenesis upon their transplantation into recipient testis [5,6,11]. Consequently, a molecular marker that can distinguish GS cells from mGS or maGS cells would be of potential value in both clinical and experimental research settings. MicroRNA (miRNA) are a class of 20–25 nucleotide-long non- coding endogenous RNAs that post-transcriptionally modulate the gene expression through canonical base pairing between the seed sequence of the miRNA (nucleotides 2–8 at its 59 end) and its complementary seed match sequence in the 39UTR of target mRNAs [12]. Imprinted miRNAs represent a family of miRNA that are mono-allelically expressed in a parent-of-origin manner and act in trans, generally outside the genomic region from where PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22481