cells for tissue engineering or studying cellular reprogramming. Prior to clinical and/or research applications, putative GS/maGS colonies may be screened for imprinting status of imprinted miRNAs using sperm and ES cells as controls. An androgenetic imprinting status comparable to those of sperm would suggest that the colony originated from GS cells. However, it must be noted that imprinted miRNA is not a marker of stemness in the testes-derived male germ-line stem cells and therefore, may not be used as a tool for their isolation and/or identification. Supporting Information Figure S1 Characterization and in vitro differentiation of male germ-line (GS) and multipotent adult germ-line (maGS) stem cells in mouse. Embryonic stem (ES) cells were used as control for comparison. A: Colony characteristic, alkaline phosphatase (AP) activity and embryoid bodies generated from GS, maGS and ES cells. Calibration bar: 100 m. B: Expression of stem cell and germ-cell marker genes in undifferentiated colonies, embryoid bodies (EB) and all-trans retinoic acid -treated embryoid bodies (EBRA) of GS, maGS and ES cells. (TIF) Figure S2 DNA methylation status of Igf2-H19 gene cluster (A) and expression of Let-7a, Let-7d and miR-194 miRNAs (B) in multipotent adult germ-line (maGS) stem cells cultured in two different culture conditions. maGS (1): maGS cells cultured in the presence of GDNF, LIF and STO feeder cell; maGS (2): maGS cells cultured in feeder-free ES cell- like culture condition. Embryonic stem (ES; open box) cells were used as controls for comparison. Values above the bars indicate relative abundance of miRNAs normalized to the expression of References 1. de Rooij DG (2006) Rapid expansion of the spermatogonial stem cell tool box. Proc Natl Acad Sci U S A 103: 7939–7940. 2. Han SY, Gupta MK, Uhm SJ, Lee HT (2009) Isolation and in vitro culture of pig spermatogonial stem cells. 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