Gsk3b Inhibition Induces Teratoma Formation Figure 2. Identification of three germ layers in teratomas. (A–E): Teratomas developed from mESCs cultured under standard conditions. (F–J): Teratomas developed from mESCs cultured in CDSF supplemented with 15% FBS (CDSF+FBS). (K–O): Teratomas developed from mESCs cultured in CDSF followed by transfer to standard conditions (CDSF to Stand). Ectoderm is represented by neural tissue and keratin pearl, mesoderm is represented by cartilage and striated muscle, and endoderm is represented by ciliated epithelium. Bars, 20 mm. doi:10.1371/journal.pone.0021355.g002 S1G). When cultured in other established CDSF media supple- mented with N2 [56], B27 [57], and either Bmp4 and LIF [14], or pharmacological inhibitors of Erk and Gsk3b [22], mESCs grew into teratomas more efficiently (see ‘‘N2B27-BL’’ and ‘‘N2B27-2i’’ in supporting information Table S1 and Fig. S4). Also, W4 mESCs exhibited similar phenotypic changes when maintained in CDSF with the Gsk3b inhibitor (Fig. 5B). Gsk3b is known to regulate the activity of the c-Myc protein in mESCs [24,58]. However, RT- PCR analysis showed that inhibition of Gsk3b did not result in upregulation of Eras and c-Myc in mESCs cultured in CDSF, whereas Bmp4 induced upregulation of c-Myc (Fig. 5D and supporting information Fig. S5). Discussion In this report, we present experimental evidence to suggest that short-term CDSF culture reduces the tumor-like growth of mESCs, which is reversed by pharmacological inhibition of Figure 3. Mouse embryonic stem cells cultured under CDSF conditions are pluripotent. (A–D): Mouse ESCs cultured under CDSF conditions were immunostained (right) with either specific antibodies against Nanog (A), Sox2 (B) and SSEA1 (C), or normal mouse serum (D). Also, phase contrast (left), and DAPI-stained (middle) images of these mESCs are shown. Bars, 20 mm. (E): Phase contrast (left) and fluorescence (right) images of a chimeric blastocyst aggregated with the Oct3/4::EGFP mESC line. Bar, 20 mm. (F): A phase contrast image of a male gonad dissected from a chimeric E13.5 embryo. The rectangle indicates the area shown in G. Bar, 100 mm. (G): Enlarged phase contrast (left) and fluorescence (right) images of the male gonad shown in F. Bar, 50 mm. doi:10.1371/journal.pone.0021355.g003 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21355