Gsk3b Inhibition Induces Teratoma Formation Figure 5. Screening of factors responsible for the tumor-like growth of mouse embryonic stem cells. (A): Cumulative numbers of mESCs were compared among ESCs cultured under each condition indicated for 3 passages. Cell counts were normalized to CDSF conditions. A value for CDSF conditions is normalized to 1. Parentheses indicate the number of biological replicates per condition. Standard errors of the means are indicated by bars. +RA, CDSF with retinoic acid; +RL, CDSF with retinol; +RL+RBP, CDSF with retinol and retinol binding protein; +Bmp4, CDSF with Bmp4; +iGsk3b, CDSF with the Gsk3b inhibitor. (B): Total numbers of biological replicates that resulted in formation of teratomas were compared among mESCs cultured under each condition indicated. Orange and yellow boxes indicate the number of biological replicates that developed into teratomas within 6 months and in more than 6 months, respectively. Blue bars indicate the number of biological replicates that failed to form teratomas for more than 6 months. Data for the Gsk3b inhibitor include results obtained with R1 and W4 mESCs. For those biological replicates indicated as (i), (ii) and (iii) in the bar chart, the number of technical replicates, the number of days needed for the experimental animals to reach their end points, and the sizes of the longest axis of the teratomas are shown per technical replicate. Asterisks (*) indicate that W4 mESCs were transplanted. n.d., not determined. (C, top): NOD-SCID mice that received transplantation of mESCs cultured in CDSF supplemented with Bmp4 (right) or the Gsk3b inhibitor (left) are shown. Bars, 1 cm. (C, bottom): Representative histological images are shown. The presence of three germ layers (see the legend for Fig. 2) is evident in teratomas developed from mESCs cultured in CDSF supplemented with the Gsk3b inhibitor. Bars, 20 mm. (D): Abundance of each transcript indicated above was examined in mESCs cultured under each condition on the right by 24 cycles of PCR. Ef1a was used as a reference. White arrowheads indicate the PCR product of c-Myc. doi:10.1371/journal.pone.0021355.g005 On the other hand, pharmacological inhibition of Gsk3b supports the self-renewal and pluripotency of ESCs [22,23,24,25]. Now, the question is what Gsk3b upstream and downstream genes are in self-renewing ESCs. Both LIF-Stat3 [73,74] and insulin pathways activate the PI3K- Akt1 signaling pathway [24,68], which mediates the inactivation of Gsk3b [24,40,64]. However, CDSF includes LIF and insulin [26], and failed to support the tumor-like growth of mESCs (Fig. 1 and 4). Thus, LIF and insulin are not the upstream of Gsk3b. Because Eras activates Akt1 [38], the downregulation of Eras in mESCs cultured in CDSF (Fig. 4C and 5D) may have resulted in the activation of Gsk3b. However, human ESCs do not express human ERAS but they do grow into teratomas [41,42]. Because this signaling cascade is not evolutionarily conserved, Eras is not the upstream of Gsk3b. Based on our results, Bmp4 poorly promoted proliferation and teratoma formation of mESCs cultured under CDSF conditions (Fig. 5A, 5B). It is well known that the Bmp4 and Wnt signals interact with each other in many morphogenetic events, which could result in either synergistic or antagonistic effects depending on cell types [69]. Because of the following two observations, we consider the effect of Bmp4 on Gsk3b or the tumor-like growth of mESCs antagonistic or indirect. First, our RT-PCR results revealed that mESCs cultured in CDSF supplemented with Bmp4 upregulated c-Myc, which was not the case in mESCs treated with the Gsk3b inhibitor (Fig. 5D and supporting information Fig. S5). Second, in one set of our experiments, Bmp4 efficiently induced formation of teratomas when ESCs at the earlier passage number (passage 7) were used for the culture (see (ii) in Fig. 5B). In contrast, when mESCs at passage 9 or later PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21355