Gsk3b Inhibition Induces Teratoma Formation Figure 4. Phenotypic changes observed in mouse embryonic stem cells cultured under CDSF conditions. (A): The sizes of the teratomas formed (orange bar, left axis) and the number of days required for the experimental NOD-SCID mice to reach their end points (blue stars, right axis) were compared among mESCs cultured under the conditions indicated (see Fig. 1A). Parentheses indicate the number of biological replicates (i.e., mESCs prepared at different passages) per culture condition. Standard errors of the means are indicated by bars. a: Only one passage in standard conditions followed CDSF culture. b: Two out of seven transplantations showed no sign of teratoma formation when paired with the standard and CDSF-Standard conditions, whereas five out of seven transplantations showed no sign of teratoma formation for 6 months. (B, top): Cell doublings were measured every 48 hours after plating 16106 cells per well onto 6-well plates (see Materials and Methods for the formula). Only CDSF conditions produced statistically-significant differences compared to standard conditions. Parentheses indicate the number of biological replicates per condition. Standard errors of the means are indicated by bars. (B, bottom): Phase contrast (top) and fluorescence (bottom) images are shown for the Oct3/4::EGFP mESC line (Fig. 1A) grown under the conditions indicated below 1 and 2 days after plating 0.16106 cells per well in 6-well plates. Bars, 50 mm. (C): Abundance of each transcript indicated above was examined in mESCs cultured under each condition on the right by 25 cycles of PCR. Ef1a was used as a reference. doi:10.1371/journal.pone.0021355.g004 Gsk3b. Our present study indicates that downstream of Gsk3b is primarily responsible for tumorigenesis in mESCs, which may involve uncharacterized gene products. Although the exact mechanism currently remains unknown, our present study provides a basis for further study to establish the signaling pathway responsible for the tumor-like property of ESCs. In general, serum provides hormones, growth factors, and steroids to cultured cells. It also contains remnants of plasma components used for the activation and processing of blood clots and substances that do not normally pass through the endothelial barrier [59,60,61]. Therefore, serum is similar but not identical to the interstitial fluid (i.e., lymph) that surrounds cells in vivo [62]. We were unable to observe any cellular mass at the injection sites that were derived from mESCs cultured in CDSF at either one week or six months after transplantation. Therefore, it is suggested with our data that interstitial fluid will not support the tumor-like growth of mESCs. Perhaps mESCs cultured under short-term CDSF conditions became more susceptible to LIF and exhibited cell death after transplantation due to the absence of a continuous supply of LIF [26]. However, because each mESC line shows a different degree of LIF dependency [54], other mouse pluripotent stem cells cultured in the serum-free medium may exhibit a capability to continuously grow after transplantation, as we have seen in embryonic carcinomas (F9; supporting information Fig. S1D) and germline-incompetent mESCs, D3 (data not shown). In addition, we found that the formation of teratomas became sporadic when we transplanted 0.56106 mESCs cultured under standard conditions for a period of 6 months. Collectively, our study demonstrated that one or more extrinsic factors or niche [43] plays an important role in the formation of a teratoma. This idea is further supported by the fact that two mESCs were sufficiently able to grow into a teratoma only when mixed with 26106 non- tumorigenic fibroblasts (MRC-5) prior to transplantation into immunocompromised mice [63]. Interestingly, the slow growth observed in Eras-null mESCs became more evident when they were cultured without feeder cells [38]. Our present data indicate that animal sera contain one or more factors that inhibit the activity of the Gsk3b protein (Fig. 1, 4 and 5). Gsk3b is involved in the canonical Wnt signaling pathway [64,65,66] and interacts with other biologically important signaling pathways such as phosphoinositide 3-kinase (PI3K)- Akt1 [40,67,68], Bmp4 [69] and hedgehog [70,71,72] signaling pathways. Although the secreted protein Wnt eventually inhibits the activity of Gsk3b, the role of Wnt in the maintenance of self- renewal and pluripotency of ESCs remains elusive [20,21,22,23]. PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21355