Gsk3b Inhibition Induces Teratoma Formation Figure 1. The tumorigenicity of mouse embryonic stem cells can be reduced by short-term serum-free culture. (A): A mouse embryonic stem cell (mESC) line harboring an EGFP reporter driven by the Oct3/4 promoter (Oct3/4::EGFP) was maintained in either standard or chemically- defined serum-free (CDSF) medium as indicated. (B–E): Phase contrast (left) and fluorescence (right) images of the ESC line under the conditions indicated above are shown. Bars, 20 mm. (F–H): After the mESCs were cultured as indicated, they were transplanted into NOD-SCID mice subcutaneously. Teratoma formation was observed by week 11. Bars, 1 cm. doi:10.1371/journal.pone.0021355.g001 soon as two days after transfer to CDSF conditions (1.91 doublings 6 S.E.M. of 0.0967 vs. 2.72 doublings 6 S.E.M. of 0.0923 for the standard condition in 48 hrs, p,0.005). Despite the longer doubling time, mESCs cultured in CDSF did proliferate steadily (Fig. 4B, bottom). Additionally, transcripts associated with cellular pluripoten- cy, Sox2, Esg1/Dppa5 and Oct3/4, were expressed in mESCs cultured in CDSF (Fig. 4C). Interestingly, Eras, which regulates the tumorigenic growth of mESCs [38], was downregulated in these mESCs (Fig. 4C), whereas it became upregulated when mESCs were maintained in CDSF+FBS or under CDSF-Standard conditions (Fig. 4C, see also supporting information Fig. S5). Collectively, the loss of tumor-like potential in mESCs cultured under CDSF is associated with a slower growth rate and the reduced expression of Eras. It is interesting to note that Eras-null mESCs can contribute to the germline in chimeric animals but show significantly reduced growth rate [38]. Pharmacological inhibition of Gsk3b reversed the effect of serum-free culture on the tumor-like growth of mouse embryonic stem cells To identify a potential serum factor responsible for inducing the tumor-like growth in mESCs, initially we focused on molecules known to sustain the self-renewing growth of mESCs, such as LIF [9,10], Bmp4 [14], vitamin A derivatives (all-trans retinoic acid, RA [15], and retinol [16,17]), and simultaneous inhibition of Erk and Gsk3b [31]. Pharmacological inhibition of Gsk3b alone promotes self-renewal of both mouse and human ESCs [23]. We excluded LIF from screening because CDSF contains LIF [26]. In the absence of LIF, mESCs undergo differentiation or cell death [26,44]. We also ruled out an inhibitor of Erk [31], which acts downstream of FGF receptors, because inhibition of Erk cannot promote the growth of mESCs [31]. Therefore, we focused on testing other molecules such as Bmp4, RA, retinol with or without retinol binding protein (RBP) [55], and an inhibitor of Gsk3b (CIHR99021) [31]. Addition of RA in CDSF induced differentiation of mESCs as evidenced by the reduced expression level of EGFP (supporting information Fig. S3A). Addition of retinol with or without RBP in CDSF did not induce differentiation of mESCs (supporting information Fig. S3B, S3C), but failed to accelerate their growth and to induce teratoma formation (Fig. 5A, 5B). In contrast, mESCs cultured in CDSF with Bmp4 or the Gsk3b inhibitor maintained Oct3/4 expression (supporting information Fig. S3D, S3E), increased the number of cell doublings (Fig. 5A) and formed teratomas in 17% or 67% of transplantations by 7 months, respectively (Fig. 5B, 5C and supporting information Fig. S1F, PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21355