were cultured in CDSF with Bmp4 and transplanted, they sporadically developed into teratomas. Further investigation is required to determine the upstream of Gsk3b in ESCs. We showed that a CDSF medium supplemented with Bmp4 and LIF supported the proliferation of mESCs that maintained transcriptional activity of Oct3/4 (supporting information Fig. S3D). In contrast, Hayashi et al. demonstrated that a CDSF medium supplemented with Bmp4 supported the differentiation of trophoblasts from mESCs [47]. However, Hayashi et al. used a basal CDSF medium that lacked oleic acid and LIF, and plated mESCs on laminin-coated dishes, but not on collagen IA-coated dishes. In addition, the presence of LIF under these CDSF conditions inhibited the differentiation of trophoblasts [47]. Therefore, it is likely that the different chemical composition of a basal medium contributed to the differentiation of trophoblasts when Bmp4 was supplemented in the culture. It is interesting to note that in concert with LIF, Bmp4 suppresses differentiation of the neural lineage in mESCs maintained under serum-free conditions [14] supplemented with N2 and B27, which were originally developed to culture a neuroblastoma cell line [56] and hippocampal neurons [57], respectively. Mouse ESCs maintained under serum-free condi- tions supplemented with LIF, Bmp4, N2 and B27 are pluripotent and can contribute to the germline in chimeric animals [14], and grew into teratomas (supporting information Table S1 and Fig. S4). On the other hand, we showed that both the CDSF medium used in the present study, which contains LIF, and the CDSF medium supplemented with Bmp4 poorly sustained the tumor- like growth of mESCs. Taken together, it is suggested with these data that the cellular pluripotency and the tumor-like growth of ESCs may be regulated by different mechanisms, and that extrinsic factors play significant roles in cell fate decisions of mESCs. It will be interesting to investigate whether ESCs need unique substrate stiffness to grow into teratomas, because our previous study showed that stiffer substrates promoted differen- tiation of mESCs [75]. Gsk3b inhibits the activity of its target c-Myc [24,76], which is involved in the self-renewal of mESCs [58] and responsible for an age-associated incidence of tumorigenesis in chimeric mice generated with mouse iPSCs [77,78]. In contrast, our RT-PCR results showed that mESCs cultured under CDSF conditions with the Gsk3b inhibitor failed to significantly upregulate transcription of Eras and c-Myc (Fig. 5D) but efficiently developed into teratomas (Fig. 5B, 5C). Thus, we propose that Gsk3b downstream genes other than c-Myc may regulate the tumor-like growth of ESCs, which play independent roles from maintaining the self-renewal of ESCs. In addition, candidate Gsk3b downstream genes responsi- ble for tumorigenesis in mESCs may act independently from ERAS in human ESCs [41,42]. Currently, our investigation is ongoing. Further study to identify the candidate genes and to test their roles in human ESCs and iPSCs will lead us to establishing a strategy to significantly improve the safety of human iPSCs. Materials and Methods Ethics statement Use of animals was approved by the Illinois Institutional Animal Care and Use Committee (protocol # 10093, approved on 6/18/ 10). Cell culture A mouse embryonic stem cell (mESC) line of R1 [79], which expresses EGFP under the Oct3/4 promoter [80], was kindly provided by Dr. William L. Stanford [51]. This Oct3/4::EGFP Gsk3b Inhibition Induces Teratoma Formation mESC line was thawed on feeders and maintained under standard [81] or chemically-defined serum-free (CDSF) [26] conditions at 37uC, 5% CO2. The exact number of passages that this mESC line has gone through is unknown. However, when these ESCs were brought to our laboratory, they were passaged on feeders two more times and frozen as a stock. When these ESCs were thawed on feeders, more frozen stocks were made at passage 5–7. These stocks were used for the current study. Under standard conditions, mESCs were maintained on 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com)-coated tissue culture dishes in high glucose-Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supple- mented with 15% fetal bovine serum (FBS; Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 2 mM GlutaMax I (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), and 1,000 U/ml LIF (Millipore, Billerica, MA, http://www.millipore.com). When the mESCs reached 80% to 100% confluence, they were routinely passaged at a ratio of 1:6 every two days using TrypLETM Express (Invitrogen). The cells were discarded after being passaged 10 times onto gelatin-coated dishes. Approxi- mately 16105/cm2 mESCs maintained under standard condi- tions were plated onto tissue culture dishes coated with 0.15 mg/ml type IA collagen (Nitta Gelatin Co., Osaka, Japan, http://www.nitta-gelatin.co.jp), which contain the ESF7 medi- um (Cell Science & Technology Institute Inc. Miyagi, Japan, http://www.cstimedia.com) [26] supplemented with 1,000 U/ ml LIF. This was counted as passage 1 under CDSF conditions. Mouse ESCs at passage 7–12 were used to start CDSF culture. Mouse ESCs grown under CDSF conditions were split every 3 or 4 days with 0.02% EDTA (Sigma-Aldrich). Similarly, mESCs of W4 (129S6, purchased at passage 9; Taconic, Hudson, NY, http://www.taconic.com) and D3 (129S2/SvPas, CRL-1934, ATCC, Manassas, VA) were used to test CDSF conditions. When CDSF culture was supplemented with FBS, 0.02% EDTA was used for passaging cells. Following serum lots were used to supplement CDSF culture: Lot. 1359246 and 726570, Invitrogen; Lot. L0228, Atlanta Biologicals (Lawrenceville, GA, http://www.atlantabio.com); Lot. A74B00Z, Gemini Bio-Prod- ucts (West Sacramento, CA, http://www.gembio.com). Images of cell morphology and fluorescence were taken under the same conditions using an inverted microscope equipped with an epi- fluorescence lamp (DMI4000B, Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com) [82,83]. To measure the frequency of cell doubling, the mESCs were plated at 16106 per one well of 6-well plates. Two days after plating, the number of cells was counted for the second and third passages. Cell doubling was calculated based on the following formula: Cell doubling = log2[(the number of cells 2 days after plating)/(16106)]. Statistical tests were performed using the Mann-Whitney’s U-test. To compare the incidence of teratoma formation, another basal culture medium was prepared by mixing the NeurobasalTM medium supplemented with B27 (Invitrogen) 1:1 with DMEM/ F12 (Invitrogen) supplemented with N2 (Invitrogen) and 50 mg/ml bovine serum albumin as described already [84]. The resulting basal medium was supplemented with either 10 ng/ml LIF and 10 ng/ml Bmp4 (R & D systems) [14], or 1 mM Stemolecule TM PD0325901 and 3 mM Stemolecule TM CHIR99021 (2i; Stemgent, Cambridge, MA, https://www.stemgent.com/) [22], and used to culture mESCs for three passages, which needed seven days before subcutaneous injection into NOD-SCID mice. PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21355