animals, is particularly useful and widely accepted for the validation of pluripotency in human ESCs and iPSCs [13,36]. However, their tumor-like growth hampers the therapeutic application of human iPSCs [37]. Little is known about the inherent tumorigenic property of ESCs, except that the oncogene Eras regulates the tumor-like growth of mESCs via activation of Akt1 [38], which may result in inactivation of Gsk3b [24,39,40]. However, human ESCs do not express human ERAS [41,42], but grow into teratomas [3]. Therefore, the underlying mechanism involved in the tumor-like growth of ESCs remains unknown. On the other hand, mESCs contribute to the development of normal chimeras, instead of forming teratomas, when mixed with mouse preimplantation embryos. This finding indicates that mESCs require proper extrinsic signals or niches [43] to differentiate normally and to contribute to the development of chimeras. In contrast, mESCs exhibit cell death when they are cultured without LIF [26,44]. This result raises the question of whether mESCs inherently possess the tumor-like property or are provided with extrinsic signals that promote their tumorigenesis. In this report, we present experimental evidence that short-term serum-free culture reduces the tumorigenicity of mESCs, which is reversed by pharmacological inhibition of Gsk3b. It is suggested with these data that the activity of Gsk3b orchestrates the tumor- like growth of ESCs, which may support a novel mechanism independent from the one regulating the pluripotency of ESCs. Results Mouse embryonic stem cells reduced their tumorigenicity but maintained their pluripotency under chemically-defined serum-free culture To examine whether mESCs are determined to grow as teratomas, we cultured mESCs in chemically-defined serum-free medium with LIF (referred to as ‘‘CDSF’’) [26] for three passages (Fig. 1A, 1C) and subcutaneously transplanted them into non- obese diabetic mice with severe combined immunodeficiency disease (NOD-SCID mice) [45]. We used a uniquely formulated serum-free medium, ESF7 (see Materials and Methods), because components in this medium is fully disclosed [26]. This medium has been used to maintain mESCs by other studies (e.g., [46,47]). However, the pluripotency of mESCs cultured under this medium has not been tested by teratoma formation assays. Surprisingly, these mESCs failed to grow as teratomas (Fig. 1F-1H), whereas mESCs maintained under standard conditions (Fig. 1A, 1B) grew into a well-developed teratoma in 5 weeks (Fig. 1F, 2A–2E, and supporting information Fig. S1A). When mESCs were cultured in CDSF supplemented with 15% FBS (referred to as ‘‘CDSF+FBS’’; Fig. 1A, 1D), they formed a well-developed teratoma in 5 weeks (Fig. 1G, 2F–2J, and supporting information Fig. S1B). Thus, tumorigenesis in mESCs is not simply inhibited by CDSF conditions. When the injections were properly performed, we did not observe blood coming out of the injection sites. Only properly performed injections were counted in the present study. We conclude that CDSF did not compromise the pluripotency of mESCs per se for the following reasons. First, throughout the culture period (three passages, 9–12 days), the transcriptional activity of the master regulator of pluripotency, Oct3/4 (Pou5f1) [48,49,50], was validated using a mESC line that expresses the enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter [51] (Fig. 1B, 1C). Unless otherwise noted, this mESC line was used throughout the present study. Next, immunofluo- rescence microscopy confirmed that the mESC line cultured under CDSF conditions maintained the expression of Nanog, Sox2, and SSEA1 (Fig. 3A–3C). In addition, when the mESC line cultured Gsk3b Inhibition Induces Teratoma Formation under CDSF conditions was aggregated with wild-type morulae, resulting chimeric blastocysts exhibited green fluorescence in the inner cell mass (Fig. 3E). Eleven days after these blastocysts were transferred to pseudopregnant females, fluorescent cells were detected in a nascent male gonad of an embryo at embryonic day 13.5 (Fig. 3F, 3G). Furthermore, when the mESC line was maintained in either CDSF+FBS (n = 4) or CDSF for three passages and brought back to the standard medium for one or two passages (referred to as ‘‘CDSF-Standard’’, n = 4; Fig. 1A), EGFP expression was maintained (Fig. 1D, 1E) and the cells became teratomas (Fig. 1G, 1H, and 2F–2O; p,0.00016, see supporting information Table S1 and Fig. S1B, S1C). Similar results were obtained using four different lots of FBS. These data demonstrate that tumorigenesis in mESCs can be suppressed by short-term culture in the serum-free medium. The plasticity of tumorigenicity in mESCs appears to be unique; embryonic carcinomas (F9) [52], which are also pluripotent stem cells, formed teratomas when cultured in CDSF and transplanted (supporting information Fig. S1D). Likewise, germline-incompetent mESCs, D3, grew into teratomas (data not shown). Mouse embryonic stem cells cultured under serum-free conditions exhibited longer doubling time while maintained expression of genes associated with cellular pluripotency When NOD-SCID mice transplanted with mESCs reached their end points, they were sacrificed and examined for teratomas. This procedure usually yielded teratomas of about 30 mm in diameter (bars in Fig. 4A). However, the number of days needed for the experimental animals to reach their end points varied (stars in Fig. 4A). Interestingly, it took 4763.1 days for mESCs cultured in CDSF+FBS to grow into 30 mm teratomas, whereas it took 3761.8 days for mESCs cultured in CDSF followed by standard conditions for two passages to reach 30 mm in diameter. However, when mESCs cultured in CDSF were passaged only once into standard conditions, it took 76 days for these mESCs to grow into a 30 mm teratoma. Although this sample size is too small to be statistically significant, it is suggested with these data that CDSF may even suppress the growth of teratomas. Among the experimental NOD-SCID mice examined in this study, two out of the seven mice had mESCs cultured in CDSF injected into one side of the animal and mESCs cultured in media containing FBS injected into the other side. Therefore, the formation of teratomas from mESCs cultured in CDSF could not be examined beyond the end point of the animals (Fig. 4A). However, in the other five animals, we were able to determine that transplanted mESCs under CDSF did not generate teratomas for up to 6 months. At one injection site, we were able to identify a tiny mass of mESCs that had been cultured under CDSF-Standard conditions one week after transplantation (supporting information Fig. S1E). The mESCs in this mass had the appearance of cells undergoing initial differentiation (supporting information Fig. S2). However, we did not 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. Mouse ESCs cultured in CDSF exhibited a significantly longer doubling time (,28 hrs) than ones cultured under standard conditions (,17 hrs, p,0.005) during the first two passages, whereas ones cultured in CDSF+FBS or under CDSF-Standard conditions took ,19 and ,20 hrs to divide, respectively (Fig. 4B, top). Similar results were obtained with another mESC line, W4 [53,54] (data not shown). The differences in doubling times of mESCs were evident as PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21355