Short-Term Serum-Free Culture Reveals that Inhibition of Gsk3b Induces the Tumor-Like Growth of Mouse Embryonic Stem Cells Yanzhen Li, Tamaki Yokohama-Tamaki, Tetsuya S. Tanaka* Department of Animal Sciences, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America Abstract Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3b. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3b, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3b in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3b governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs. Citation: Li Y, Yokohama-Tamaki T, Tanaka TS (2011) Short-Term Serum-Free Culture Reveals that Inhibition of Gsk3b Induces the Tumor-Like Growth of Mouse Embryonic Stem Cells. PLoS ONE 6(6): e21355. doi:10.1371/journal.pone.0021355 Editor: Martin Pera, University of Southern California, United States of America Received October 28, 2010; Accepted May 31, 2011; Published June 23, 2011 Copyright: 2011 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the USDA Hatch Project (ILLU-538-323), University of Illinois and the Illinois Regenerative Medicine Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ttanaka@illinois.edu Introduction Embryonic stem cells (ESCs) [1,2,3] and induced pluripotent stem cells (iPSCs) [4,5,6,7] are very promising tools for use in drug screening and customized tissue replacement [8] because they are capable of self-renewal that sustains pluripotency. The self-renewal and pluripotency of mouse stem cells (ESCs and iPSCs) are maintained by extrinsic factors, such as supplementing basal culture medium with leukemia inhibitory factor (LIF) [9,10,11,12,13] and fetal bovine serum (FBS). FBS further facilitates their self-renewal by offering other factors, such as bone morphogenetic protein 4 (Bmp4) [14], retinoids [15,16,17], threonine [18] and glutathione [19]. However, FBS also provides cultures with many other uncharacterized components that may affect the capability of ESCs and iPSCs to self-renew and differentiate. Undefined culture conditions using animal sera may have contributed to results finding contradictory roles of the Wnt signaling pathway in ESCs [20,21,22,23]. However, it is now firmly established that pharmacological inhibition of glycogen synthase kinase 3b (Gsk3b) promotes the self-renewal of both mouse [22,24] and human ESCs [23], and derivation of mouse ESCs [25]. To eliminate the effects of unknown components in animal sera as well as the contamination of animal products, chemically- defined serum-free culture methods have been established [14,26,27,28]. Typically, defined culture media are composed of critical growth factors (e.g., LIF and/or Bmp4) and other factors present in animal sera, such as hormones (e.g., insulin and transferrin), vitamins, fatty acids and minerals. Commercially- made serum replacements that may contain these components in animal sera [29] are often used to maintain ESC culture (e.g., [30]), although the exact components cannot be disclosed by their patents [28]. The maintenance of the undifferentiated state of mouse ESCs (mESCs) using defined culture media has been well documented [31,32]. Furthermore, the pluripotency of these mESCs has been validated by their differentiation in vitro [26,32] or by the development of chimeric mice [14]. Another way to validate the pluripotency of ESCs and iPSCs is to examine the ability of these cells to develop into tumors called teratomas after their transplantation into immunocompromised mice [33,34,35]. Such teratoma formation assays have validated the pluripotency of mESCs maintained in the presence of a Gsk3b inhibitor [23,25]. This method, which requires no special technique or equipment and reduces the use of experimental PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21355