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. Two transplantations for ESF7 showed no sign of teratoma formation when paired with the standard conditions. Standard, Standard conditions; CDSF, CDSF conditions; BL, other established CDSF conditions supplemented with N2, B27, Bmp4 and LIF; 2i, other established CDSF conditions supplemented with N2, B27, and pharmaco- logical inhibitors of Erk and Gsk3b. (TIF) Figure S5 Expression levels of markers were compared among different culture conditions. Abundance of each transcript indicated above was examined in mESCs cultured under each condition on the right by 19, 21 and 23 cycles of PCR (indicated by triangles). Ef1a was used as a reference. White arrowheads indicate the PCR product of c-Myc. (TIF) References 1. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156. 2. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78: 7634–7638. 3. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147. 4. Takahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126: 663–676. 5. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al. (2007) Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 318: 1917–1920. 6. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131: 861–872. 7. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, et al. (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451: 141–146. 8. Gurdon JB, Melton DA (2008) Nuclear reprogramming in cells. Science 322: 1811–1815. 9. Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, et al. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684–687. 10. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, et al. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336: 688–690. 11. Niwa H (2007) How is pluripotency determined and maintained? Development 134: 635–646. 12. Ohtsuka S, Dalton S (2008) Molecular and biological properties of pluripotent embryonic stem cells. Gene Ther 15: 74–81. 13. Tanaka TS (2009) Transcriptional heterogeneity in mouse embryonic stem cells. Reprod Fertil Dev 21: 67–75. 14. Ying QL, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115: 281–292. 15. Wang R, Liang J, Yu HM, Liang H, Shi YJ, et al. (2008) Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism. Differentiation 76: 931–945. 16. Chen L, Khillan JS (2008) Promotion of feeder-independent self-renewal of embryonic stem cells by retinol (vitamin A). Stem Cells 26: 1858–1864. 17. Chen L, Khillan JS (2010) A novel signaling by vitamin A/retinol promotes self renewal of mouse embryonic stem cells by activating PI3K/Akt signaling pathway via insulin-like growth factor-1 receptor. Stem Cells 28: 57–63. 18. Wang J, Alexander P, Wu L, Hammer R, Cleaver O, et al. (2009) Dependence of mouse embryonic stem cells on threonine catabolism. Science 325: 435–439. 19. Yanes O, Clark J, Wong DM, Patti GJ, Sanchez-Ruiz A, et al. (2010) Metabolic oxidation regulates embryonic stem cell differentiation. Nat Chem Biol 6: 411–417. 20. Lindsley RC, Gill JG, Kyba M, Murphy TL, Murphy KM (2006) Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm. Development 133: 3787–3796. 21. Dravid G, Ye Z, Hammond H, Chen G, Pyle A, et al. (2005) Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23: 1489–1501. Gsk3b Inhibition Induces Teratoma Formation Table S1 Effects of serum mouse embryonic stem cells. (DOCX) Acknowledgments on the tumorigenicity of We are grateful to Drs. Matthew B. Wheeler, Miho Kusuda-Furue, Yuki Kimura, and William L. Stanford for the critical reading of this manuscript. YL was a recipient of AYRE International Research and Learning Fellowships, UIUC. The anti-SSEA1 monoclonal antibody developed by Drs. Solter and Knowles was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Author Contributions Conceived and designed the experiments: YL TST. Performed the experiments: YL TYT TST. Analyzed the data: YL TST. Contributed reagents/materials/analysis tools: TST. Wrote the paper: YL TST. 22. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, et al. (2008) The ground state of embryonic stem cell self-renewal. Nature 453: 519–523. 23. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10: 55–63. 24. Bechard M, Dalton S (2009) Subcellular localization of glycogen synthase kinase 3beta controls embryonic stem cell self-renewal. Mol Cell Biol 29: 2092–2104. 25. Umehara H, Kimura T, Ohtsuka S, Nakamura T, Kitajima K, et al. (2007) Efficient Derivation of Embryonic Stem Cells by Inhibition of Glycogen Synthase Kinase-3. Stem Cells 25: 2705–2711. 26. Furue M, Okamoto T, Hayashi Y, Okochi H, Fujimoto M, et al. (2005) Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells. In Vitro Cell Dev Biol Anim 41: 19–28. 27. Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, et al. (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24: 185–187. 28. Furue MK, Na J, Jackson JP, Okamoto T, Jones M, et al. (2008) Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci U S A 105: 13409–13414. 29. Goldsborough MD, Price PJ, Lobo-Alfonso J, Morrison JR, Stevens ME, et al. (1998) Serum-free culture of murine embryonic stem (ES) cells. Focus 20: 8–12. 30. Suemori H, Tada T, Torii R, Hosoi Y, Kobayashi K, et al. (2001) Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 222: 273–279. 31. Ying Q-L, Wray J, Nichols J, Batlle-Morera L, Doble B, et al. (2008) The ground state of embryonic stem cell self-renewal. Nature 453: 519–523. 32. Hayashi Y, Furue MK, Okamoto T, Ohnuma K, Myoishi Y, et al. (2007) Integrins Regulate Mouse Embryonic Stem Cell Self-Renewal. Stem Cells 25: 3005–3015. 33. Solter D (2006) From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet 7: 319–327. 34. Chambers I, Smith A (2004) Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 23: 7150–7160. 35. Jaenisch R, Young R (2008) Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming. Cell 132: 567–582. 36. Yu J, Thomson JA (2008) Pluripotent stem cell lines. Genes Dev 22: 1987–1997. 37. Yamanaka S (2009) A fresh look at iPS cells. Cell 137: 13–17. 38. Takahashi K, Mitsui K, Yamanaka S (2003) Role of ERas in promoting tumour- like properties in mouse embryonic stem cells. Nature 423: 541–545. 39. Wu D, Pan W (2010) GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem Sci 35: 161–168. 40. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995) Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789. 41. Kameda T, Thomson JA (2005) Human ERas gene has an upstream premature polyadenylation signal that results in a truncated, noncoding transcript. Stem Cells 23: 1535–1540. 42. Tanaka Y, Ikeda T, Kishi Y, Masuda S, Shibata H, et al. (2009) ERas is expressed in primate embryonic stem cells but not related to tumorigenesis. Cell Transplant 18: 381–389. 43. Voog J, Jones DL (2010) Stem cells and the niche: a dynamic duo. Cell Stem Cell 6: 103–115. 44. Duval D, Reinhardt B, Kedinger C, Boeuf H (2000) Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival. FASEB J 14: 1577–1584. PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e21355