panels) or anti-Oct4 antibodies (lower panels). Same microscope fields labeled with DAPI are shown (right panels). (TIF) Data S9 characterization of PAI-1+/+ and 2/2 iPSCs: Sox2 and SSEA1 immunofluorescence. Immunofluorescence staining of cell cultures at the undifferentiated state of: CGR8 wt ESCs, iPSC wt clone, iPSC PAI-1 KO clone 3 and MEFs, as indicated. Cells were labeled with anti-Sox2 antibodies (upper panels) or anti-SSEA1 antibodies (lower panels). Same microscope fields labeled with DAPI are shown (right panels). (TIF) Data S10 histological characterization of PAI-1+/+ and 2/2 iPSC teratoma. CGR8 wt ESCs (A), iPSC wt clone (B), and iPSC PAI-1 KO clone 3 (C) were injected subcutaneously into nude mice. After four weeks, sections of the different teratomas were histologically analyzed. All teratomas presented differentiated tissues deriving from the three embryonic layers. Representative fields of well-differentiated tissues are shown. Arrows indicate examples of the three germ layer derivative structures: Cart = cartilage (mesoderm derivative), Epit = ciliated epithelium (endoderm derivative), and neural structures (ectoderm derivative): neural rosettes and nerve fibers. (TIF) Data S11 characterization of PAI-1+/+ and 2/2 iPSCs: in vitro differentiation. Characterization of the expression of ESC commitment master genes during the differentiation process References 1. Lensch M, Daheron L, Schlaeger T (2006) Pluripotent stem cells and their niches. Stem Cell Reviews and Reports 2: 185–201. 2. Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, et al. (2007) Niche- mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J 26: 4744–4755. 3. Mondino A, Blasi F (2004) uPA and uPAR in fibrinolysis, immunity and pathology. Trends in Immunology 25: 450–455. 4. Irigoyen J, Munoz-Canoves P, Montero L, Koziczak M, Nagamine Y (1999) The plasminogen activator system: biology and regulation. Cell Mol Life Sci 56: 104–132. 5. Rifkin DB, Mazzieri R, Munger JS, Noguera I, Sung J (1999) Proteolytic control of growth factor availability. APMIS 107: 80–85. 6. Chapman HA, Wei Y (2001) Protease crosstalk with integrins: the urokinase receptor paradigm. Thromb Haemost 86: 124–129. 7. Stefansson S, Lawrence DA (1996) The serpin PAI-1 inhibits cell migration by blocking integrin [alpha]v[beta]3 binding to vitronectin. Nature 383: 441–443. 8. Resnati M, Pallavicini I, Wang JM, Oppenheim J, Serhan CN, et al. (2002) The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proceedings of the National Academy of Sciences of the United States of America 99: 1359–1364. 9. Liang X, Kanjanabuch T, Mao SL, Hao CM, Tang YW, et al. (2006) Plasminogen activator inhibitor-1 modulates adipocyte differentiation. Am J Physiol Endocrinol Metab 290: E103-E113. Epub 2005 Sep 2006. 10. McMahon GA, Petitclerc E, Stefansson S, Smith E, Wong MK, et al. (2001) Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem 276: 33964–33968. 11. Isogai C, Laug WE, Shimada H, Declerck PJ, Stins MF, et al. (2001) Plasminogen Activator Inhibitor-1 Promotes Angiogenesis by Stimulating Endothelial Cell Migration toward Fibronectin. Cancer Res 61: 5587–5594. 12. Balsara RD, Castellino FJ, Ploplis VA (2006) A Novel Function of Plasminogen Activator Inhibitor-1 in Modulation of the AKT Pathway in Wild-type and Plasminogen Activator Inhibitor-1-deficient Endothelial Cells 10.1074/ jbc.M512819200. J Biol Chem 281: 22527–22536. 13. Bonavaud S, Charriere-Bertrand C, Rey C, Leibovitch MP, Pedersen N, et al. (1997) Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion. J Cell Sci 110: 1083–1089. 14. Suelves M, Lopez-Alemany R, Lluis F, Aniorte G, Serrano E, et al. (2002) Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo. Blood 99: 2835–2844. 15. Fibbi G, Barletta E, Dini G, Del Rosso A, Pucci M, et al. (2001) Cell Invasion Is Affected by Differential Expression of the Urokinase Plasminogen Activator/ Urokinase Plasminogen Activator Receptor System in Muscle Satellite Cells from Normal and Dystrophic Patients. Lab Invest 81: 27–39. The PA System Modulates ESC Differentiation of iPSCs. Retinoic acid-treated (RA) or not (NoRA) EBs from wild type CGR8 ESCs and iPSC clones were induced to differentiate. mRNAs were extracted at day 10 and analyzed by real time RT- PCR for the expression of FoxA2, Mesp1 and Tbx1. Results are expressed in arbitrary units, with the values of wild type CGR8 at day 0 taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. (TIF) Data S12 Forward and reverse primer sequences of the various genes analyzed by real-time RT-PCR. (TIF) Acknowledgments The authors thank Dr. Declerck for providing us with reagents, P. Cau for helpful discussions and J. Fiteni for histological preparations. We thank Gise`le Froment and Caroline Costa from the lentivectors production facility/SFR BioSciences Gerland - Lyon Sud (UMS3444/US8). O.H. was supported by fellowships from the Association Franc¸aise contre les Myopathies, the Fondation pour la Recherche Me´dicale, and the Socie´te´ Francophone du Diabe`te. Author Contributions Conceived and designed the experiments: BB. Performed the experiments: OH EB FP MV DB YH DN AR AM CEY. Contributed reagents/ materials/analysis tools: MI MK MCA. Wrote the paper: BB. 16. Lluis F, Roma J, Suelves M, Parra M, Aniorte G, et al. (2001) Urokinase- dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo. Blood 97: 1703–1711. 17. Koh TJ, Bryer SC, Pucci AM, Sisson TH (2005) Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration. Am J Physiol Cell Physiol 289: C217–223. Epub 2005 Feb 2016. 18. Wobus AM, Boheler KR (2005) Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 85: 635–678. 19. Kashyap V, Rezende N, Scotland K, Shaffer S, Persson J, et al. (2009) Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cell and Development 18: 1093–1108. 20. Wobus AM (2001) Potential of embryonic stem cells. Mol Aspects Med 22: 149– 164. 21. Bost F, Caron L, Marchetti I, Dani C, Le Marchand-Brustel Y, et al. (2002) Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage. Biochem J 361: 621–627. 22. Iacovino M, Bosnakovski D, Fey H, Rux D, Bajwa G, et al. (2011) Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells. Stem Cells in press. 23. Caron L, Bost F, Prot M, Hofman P, Binetruy B (2005) A new role for the oncogenic high-mobility group A2 transcription factor in myogenesis of embryonic stem cells. Oncogene 24: 6281–6291. 24. Aouadi M, Bost F, Caron L, Laurent K, Le Marchand Brustel Y, et al. (2006) p38 Mitogen-Activated Protein Kinase Activity Commits Embryonic Stem Cells to Either Neurogenesis or Cardiomyogenesis. Stem Cells 24: 1399–1406. Epub 2006 Jan 1319. 25. Nar H, Bauer M, Stassen J-M, Lang D, Gils A, et al. (2000) Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation. Journal of Molecular Biology 297: 683–695. 26. Barruet E, Hadadeh O, Peiretti F, Renault VM, Hadjal Y, et al. (2011) p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells. Stem Cells Dev 20: 1233– 1246. 27. Morange PE, Lijnen HR, Alessi MC, Kopp F, Collen D, et al. (2000) Influence of PAI-1 on adipose tissue growth and metabolic parameters in a murine model of diet-induced obesity. Arterioscler Thromb Vasc Biol 20: 1150–1154. 28. Carey BW, Markoulaki S, Hanna J, Saha K, Gao Q, et al. (2009) Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A 106: 157–162. 29. Halvorsen B, Staff AC, Henriksen T, Sawamura T, Ranheim T (2001) 8-iso- Prostaglandin F2Iˆ6 Increases Expression of LOX-1 in JAR Cells. Hypertension 37: 1184–1190. PLOS ONE | www.plosone.org 14 November 2012 | Volume 7 | Issue 11 | e49065