The PA System Modulates ESC Differentiation Figure 5. Doxycycline-induced human PAI-1 expression inhibits ESC adipogenesis of the A2lox.cre mESC clone3. (A and B) Retinoic acid-treated (RA, panel B) or not (NoRA, panel A) EBs from A2lox.cre mESC clone3 cells treated (Dox, red lines) or not (Ct, black lines) by 0.5 mg/ml of doxycycline were induced to differentiate and analyzed at various time, as indicated, between days 0 (d0) to 24 (d24). Endogenous mouse (mPAI- 1, blue lines) and ectopic human (hPAI-1) PAI-1 protein expressions in conditioned medium were quantified by ELISA technique. Values are given in ng of PAI-1 amounts and expressed as means of at least three independent experiments 6 S.E.M. (C) Evaluation of the turnover of Dox-induced intracellular human PAI-1 by ELISA after doxycycline removal. ESCs were treated with doxycycline either all the time (blue line) or between days 0 to 3 (red line) and hPAI-1 protein was analyzed until day 7. Values are given in ng of human PAI-1 amounts/mg of total proteins. Retinoic acid-treated EBs from A2lox.cre mESC clone3 cells were induced to differentiate to adipocyte and treated or not by doxycycline for different period of time: from days 0 to 24 [Dox], from days 0 to 3 [Dox(d0–d3)], from days 3 to 7 [Dox(d3–d7)], from days 7 to 24 [Dox(d7–d24)]. (D) Scheme of the different doxycycline treatments. (E to G) mRNAs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers adiponectin (E), aP2 (F) and PPARgamma (G). Results are expressed in arbitrary units, with the values of untreated A2lox.cre mESC clone3 cells at day 24 taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. Significance to Ct values is given as: N.S. non significant, *P,0.05, **P,0.01 and ***P,0.001. (H) Adipocyte formation of A2lox.cre mESC clone3 cells treated or not, as indicated, by doxycycline was visualized by Oil RedO staining, representative fields are shown. doi:10.1371/journal.pone.0049065.g005 Discussion Regulation of proteolytic activity generated by the plasminogen activation system is involved in many physiopathological pro- cesses; in this study we demonstrate that it controls several distinct and successive steps of the in vitro differentiation of embryonic stem cells. We found that, during the terminal differentiation of ESCs, either uPA inhibition or PAI-1 overexpression strongly and concomitantly decrease adipogenesis and skeletal myogenesis. Likewise, inhibitory effects were observed on adipogenesis when interfering with the PA system during the early commitment of ESCs. By contrast, skeletal myogenesis is stimulated by an early uPA inhibition or PAI-1 overexpression. Although we cannot exclude that tPA plays a role in ESC differentiation, we reproduced most of the PAI-1 overexpression effects by inhibiting uPA with amiloride, suggesting that, in our experimental model, PAI-1 is mainly acting via inhibition of uPA. Interestingly these regulations are specific to the adipogenic and skeletal myogenic lineages; indeed neither endothelial cell formation, smooth muscle myogenesis, cardiomyogenesis nor neurogenesis are affected. The decrease of adipogenesis resulting from inhibition of uPA or from PAI-1 overexpression, during the terminal differentiation of ESCs is in agreement with the previous in vitro roles described for uPA and PAI-1 in adipocyte differentiation [9]. It is also consistent with in vivo data showing that PAI-12/2 mice are prone to obesity [27]. Between days 0 to 7 of the differentiation protocol of ESCs, embryoid bodies recapitulate the formation of the three PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49065