The PA System Modulates ESC Differentiation Figure 8. characterization and adipogenic capacities of PAI-12/2 pluripotent cells. (A) In comparison to primary MEFs and wt CGR8 ESCs, the degree of reprogrammation of wt and PAI-1 KO, clones 1 and 3, iPSCs was characterized by the mRNA levels of Oct4 and Nanog genes and by the extinction of the expression of GFP gene. Results are expressed in arbitrary units, with the values of CGR8 mESCs taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. Significance is given as: *P,0.05, **P,0.01 and ***P,0.001. (B) Characterization, during the differentiation process of iPSCs, of the expression of ESC commitment master genes. Retinoic acid-treated (RA) or not (NoRA) EBs from wild type CGR8 ESCs and iPSC clones were induced to differentiate. mRNAs were extracted at various time, as indicated, and analyzed by real time RT-PCR for the expression of Pax6, Sox17 and Brachyury. Results are expressed in arbitrary units, with the values of wild type CGR8 taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. (C) Retinoic acid-treated (RA) or not (NoRA) EBs from either wild type (WT) pluripotent cells or PAI- 12/2 iPSCs (PAI-1 KO) were induced to differentiate to adipocytes. mRNAs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers PPARgamma and adiponectin. Results are expressed in arbitrary units, with the values of WT NoRA condition at day 24 taken as 1, and are the means 6 S.E.M. of at least 3 independent experiments. Significance is given as: N.S. non significant, *P,0.05, **P,0.01 and ***P,0.001. doi:10.1371/journal.pone.0049065.g008 modulated very early during differentiation. Whether interfering with the PA system at that step, targets directly or indirectly putative skeletal myogenic precursors needs to be investigated. Interestingly, the expression of the transcription factors Pax3, Pax7 and MyoD that are known to be necessary during the early steps of myogenesis, were undetectable at that time, appearing only after day 14 of the differentiation protocol (data not shown); suggesting that our effect targets earlier myogenic precursors. Therefore, by pharmaceutical and genetic experiments we found that early uPA inhibition, between days 0 to 3 of differentiation, augments ESC myogenesis. To our knowledge, this is the first time that such augmentation of in vitro myotube formation in the mouse ESC model is described. Other ESC myogenic stimulations described in the literature are limited to the formation myogenic precursors, as for example, the treatment of EBs with low doses of retinoic acid [36]. Since full skeletal myogenesis from ESCs is a complex, long and difficult process to reproduce in culture, especially within the human ESC model, we believe that our results represent an important contribution in order to fully differentiate ESCs towards this lineage. Our results show that the PA system is capable to specifically and concomitantly regulate two complex multi-steps ESC lineages, adipocyte and skeletal muscle. Particularly, the very early opposite regulation evidenced here, between days 0 to 3, could suggests that, during that period, uPA activity induces a switch augment- ing, on the one hand, adipogenesis and inhibiting, on the other hand, skeletal myogenesis. This is reminiscent to a comparable switch regulation between neurogenesis and cardiomyogenesis induced by the p38MAPK that we already described in this model [24]. To better understand the molecular mechanisms involved in such switch, we need to identify the cellular precursors affected by these processes. PLOS ONE | www.plosone.org 12 November 2012 | Volume 7 | Issue 11 | e49065