(Fig. 5A and B, red curves). Importantly, in undifferentiated ESCs and during the first 10 days of differentiation, whereas endogenous mouse PAI-1 is not expressed (Fig. 5A and B, blue curves), significant amounts of exogenous human PAI-1 protein are produced. Activity of the exogenous human PAI-1 protein was verified on supernatants at day 3 using a PAI-1 activity assay. No activity was found in untreated cells, whereas a strong human PAI- 1 activity was detected in doxycycline treated cells (data not shown), demonstrating that the secreted exogenous protein was functional. We then evaluated the turnover of Dox-induced human PAI-1 by quantifying by ELISA assay the remaining protein at different time after doxycycline removal (Fig. 5C). ESCs were treated with doxycycline either all the time or between days 0 to 3 and hPAI-1 protein was analyzed until day 7. Starting from a plateau at day 3, a similar PAI-1 protein amount is found at day 4, 50% is found at day 5 and 25% at day 7. These results suggest that the half-life of hPAI-1 protein in our cells is around 48 hrs. PAI-1 Overexpression in A2lox.cre ESC Clone3 Decreases ESC Adipogenesis Doxycycline was applied during the differentiation of A2lox.cre ESC clone3 in four different conditions: i) all the time of differentiation, from days 0 to 24 [Dox], ii) only from days 0 to 3 [Dox(d0–d3)], iii) from days 3 to 7 [Dox(d3–d7)], iv) from days 7 to 24 [Dox(d7–d24)] (Fig. 5D). The [Dox] and [Dox(d7–d24)] treatments significantly decrease by 50% the expression of adipogenic markers measured at day 24: adiponectin (Fig. 5E), aP2 (Fig. 5F) and PPARgamma (Fig. 5G). By contrast the [Dox(d0–d3)] and [Dox(d3–d7)] treatments have no effect. These regulations of adipocyte formation are also detected after Oil redO staining of the cultures (Fig. 5H). uPA Inhibition or PAI-1 Overexpression Modulates Successively Positively then Negatively ESC Skeletal Myogenesis In parallel experiments, we investigated the role of the PA system in ESC skeletal myogenesis. The amiloride uPA inhibitor was applied on CGR8 ESCs in the same conditions as for the adipogenesis study (Fig. 4A). The [A] and [A(d7–d24)] amiloride treatments of CGR8 cells strongly decrease the expression of MyH1 and Myogenin skeletal muscle markers measured at day 24 (Fig. 6A). Although to a lesser extent, the [A(d3–d7)] treatment also inhibits skeletal myogenesis. Surpris- ingly, we found that the [A(d0–d3)] treatment of CGR8 cells leads to a 3.6 fold increase in expression of the MyH1 myogenic marker, and a 2.6 fold increase in Myogenin expression (Fig. 6A). Similar results are obtained by studying muscle proteins by western blot analysis (Fig. 6B), immunofluorescence experiments with anti-Myosin Heavy Chain1 antibodies (Fig. 6C), and flow cytometry with anti-myogenin antibodies (Fig. 6D). The effects of human PAI-1 overexpression on skeletal myogenesis of A2lox.cre ESC clone3 were investigated by applying doxycycline in the same conditions as for the adipogen- esis study (Fig. 5D). Although less effective than in CGR8 ESCs, myogenesis is readily detectable in A2lox.cre ESCs and in its derivative clone3. We found results that parallel the effects of amiloride on CGR8 cells: [Dox] and [Dox(d7–d24)] treatments inhibit myogenesis, whereas [Dox(d0–d3)] activates it (Fig. 7). The [Dox(d3–d7)] condition gives a modest activation of MyH1 and no effect on Myogenin expression (Fig. 7A). Interfering with the PA system does neither affect neurogenesis nor the other mesodermal ESC differentiations. Amiloride The PA System Modulates ESC Differentiation treatments have no effect on neurogenesis (Data S6A) and cardiomyogenesis (Data S6B) and doxycycline-induced over- expression of human PAI-1 does not interfere with neurogenesis (Data S6C), cardiomyogenesis (Data S6D,E), endothelial (Data S6F, G, H) and smooth muscle (Data S6I) differentiations of A2lox.cre mESC clone3. Particularly, interfering with the PA system during the d0–d3 critical period did not affect these differentiations. PAI-12/2 Pluripotent Cells are Prone to Form Adipocytes To investigate the effect of the absence of PAI-1 during ESC differentiation we isolated and characterized pluripotent iPSCs from PAI-12/2 mouse embryo fibroblasts (MEFs). Control MEFs and MEFs knock-out for the PAI-1 gene [27] were transduced with the reprogramming virus encoding the Klf4, Oct4, Sox2 and c-Myc transcription factors together with GFP in a single poly- cistronic transcript [28]. Several colonies presenting an ESC-like phenotype (see an example in Data S7A) were cloned and characterized for: i) mRNA and protein expression of the Nanog and Oct4 pluripotent factors, which should be comparable to ESC; ii) transgene extinction of gene expression -measured by GFP expression and by specific primers-, which signs complete epigenetic reprogrammation; and iii) in vitro and in vivo pluripotent properties. Among the pluripotent factors, Nanog is not trans- duced by the reprogramming vector; therefore its de novo expression signs a reprogrammation process. As expected, the analyzed colonies reached various degrees of reprogrammation; only some of them are completely reprogrammed. An example of this characterization is given in Fig. 8A and Data S7, S8, S9. To confirm in vivo their pluripotent potential, ESCs, fully repro- grammed control PAI-1+/+ and PAI-1 KO iPSCs (clone 3) were injected subcutaneously into nude mice. Four animals were injected for each cell line and tumours developed after four weeks in all animals; sections of the different teratomas were histolog- ically analyzed. All teratomas presented differentiated tissues deriving from the three embryonic layers (Data S10). We then chose to compare the in vitro differentiation potential of both fully (clone 3) and partially (clone 1) reprogrammed PAI-1 KO iPSC clones to wild-type ESCs and fully reprogrammed control PAI-1+/+ cells. We first checked, during the differentiation process, the expression of known master genes of the ESC commitment towards the embryonic cell lineages. We analyzed the expression of Pax6 for the ectoderm (Fig. 8B), Sox17 for the endoderm (Fig. 8B), Brachyury (Fig. 8B) Tbx1 and Mesp1 (Data S11) for the mesoderm, and FoxA2 for the mesendoderm (Data S11). Upon induction of ESC differentiation, the expression of these genes is characterized by early peaks, variable between days 4 to 10, which are regulated by RA. As expected, the iPSCs+/+ gives an expression profile of these genes very similar to the wild type CGR8 ESC control. Likewise, the partially reprogrammed iPSC clone 1 gives aberrant level of expression and RA responses in each case, demonstrating that its incomplete reprogrammation interferes with its differentiation capacities. Interestingly, the fully reprogrammed iPS PAI-1 KO clone 3 gives in majority similar expression as wt ESCs and iPSCs, except for the ectoderm Pax6 and the mesoderm Tbx1 master genes. We found no difference in the capacities of PAI-12/2 iPSCs to form cardiomyocytes and myotubes (data not shown). By contrast, at the opposite of control wild-type cells which necessitate an early retinoic acid treatment to form adipocytes by day 24, the PAI-12/ 2 iPSCs form adipocytes spontaneously and do not respond to the retinoic acid treatment (Fig. 8C). PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49065