ECM was incubated with anti-rabbit ALEXA468 (Invitrogen, France) for 1 hour at RT. For iPSC characterization, fixed cells at day 0 were permeabilized in PBS-0.2% Triton X-100 for 15 min at RT and incubated with either anti-SSEA1 (MC480) (DSHB, Iowa City, USA), anti-Sox2 (AB5603) (Millipore, France), anti-Oct4 (ab19857) (Abcam, France), or anti- Nanog (SC30328) (Santa Cruz Biotechnology, California, USA) in PBS-10% FBS for 2 hours. After three washes in PBS, cells were incubated with either anti-mouse ALEXA488 (Invitrogen, France), anti-rabbit ALEXA468 (Invitrogen, France), or anti-goat ALEXA568 (In- vitrogen, France) for 1 hour at RT. For Oil Red O Staining, fixed cells at day 24 were immersed 30 min at RT in a solution of Oil Red O [29], and then washed 5 times with deionised water. Flow Cytometry Cells were trypsinized, washed with 10%FBS/DMEM, fixed in 0,5% paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% Triton/PBS buffer for 10 min at RT. Cells were then stained with an a-Myogenin (FD5) antibody at 1:10 dilution in 5%FBS/PBS (DSHB, Iowa City, USA) for 1 hour under agitation, washed with 5%FBS/PBS, and incubated with an anti-mouse IgG secondary antibody conjugated to PE (1:100 dilution in 5%FBS/PBS) (Beckman Coulter, France) for 30 min under agitation. After additional washes in PBS, cells were resuspended in 500 ml PBS and analyzed on a FC500 Flow cytometer using CXP software (Beckman Coulter, France). For each sample, at least 5000 cells were analyzed. Cursor for positive fluorescent cell determination was placed, such as 75% of events were outside (first decade) the cursor region when an isotype IgG antibody was used. Results are given as percentage corresponding to positive fluorescent cells included in the region defined by the cursor and after subtracting the irrelevant’s percentage. ELISA Assays Murine uPA and tPA antigens were quantified using respectively the MUPAKT-TOT and MTPAKT-TOT immuno- assay kits from Gentaur, France, according to supplier’s recommendations. A standard calibration curve was prepared using dilutions of purified uPA from 0.025 to 2 ng/ml and of purified tPA from 0 to 50 ng/ml. Murine uPA and tPA activities were quantified using respectively the MUPAKT and MTPAKT immunoassay kits from Gentaur, France, according to supplier’s recommendations. A standard calibration curve was prepared using dilutions of purified uPA from 0.025 to 2 ng/ml and of purified tPA from 0 to 50 ng/ ml. Human and mouse PAI-1 antigens were quantified by immunoassays, wells of microtiter plates were coated overnight at 4uC with 2 mg/ml human PAI-1 antibody (15H12, provided by Dr P. Declerck), or 48 h at 4uC with 2 mg/ml rabbit anti-mouse PAI- 1 (H34G6D10 provided by Dr P. Declerck), washed with PBS 0,004% Tween 80, and blocked for 2 h with a solution of 1% BSA in PBS. Cell lysates, diluted five times, or undiluted conditioned media, prepared from 24 hours cell supernatants cultivated without serum, were added to each well and incubated overnight at 4uC. After four washes with PBS 0,004% Tween 80, biotinylated anti-mPAI-1 antibody Ra-mPAI-1(1:800) was added for 1 h at RT, and for another hour with vectastain ABC (Vector, UK), or anti-human antibody 12A4A6 (1:800) was added for 2 h at RT. After four washes with PBS 0,004% Tween 80, ultra-TMB solution (Thermo scientific, France) was added. The reaction was stopped by adding H2SO4 (2M final concentration), and the The PA System Modulates ESC Differentiation optical density was read at 450 nm. A standard calibration curve was prepared using dilutions of purified PAI-1 from 0 to 10 ng/ ml. Human PAI-1 activity was quantified by using the Zymutest PAI-1 Activity immunoassay kit (HYPHEN Biomed, France); according to supplier’s recommendations. A standard calibration curve was prepared using dilutions of purified PAI-1 from 0 to 20 ng/ml. Teratoma Formation and Histological Analyses CGR8 ESCs, and wt and PAI-12/2 iPSCs were trypsinized and washed, and 56106 cells resuspended in 200 ml of PBS were injected subcutaneously into both sides of the back of 6-week-old athymic nude female mice (Harlan, France). At 5 weeks following injection, tumours were surgically excised, fixed in formalin, paraffin-embedded, and 5 mm thick sections were analyzed and photographed after hematoxylin–eosin staining. Statistical Analyses All experiments were performed at least three times. All data are expressed as the mean 6SEM. Treatments were compared with their respective controls and significant differences among the groups were determined using unpaired Student’s t-test. A value of p,0.05 was taken as an indication of statistical significance. Results Gene Expression of uPA, tPA,uPAR and PAI-1 before and after ESC Differentiation Mouse ESCs are differentiated by removing the LIF from the medium and forming aggregates in suspension, called embryoid bodies (EBs), for one week. After this crucial commitment phase, cells are allowed to terminally differentiate by replating EBs on Petri dishes. This experimental protocol leads to the spontaneous formation, into the same culture dishes and in function of time, of several well-differentiated cells from a fraction of the starting ESC population. Neuron and adipocyte formation necessitates a critical early three days treatment of EBs between days 3 to 5 of the protocol with retinoic acid (hereafter referred as RA or adipogenic conditions); conversely, not treated RA EBs will form spontane- ously endothelial cells, cardiomyocytes, and smooth and skeletal myogenic cells (hereafter referred as NoRA or myogenic conditions). Routinely, at day 12 of differentiation, we followed neurogenesis by the expression of the neuronal marker MAP2; endothelial cell differentiation by the expression of Flk-1, PECAM and von Villebrand Factor (vWF) genes; smooth muscle cells by the alpha-smooth muscle actin (a-SMA) marker; and cardiomyo- genesis by the TroponinT and GATA4 markers. At day 24 of differentiation, we followed adipogenesis by the expression of aP2, PPARgamma and adiponectin genes and skeletal myogenesis by the Myogenin and Myosin heavy Chain-1 (MyH1) markers [26,30,31]. mRNAs, proteins and enzymatic activities of uPA and tPA have been analyzed by quantitative RT-PCR and ELISA techniques at various times during the differentiation of CGR8 mouse ESCs. uPA and tPA expressions and activities are almost undetectable in undifferentiated ES cells (Fig. 1 and 2). Upon differentiation, with or without RA treatment, uPA mRNA expression increases gradually, reaching a maximum at day 14 (Fig. 1A). A similar expression profile is found for uPA proteins in cell supernatants, except for the NoRA myogenic condition where it continues to augment till day 24 (Fig. 1B). In- terestingly, uPA activities in supernatants, although following an identical profile until day 14, are then lower. This latter PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49065