[13,14] and uPA is able to stimulate proliferation, migration and fusion of satellite cells, whereas uPA inhibition abrogated myogenesis [15]. In addition, genetic approaches demonstrated that uPA, but not tPA, activity is required for efficient skeletal muscle regeneration in vivo [16] and that this process is accelerated in PAI-1-deficient mice [17]. However the precise role of the PA system and more generally the role of ECM during the development and the commitment of pluripotent cells in in vitro experimental models such as the differentiation of Embryonic Stem Cells (ESCs) are still poorly known. ESCs are pluripotent and can be induced to differentiate into a variety of cell types [18]. Transplantation of ESCs has been proposed as a future therapy for various human diseases. However only variable fractions of cells reaches the desired differentiated phenotype, therefore their therapeutic use necessitates a better understanding of the molecular mechanisms that control their commitment. Self-renewal of ESCs is dependent on a complex interplay between specific stimuli (for example, the growth factor LIF for mouse ESCs) specific epigenetic processes, miRNAs and transcription factors involved in the development of the embryo. Appropriate culture conditions result in their commitment into mature differentiated cell types (see for reviews [18,19,20]). In order to approach the potential regulatory role of extracellular matrix, we studied the plasminogen activation (PA) system during ESC differentiation. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. We then characterized the role of the PA system in ESC differentiation by different approaches and found that interfering with uPA activity leads, on the one hand, to inhibition of adipogenesis and, on the other hand, to positive and negative regulations of skeletal myogenesis by successive different molecular mechanisms. Materials and Methods Differentiation of ESCs CGR8 mouse ESCs [21] were grown on gelatin-coated plates and A2lox.cre ESCs [22] were grown on MEF feeders treated with 10 mg/ml mitomycin C for 3 hours. 16103 ESCs were aggregated without LIF to form embryoid bodies (EB) at day 0, cultivated in suspension until day 7, treated or not with 1027 M All-Trans Retinoic Acid (Sigma, France) daily between days 3 to 5, plated at day 7 on gelatin-coated 6-well plate and treated with 85 nM insulin (Sigma, France), 2 nM 3,39,5-tri-iodothyronine (Sigma, France) to induce terminal differentiation. Fresh medium contain- ing the inducers was applied every 2 days. EBs were analyzed for cardiomyocytes, endothelial cells or neurons at day 12, and adipocytes or myotubes at day 26, as described previously [21,23,24]. 100 mmol/L of the uPA inhibitor Amiloride (Sigma, France) or 0.5 mg/ml of doxycycline (Sigma, France) were added to the culture media at different period of time as indicated. Plasmid and Generation of ESC Lines After addition of two HindIII sites, the human PAI-1 cDNA isolated from the PIGE20-hPAI-1-stable [25] was inserted into the p2Lox [22], generating the p2Lox-PAI-1 plasmid. The A2lox.cre ESC line was electroporated with 2 mg of p2Lox-hPAI-1 (Amaxa Biosystems, A24 program for ESC), according to the manufac- turer’s protocol, and selected with 500 mg/ml G418 (PAA, France). Transfection was realized onto mitomycin C-treated stable neomycin resistant MEF feeder cells obtained by trans- duction with the CHC-vector, a MLV retrovirus-vector coding the The PA System Modulates ESC Differentiation Neo cDNA using an MOI of 6 UI/cell, followed by Neomycin selection (100 mg/ml) as described previously [26]. Generation of iPSCs 16105 wt or PAI-12/2 MEFs [27] cultivated in DMEM supplemented with 10% fetal bovine serum were infected, at 30xMOI, with the KOSM polycistronic lentivirus vector encoding the murine transcriptor factors Klf4, Oct4, Sox2 and Myc [28]. The day after, medium was changed for the ESC medium. Colonies presenting an ESC phenotype were visible after 2 weeks and picked at day19. RNA analysis Total RNA was prepared using Trizol reagent (Invitrogen, France). Real time Reverse Transcriptase-PCR was performed with the ABI Prism7300 (Applied BioSystems, France) and Mesa Green MasterMix (Eurogentec, France). The relative amounts of the different mRNAs were quantified by using the comparative CT method (2-(DDCT)). 36B4 was used as housekeeper transcripts and gene expressions were normalized using 36B4 RNA levels. Primer sequences are given in Data S12. Western Blot Analysis ESCs were lysed as described [21] at days 0 (for Oct4 analysis) or 24 (for MyH1) of the differentiation protocol. Samples at day 0 (20 mg) were separated by SDS-polyacrylamide gel electropho- resis on a 10% gel (Invitrogen, France), transferred onto polyvinylidene difluoride membranes (Millipore, France) and incubated with antibodies against Oct-4 (1:1000, Abcam, France) or ERK (1:2000, Santa Cruz, California, USA). Samples at day 24 (20 mg) were separated by SDS-polyacrylamide gel electrophoresis on a 3–8% gel (Invitrogen, France), transferred onto polyvinyli- dene difluoride membranes (Millipore, France) and incubated with antibodies against Myosin (MF20) (1:500, DSHB, Iowa City, USA) or ERK. Immunocytochemistry, Immunofluorescence and Oil Red O Staining For PAI-1 immunocytochemistry studies, cells were washed at day 24, fixed in 4% paraformaldehyde for 15 min at room temperature (RT), permeabilized in PBS-0.2% Triton X-100 for 15 min at RT, blocked by serum for 30 min, and then incubated with 23.4 mg/ml rabbit anti-mouse PAI-1 (H34G6D10, provided by Dr P. Declerck) for 1 h. After rinsing twice with PBS, anti- rabbit IgG secondary antibody (Vector, UK) was added, incubated for 40 min, followed by two washes in PBS, incubated with vectastain ABC (Vector, UK) for 30 min, washed with PBS, and stained with DAB (Dako, France). Slides were counterstained with hematoxylin (1:2 dilution in distilled water). For MyH1 immunofluorescence studies, fixed cells at day 24 were permeabilized in PBS-0.2% Triton X-100 for 15 min at RT and incubated with anti-Myosin (MF20) (DSHB, Iowa City, USA) in PBS-10% FBS for 2 hours. After three washes in PBS, cells were incubated with anti-mouse ALEXA488 (Invitrogen, France) for 1 hour at RT. For ECM characterization, cell cultures were first acellularized by using a cultured cells Acellularization kit (Sigma, France). Briefly, EBs at days 12 and 24 were incubated for 10 min with the Acellularization buffer and gently shaked at RT. After carefully aspirating the Acellularization buffer, cultures were carefully rinsed twice with PBS. The ECM was then incubated with anti-Collagen I (1:100, Abcam, France) in PBS-1% BSA for 2 hours at RT. After three washes in PBS, PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49065