Plaidoyer pour que le Conseil constitutionnel devienne une cour constitutionnelle

Plan de l'article I – La dimension formelle : motiver pour convaincre II – La dimension organique : une autonomie renforcée du Conseil constitutionnel III – La dimension substantielle : la défense d’une jurisprudence courageuse au nom du respect des droits et libertés formellement garantis par les textes constitutionnel

Inhibition of the Fgfr disrupted the normal localisation of polarity and junction proteins in the outer layer of embryoid bodies.

<p>Embryoid bodies were treated with the Fgfr inhibitor 4 µM AZD-4547, or 0.04% DMSO for 7 days. Whole-mount immunostaining demonstrated the localisation of proteins normally polarised in the primitive endoderm. (A) aPkcζ/λ a member of a polarity complex, (B) Zo-1 a tight junction protein, (C) β-catenin a protein in the adherens junction and (D) the basement membrane protein Fibronectin, were shown to lose their apico-basolateral polarised localisation when embryoid bodies were grown with an Fgfr inhibitor suggesting a disruption in the apico-basolateral polarity of these cells. A representative image from 3 independent experiments is shown. Scale bars 10 µm. Dotted lines represent position that the relevant orthogonal or aerial images were taken.</p

The outer-layer of embryoid bodies gradually developed apico-basolateral polarity.

<p>Localisation of proteins which show apico-basaolateral polarity in epithelia were examined in the outer, primitive endoderm layer of embryoid bodies using whole-mount immunostaining. (A) The polarity complex protein aPkcζ/λ showed cytoplasmic localisation on day 3, but was apically localised from day 5. (B) The tight-junction protein Zo-1 showed a polarised localisation from day 3 onwards. (C) The adherens-junction protein β-catenin showed apical and basolateral localisation at day 3, but by day 5 became more restricted to the lateral sides of cells. (D) The basement membrane protein Fibronectin formed aggregates on day 3, but from day 5 to day 10 showed gradually increasing staining at the basal side of the outer layer of cells. The outer cells remained polarised at 10 days. Representative images from 3 independent experiments are shown. Dotted lines represent position that the relevant orthogonal or aerial images were taken. Scale bars 10 µm.</p

Inhibition Mek or the Fgfr did not significantly alter the expression levels of polarity and junction proteins.

<p>Embryoid bodies were grown in different concentration of the (A, C, E), Mek inhibitor PD-0325901 and 0.04% DMSO or the (B, D, F) Fgfr inhibitor AZD-4547 and 0.08% DMSO for 7 days. Expression levels of polarity and junction proteins were assessed using western blotting (A&B) aPkcζ/λ, (C&D) β-catenin, and (E&F) Fibronectin. A representative blot and quantification from 3 independent experiments is shown for each marker. No statistically significant change was seen in any of the markers observed. Statistical analysis is a one-way Anova with a Dunnett’s post-hoc test. Error bars represent SEM. (*P = 0.1–0.5, **p = 0.001–0.01, ***p<0.001).</p

expression of primitive endoderm cell fate markers gradually increased and nanog decreased in developing embryoid bodies.

<p>Embryoid bodies were produced from R63 mES cells using the hanging drop method. Development of the embryoid body was monitored over ten days. (A) Light microscopy images show the gradual increase in size of the embryoid bodies as well as increased heterogeneity, loss of circularity and formation of cystic cavities at later timepoints. Scale bars 200 µm. (B) Whole-mount immunostaining showing nuclear localisation of Gata6, Gata4, and Hnf4α on days 3, 5, 7, and 10 of embryoid body development. The percentage of positive nuclei for each protein is shown graphically. The number of positive nuclei increased, reaching a maximum on day 7. (C) Whole-mount immunostaining showing nuclear localisation of Nanog. The number of positive nuclei rapidly decreased, with no positive cells seen on days 7 or 10. The percentage of positive nuclei for each protein is shown graphically. Data is from at least 3 independent experiments, error bars are standard error of the mean (SEM). Statistical analysis is a one-way Anova with a Tukey’s post-hoc test, (*P = 0.1–0.5, **p = 0.001–0.01, ***p<0.001). Dotted lines represent position that the relevant orthogonal or aerial images were taken. Scale bars 10 µm.</p

Inhibition of the Fgfr or Mek caused a loss in barrier function of the cells in the outer layer of the embryoid body.

<p>Embryoid bodies were treated with (A) 0.04% DMSO, or 4 µM PD-0325901(Mek inhibitor), (B) 0.04% DMSO or 4 µM AZD-4547 (Fgfr inhibitor) for 7 days. A membrane impermeable biotin which covalently links to amino groups was added to the embryoid bodies and subsequently visualised using Alexa-fluor-488-conjugated Streptavidin. The biotin was largely restricted to the apical surface of the embryoid body in DMSO controls. Following treatment with either a Mek or an Fgfr inhibitor, biotin also bound to basolateral membrane proteins of the outer cell layer and the membranes of cells under this layer. This shows that the normal epithelial barrier has been disrupted. A representative image from 3 independent experiments is shown. Scale bars 10 µm. Dotted lines represent position that the relevant orthogonal or aerial images were taken.</p

Addition of AZD-4547 inhibited Erk phosphorylation and resulted in smaller and more circular embryoid bodies.

<p>Embryoid bodies were grown in different concentrations of AZD-4547, 0.08% DMSO for 7 days. (A) Light microscopy images show a change in morphology of the embryoid bodies. Scale bars 100 µm. (B) Western blotting demonstrates that AZD-4547 reduced levels of diphosphorylated Erk1/2. A representative blot and quantification from 3 independent experiments is shown for each marker. (C) Inhibition of the Fgfr caused a significant reduction in size of the embryoid bodies. (D) Inhibition of the Fgfr caused a statistically significant increase in circularity. (E) Whole-mount immunostaining of cleaved Caspase-3 in embryoid bodies treated with 4 µM AZD-4547 or 0.04% DMSO. A small non-statistically significant increase in the number of cleaved Caspase-3 nuclei was observed upon treatment with AZD-4547 suggesting that more apoptosis may occur in the outer-layer of these embryoid bodies. A representative image from 3 independent experiments is shown. Data is from 3 independent experiments, error bars represent SEM. Statistical analysis is (B–D) a one-way Anova with a Dunnett’s post-hoc test, (E) a paired t-test (*P = 0.1–0.5, **p = 0.001–0.01, ***p<0.001).</p

Inhibition of the Fgfr with PD-173074 or the Fgfr and Mek disrupted the normal localisation of polarity and junction proteins in the outer layer of embryoid bodies.

<p>Embryoid bodies were treated with 0.1 µM of the Fgfr inhibitor PD-173074, 0.1 µM PD-173074 and 1 µMPD-0325901 or 0.02% DMSO for 7 days. Whole-mount immunostaining demonstrated the localisation of proteins normally polarised in the primitive endoderm. A) Zo-1 a tight junction protein, and (B) β-catenin a protein in the adherens junction were shown to lose their normal polarised localisation when embryoid bodies were incubated with 0.1 µM PD-173074 or 0.1 µM PD-173074 and 1 µM PD-0325901. This suggests a disruption in the apico-basolateral polarisation of these cells. A representative image from 3 independent experiments is shown. Scale bars 10 µm. Dotted lines represent position that the relevant orthogonal or aerial images were taken.</p

Expression and phosphorylation status of key signaling intermediates in ESCs and iPSCs.

<p><b>A</b> and <b>B</b> ESCs or iPSCs were seeded 8000 cells/cm² in KO SR plus LIF. After 24 hours, 10% (v/v) Hyclone serum (HY) was added where indicated (<b>A</b>), or cells were washed 3 times with PBS before LIF-containing or LIF-free KO SR was added to the cultures as indicated (<b>B</b>). After a further 24 hours incubation, protein extracts were prepared, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using the antibodies indicated. Blots in <b>A</b> and <b>B</b>(i) were all generated using cell extracts from one replicate and those in <b>B</b>(ii) from an independent experimental replicate. <b>C</b> ESCs or iPSCs were seeded at 8500 cells/cm² in GMEM 10% (v/v) Hyclone serum (HY) plus LIF, then 24 h later washed and deprived of LIF and serum for 4 h. 5000 U/ml of LIF or 10% (v/v) HY were added and proteins extracted following 0, 5, 30 min treatment with LIF or 30 min treatment with serum. The phospho-proteins indicated were detected by immunoblotting of the same membrane, which had been cut referring to the size of the protein marker, then stripped and re-probed with the corresponding pan antibody. Results generated from the same blots are grouped, with each series terminating with the respective Gapdh as loading control.</p

Culture conditions influence iPSC self-renewal and proliferation.

<p><b>A</b> ESCs or iPSCs (passage 21) were plated onto gelatin-coated dishes in KnockOut-DMEM (KO) supplemented with KO Serum Replacement (SR) and LIF in the presence or absence of 10%(v/v) Hyclone serum (HY) or GMEM supplemented with LIF and 10% (v/v) HY, as indicated. 48 hours after plating cultures were observed by light microscopy and photographs taken. Representative images are shown, scale bar = 200 µm. <b>B</b> The self-renewal capacity of ESCs and iPSCs were evaluated by alkaline phosphatase (AP) expression in clonal assays following 4 days culture in different conditions described in <b>A</b>. The average percentage of AP positive colonies ± SEM are shown from three independent experiments. Two-tailed paired t-tests indicate the following significance *** = p<0.005. <b>C</b> and <b>D</b> Cells where plated at day 0 in KO SR+LIF (<b>C</b>) or the same media supplemented with 10% (v/v) HY (<b>D</b>). Rapamycin (5 nM) or DMSO (1∶10000, as control) were added 24 hours after seeding, and kept in the medium for the duration of the experiment. Cells were harvested after 48 (D2) 72 (D3) and 96 (D4) hours and counted in triplicate. The average values ± SEM are shown from three independent experiments. <b>E</b> ESCs or iPSCs were seeded in KO SR+LIF at 8000 cells/cm². After 24 h DMSO 1∶10000 (ctrl), 5 nM Rapamycin (Rap), 10% (v/v) Hyclone serum (HY) or serum and rapamycin together (HY Rap) were added to the cultures. After 24 h treatment protein extracts were prepared, separated through 10% acrylamide gels using SDS PAGE and immunoblotted using the antibodies indicated.</p