pESCs present functional iGluR receptors and similar Lrp5/6 levels to ESCs but exhibit impaired polarization of Wnt pathway components upon contact with a Wnt source; pESC polarization is recovered by kainate addition. (A) Whole-cell time course Ca²⁺ measurements of ESCs (blue, left) or pESCs (orange, right) expressing GCaMP6s. Lines indicate Ca²⁺ response to the addition of control (CNTRL) solution (green), 100 µM kainate (pink), or 100 µM kainate to cells pretreated with 10 µM CNQX (orange). GCaMP6s intensity is expressed as fold-change to basal intensity before addition (ΔF/F₀). Points are mean of n ≥ 4, and error bars are SEM. Black arrow indicates time of addition. (B)Lrp5 and Lrp6 RNA expression levels in ESCs (blue) or pESCs (orange), presented as normalized expression to GAPDH. Bars are mean of n = 3, and error bars are SEM. ns is not significant, calculated by two-way ANOVA with Šídák’s multiple comparison test. (C and D) Examples of the quantification in E. Left: Representative image of a cell contacting a Wnt3a bead and exhibiting a polarized (C) or nonpolarized (D) distribution of Lrp6. Yellow line and arrow represent a 10-pixel-wide, 20-µm-long line used to measure the intensity profile. Wnt3a bead is highlighted with white dashed line. Scale bar, 20 µm. Right: Normalized quantification of the Lrp6 intensity profile, expressed as fold-change to maximum value across the distance from the Wnt3a bead. (E) Intensity profile of LRP6 (top) and β-catenin (bottom) in ESCs (CNTRL, blue), 10 µM CNQX-treated ESCs (green), control pESCs (CNTRL, orange) or 100 µM kainate (KA)-treated pESCs (pink) contacting Wnt3a beads. Background-normalized intensity is plotted relative to the distance from Wnt3a bead and reported as fold-change to maximum intensity value (expressed as ratio). Gray lines represent individual intensity measurements, colored lines represent mean, and error bars are SEM. n ≥ 41 cells. (F) Representative images of ESCs expressing FZD1-GFP and contacting a Wnt3a bead. Wnt3a bead is black sphere in brightfield (BF) panel, highlighted by white dashed circle. FZD1-GFP intensity is presented using the Fire LUT (ImageJ), and the calibration bar is shown in the figure. Scale bars, 20 µm. (G) Intensity profile of FZD1-GFP in ESCs (top) or pESCs (bottom) contacting a Wnt3a bead. Background-normalized intensity is plotted relative to the distance from Wnt3a bead and reported as fold-change to maximum intensity value (expressed as ratio). Gray lines represent individual intensity measurements, colored lines represent mean, and error bars are SEM. n ≥ 12 cells. (H) Percentage of ESCs or pESCs contacting Wnt3a beads that show polarized FZD1-GFP. n ≥ 12 cells. Statistical significance calculated by Fisher’s exact two-sided test: *, P < 0.05.