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. 2020 Mar 10;14(3):374–389. doi: 10.1016/j.stemcr.2020.02.004

Figure 2.

Figure 2

HESC-Derived RPE Recapitulate Dynamic POS Ensheathment In Vitro

(A–D) SEM of hESC-RPE without POS. Scale bars, 50 μm (A), 10 μm (B), 1 μm (C), and 10 μm (D).

(E) A schematic of the experiment. HESC-RPE cells were primed with 30% serum for 1 h before addition of F-POS or left untreated. After seeding F-POS, cells were incubated for 1, 2, or 3 h. After 3 h unbound POS were washed off with medium containing serum and cells were fixed at 4, 5, or 6 h. Samples without serum were incubated for 3 and 24 h before they were fixed and processed for SEM.

(F–H) SEM images of hESC-RPE with F-POS. F-POS are artificially colored yellow. Scale bar, 5 μm (F). Cells that were treated with POS and serum for 1 h show smaller and fewer sheets. Yellow arrow points to small sheets with POS. (G) Cells that were treated with POS and serum for 3 h show larger and numerous sheets. Red arrow points to the big sheets surrounding POS. See also Figures S1 and S2. (H) Cells that were treated with POS without serum for 3 h did not form sheets.

(I–K) Ten images containing around ten cells from each condition were analyzed. (I) The presence of serum and POS increases the number of total sheets per cell, and the number of sheets associated with POS. In the absence of serum very few sheets are observed. The number of sheets/cell is highest at 3 h after POS addition (pulse) and significantly decreases 3 h after POS pulse. Significance was calculated using two-way ANOVA comparing all samples to 3 h. (J) The percentage of cells that extend sheets in response to POS and serum increases significantly at 3 h and decreases again after 6 h. In contrast, cells that received POS without serum do not show ensheathment either at 3 h or at 24 h. (K) In the absence of serum most of the bound POS are unensheathed. Statistical significance for (J and K) was calculated using one-way ANOVA comparing all samples to 3 h. Data are represented as means ± SD. N = 3 biological repeats. ns > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(L–N) Examples for quantifying F-POS, sheets, and cells within a field of view. (L) Manual segmentation of F-POS. Six POS fragments were counted. Scale bar, 5 μm. (M) Manual segmentation of sheets. Twelve sheets were counted. Scale bar, 5 μm. (N) Manual quantification of the cells. Fourteen cells were counted. Scale bar, 10 μm.