Figure 1
From: Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE
Adhesion of hESC-RPE on different protein coatings (all data shown for hESC-08/017). (a) Confocal single plane images showing the subcellular distribution of vinculin and filamentous actin 24 h after seeding. In the overlay, vinculin is shown in green, actin filaments are stained by phalloidin (red) and nucleus by DAPI (blue). Scale bar 50 µm. (b) Quantification of the cell area 24 h after seeding was done by outlining the periphery of cells based on F-Actin staining using ImageJ software. The analysed cell numbers were 80 (Col), 241 (LN), 168 (Col + LN) and 265 (Col + LN + Nid) and the results are given as mean ± SD. (c) Adhesion force (Fad) correlates with the force (g) needed to detach half of the adherent cells after centrifugation, values are given as mean ± SD. Measurements were done 24 h after seeding from 3 biological and 2–3 technical replicates for each coating. (d) Phase contrast micrographs of RPE cell morphology on different coatings 3 and 10 weeks after seeding. *p < 0.05, ***p < 0.001, ****p < 0.0001. (e) Pigmentation analysis of hESC-RPE after 8 weeks of seeding. Each image and bar represent the average intensity of five differential interference contrast (DIC) images (LSM800, Carl Zeiss, air immersion objective 20x). Brightness from each image was calculated using ImageJ and represents as mean ± SD. Difference in brightness was statistically significant between LN and all other coatings; LN + Col (p = 0.0079), LN + Col + 1xNid (p = 0.0079) and LN + Col + 10xNid (p = 0.0079). In addition, difference in brightness of LN + Col was statistically significant compared to LN + Col + 1xNid (p = 0.0079). Scale bar 20 µm. All statistics were performed with Mann–Whitney U. Col—collagen IV, LN—laminin, Nid—nidogen-1.
