Figure 3.

Immobilized WNT3A Surfaces Enrich/Maintain Stem Cell Cultures

(A–F) Comma Dβ-Geo cells (7xTCF-GFP/SV40-mCherry) were seeded selectively onto WNT3A DTT or WNT3A surfaces for 30 min. The adherent cells were cultured for 18 hr and then stained live for SCA1 (A–C) or integrin α₆ (D–F). The single-cell maximum expression levels (representative of three independent experiments showing same trend) of SCA1 (A–C) or integrin α₆ (D–F) on immobilized WNT3A or DTT-treated surfaces (A and D). n > 1,000 for each condition; statistical significance determined using Mann-Whitney test (nonparametric); ^∗∗∗∗p < 0.0001. The single-cell spread of the same population when the cells have been separated into (−), (low) or (high) GFP expression (B and E). Statistical significance determined using the Kruskal-Wallis test (nonparametric) comparing distributions to the WNT3A DTT GFP (−) control; ^∗∗∗∗p < 0.0001. Tables summarizing the percentage of cells in each category (C and F).

(G and H) Mouse ESC colonies grown and passaged onto freshly immobilized surfaces (BSA ± soluble WNT3A or WNT3A ± DTT) for up to three passages under serum-free conditions. Between each passage, duplicate wells were stained for alkaline phosphatase (ALP), colonies were counted and split into three categories depending on stain intensity (determined by eye by two researchers); representative color images of three defined levels (scale bar represents 10 μm) (G). Three graphs summarizing the percentages of cells in each category for each passage (H). n = 3 independent experiments; statistical significance determined by comparing the observed population to an expected population equal in each category; ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001.