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. 2016 Jul 12;7(1):126–137. doi: 10.1016/j.stemcr.2016.06.004

Figure 4.

Figure 4

Immobilized WNT3A Surfaces Can Be Adapted to 3D-Culture to Control Human Mesenchymal Stem Cell Differentiation

(A) hMSCs were seeded onto immobilized WNT3A ± DTT to form a confluent monolayer before overlaying a collagen gel. After 7 days in culture, cells were fixed and stained with DAPI to mark individual cells. The percentage of cells per layer of the collagen gel (lower level, up to 72 μm from the gel base; middle, 72–132 μm; and upper, 132–179 μm) normalized to the number of cells at the base. A representative bottom-up max projection with DAPI (white) marking each nucleus; example cells in each layer marked with arrows. n = 3 independent experiments, mean ± SEM. The scale bar represents 10 μm.

(B and C) hMSC in collagen gels were fixed after 7 days and immunostained for STRO1 (B) and osteocalcin (C). Expression levels were compared between immobilized WNT3A and DTT-treated. Quantification of normalized (subtracted background) image pixel intensity relative to cell number was plotted. n = 3 independent experiments, mean ± SEM; statistical significance between groups determined by post hoc Mann-Whitney tests; ∗p < 0.05.

(D) Representative histological staining of hMSC gels for calcium deposition (Alizarin red staining) after 7 days of culture. The scale bar represents 100 μm.

(E) hMSCs were grown on immobilized WNT3A ± DTT, BSA ± soluble WNT3A (50 ng/ml). Migrating cells reported as the percentage of total cells in each defined layer of the gel (lower level, up to 80 μm from the gel base; middle, 80–140 μm; and upper, 140–200 μm. n = 3 independent experiments, mean ± SEM.

(F) The percentage of migratory cells in each layer when grown on immobilized WNT3A surfaces ± IWR treatment. n = 3 independent experiments, mean ± SEM.

(G) After 7 days, hMSCs grown on BSA with soluble WNT3A, immobilized WNT3A or immobilized WNT3A with IWR treatment were fixed and immunostained for STRO1. Staining across the middle of the well at the base layer (4× magnification) was quantified. n = 3 independent experiments, mean ± SEM; statistical significance determined by a one-way ANOVA test; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001) (for representative confocal images, see Figure S4C).

(H) After 7 days, hMSCs grown on immobilized WNT3A ± IWR treatment were fixed and immunostained for osteocalcin. A representative image of an osteocalcin-expressing cell within the collagen gel (left) and the quantification of osteocalcin in the layers of the collagen gel (right). n = 3 independent experiments, mean ± SEM; statistical significance between groups was determined by post hoc Mann-Whitney tests; ∗p < 0.05. The scale bar represents 50 μM.

(I) Before fixation, 2 days or 7 days with the collagen gel, hMSC cells were stained with EdU and the percentage of EdU+ cells was quantified. n = 3 independent experiments, mean ± SEM.