Hydrogel culture of stem cell‐derived disease models exhibits features of LAM. A) Representative immunofluorescence images of WT and TSC2−/− cells. Inset showing punctate PMEL and fibril ACTA2 staining. Scale bars of 100. B) VEGF‐D secreted into conditioned media measured by ELISA, following 16 h incubation in serum‐free media ± 20 × 10−9m rapamycin (mean ± SD; * = p < 0.05 by one‐way analysis of variance (ANOVA); n = 9–10). C–E) Visualization of LAM cell invasion after 3 days in hydrogel culture, as C) a schematic, D) brightfield image of single Z plane, scale bars of 250 µm, and E) computational reconstruction of cellular spatial positions. F) Median invasion distance of cellular populations plated on the hydrogel and cultured for 3 days ± 20 × 10−9m rapamycin (mean ± SD; * = p < 0.05 by one‐way ANOVA with Bonferroni post hoc comparisons; n = 124). G) Percentage of cells invaded past fixed threshold set by median invasion distance of genotype‐matched vehicle control. Cells were cultured and treated for 3 days (10 × 10−6m GM6001, a pan‐MMP inhibitor, and 20 × 10−6m Y27632, a ROCK inhibitor, mean ± SD; * = p < 0.05 by one‐way ANOVA with Bonferroni post hoc comparisons; n = 4). H) Schematic of the sample conditions tested in the bulk RNA‐seq experiment. NT = no treatment, Rapa = rapamycin treatment (20 × 10−9m, 72 h). I) Principal components analysis (PCA) of bulk RNA‐seq samples. J) Heatmap and hierarchal clustering of differentially expressed genes (DEGs) between TSC2−/− and WT samples, and between hydrogel and plastic samples, while controlling for the reciprocal covariate. Left panel: transcript expression for plastic and hydrogel cultures was averaged. Right panel: transcript expression for WT and TSC2−/− samples was averaged. DEG analysis was performed with no treatment samples; genes noted as differentially expressed if FDR < 0.05 and |log2FC| > 1. K) Overlap in DEG between genotype and ECM gene lists and LAM cell signature gene list.[23] Genes noted as DE if FDR < 0.05. L) Percentage of EdU+ (proliferating) cells from 3 h pulse (5 × 10−6m), after 3 days cultured on plastic or hydrogel ± 20 × 10−9m rapamycin (mean ± SD; * = p < 0.05 by one‐way ANOVA with Bonferroni post hoc comparisons; n = 5).
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