Fig. 6: RNA-sequencing analysis.

a Whole transcriptome analysis was performed for BMSC and ACh cultures treated with TGF-β1 (blue lines) and harvested on days 0, 1, 3, 7, and 21 as indicated by the solid red arrows (BMSC donors 1, 2, and 3, and ACh donor 1 and 2). Additionally, for BMSC donor 1 and ACh donor 2, RNA-seq analysis was performed on day 21 following TGF-β1 withdrawal on days 1, 3, and 7, represented by dashed red arrows. b MDS plot reveals the convergence of BMSC cultures on a common gene expression profile by day 21. This gene expression profile was common in 21-day cultures, despite being exposed to exogenous TGF-β1 for different durations (1, 3, 7, or 21 days). BMSC and ACh gene expression was dissimilar in samples treated in the same way. Solid arrows in the MDS plot show the progression of gene expression profiles from day 0 cultures maintained continuously in TGF-β1, and analyzed on day 1, 3, 7, or 21. Dashed arrows point to the changes in global gene expression on day 21, following TGF-β1 withdrawal from cultures on day 1, 3, or 7. c Differentially expressed genes related to osteochondral transcription factors (left), soluble factors and receptor signaling (middle), and ECM molecules and ECM biosynthesis (right). Each timepoint in the heatmap is represented by a color shown on the bottom row of the heatmap, which corresponds to the color key on the bottom left of the figure. Each BMSC timepoint has three columns, representing three unique BMSC donors, and each ACh timepoint has two columns, representing two unique ACh donors; only one BMSC donor and one ACh donor was sequenced on day 21 with TGF-β1 withdrawal on day 1, 3, or 7, and are represented by the lighter pink shades; heatmap colors represent the average of normalized logCPM values from three culture wells.