Fig. 2

Three-dimensional microenvironment regulates iBMEC gene expression. A To elucidate the role of 3D microenvironment on iBMEC gene expression, 2D and 3D models were compared by paired bulk RNA sequencing. These findings are benchmarked to previous work on the role of shear stress (SS) [28] and to datasets for brain microvessels, endothelial cells, and epithelial cells. B Volcano plots depicting significantly (adjusted p < 0.05, Wald test with Benjamini–Hochberg correction) upregulated genes (blue) and downregulated genes (red) in iBMECs in 3D microvessels versus 2D monolayers (n = 5 per condition). The five paired 2D/3D replicates encompass three WTC iBMECs (one for each WTC iPSC source) and two BC1 iBMECs (an independent iPSC source). Selected genes are labeled. C Semi-quantitative validation of protein enrichment in 3D microvessels. Representative immunofluorescence images for claudin-5, ICAM-1, and MT2A in 2D and 3D are shown. Data are normalized to the nuclei signal and endothelium area, and presented as mean ± SD (n = 2 microvessels across independent differentiations). D Comparison of upregulated genes between the 3D microenvironment and 2D monolayers under shear stress. The Venn diagram shows overlap of genes upregulated by the 3D microenvironment or shear stress by log₂FC > 1. The lollipop plot highlights normalized enrichment scores (NES) of select Hallmark gene sets by 3D microenvironment and shear stress (see Additional file 1: Fig. S4A for all 50 gene sets). E, F Heatmaps of changes in endothelial and epithelial transcript abundance due to 3D microenvironment versus shear stress; DEGs are labeled with an asterisk. G iBMECs were benchmarked to datasets for brain microvessels, endothelial cells, and epithelial cells (see Additional file 1: Fig. S5 for complete analysis). 3D microenvironment increases the correlation to brain microvessels and endothelial cells, while shear stress (SS) alone does not. Changes in correlation (%) are calculated as the Spearman correlation coefficient between 3D or SS iBMECs versus each dataset minus the Spearman correlation coefficient between 2D or static iBMECs versus each dataset (positive values indicate increased transcriptomic similarity to an external dataset). Each dataset is shown as a black dot; significance calculated using a Mann–Whitney test. Data are presented as mean ± SD. ** p < 0.01. See also Additional file 1: Fig. S4–S6) and Additional file 2