Fig. 1.

Expression of SARS-CoV-2 host factors in brain barrier cells. (A) qRT-PCR analysis of ACE2, TMPRSS2, neuropilin 1 (NRP1) and basigin (BSG) as expression fold change relative to Calu-3 cells is shown. β-actin and GAPDH were used as reference genes. Each dot represents an independent differentiation for EECM-BMECs from 5, DMM-BMECs from 6 and BPLCs from 5 iPSC clones, or an independent replicate of HIBCPP, VeroE6 and Calu3. P-values were determined using one-way Anova followed by Tukey’s test to correct for multiple comparison. (B) Western Blot analysis of ACE2 using ab15348 raised against the intracellular domain of ACE2 with β-actin as a loading control is shown. Glycosylated ACE2 is expected at 130 kDa (indicated with arrowhead). Each lane represents an independent differentiation of EECM-BMECs, DMM-BMECs or BPLCs with the clone ID of the iPSC indicated and a replicate of HIBCPP with displayed passage number. 25 µg of protein was loaded per lane. (C) Quantification of relative ACE2 expression by Western blotting is shown normalized to the expression in Calu-3 cells. Each dot represents a clone for EECM-BMECs, DMM-BMECs or BPLCs (3 iPSC clones each) and a replicate of HIBCPP cells. P-values were determined by one-way Anova with Tukey’s test to correct for multiple comparison. (D) Confocal images from immunofluorescence staining for TMPRSS2 (orange) and ACE2 (green) using AG-20 A-0037PF antibody in DMM-BMEC and HIBCPP cells are shown. Nuclei were stained with DAPI (blue). A single Z-plane and for the merge in addition the orthogonal sections are shown. Scale bar = 20 μm. The images are representative of DMM-BMEC differentiations from 3 clones and 3 replicates of HIBCPP. (E) Representative histogram overlays from flow cytometry analysis for NRP1 or BSG on EECM-BMEC (from 4 differentiations, purple), BPLC (from 2 differentiations, petrol), DMM-BMEC (1 differentiation, dark red) and HIBCPP (3 replicates, blue), of Calu-3 (1 replicate, green) with isotype control (grey) are shown