FOSB and JUND transcription factors induce ABCB1 expression and regulate transport in human pluripotent stem cell-derived endothelial cells

Abstract

The blood-brain barrier endothelium is distinguished by specialized transport functions, including high expression of the efflux transporter ABCB1 (P-glycoprotein). However, the transcriptional mechanisms that induce ABCB1 expression in human brain microvascular endothelial cells (BMECs) remain poorly defined, and most human pluripotent stem cell (hPSC)-derived BMECs lack robust ABCB1 expression. Here, we sought to identify transcription factors (TFs) capable of inducing functional ABCB1 expression in hPSC-derived endothelial cells. We generated a CRISPR-engineered ABCB1-EGFP reporter hPSC line and differentiated this line into central nervous system (CNS)-like endothelial cells (CECs). A pooled overexpression screen of a TF library was performed, followed by fluorescence-activated cell sorting and barcode sequencing to identify candidate regulators of ABCB1 expression. Candidate TFs were tested by lentiviral overexpression and flow cytometry for ABCB1, and lead TFs were validated across multiple hPSC lines including inducible ETV2 forward-programmed endothelial cells. Bulk RNA sequencing was performed on CD144-sorted endothelial populations, followed by differential expression, pathway enrichment, and motif analyses. Functional transport remodeling was assessed using Rhodamine 123 efflux, albumin uptake and transferrin uptake. The screen identified AP-1 family TFs as candidate ABCB1 regulators. Individual AP-1 family TFs were insufficient to induce ABCB1 expression, whereas co-expression of FOSB and JUND robustly induced ABCB1 expression across multiple stem cell lines. ABCB1 induction occurred in a subset of CD144 + endothelial cells and was associated with reduced CD144 expression, which was partially rescued by ETV2 reinforcement and, to a lesser extent, by SOX17 overexpression. Transcriptomic profiling demonstrated that FOSB+JUND activated inflammatory responses and signaling programs associated with MAPK, PI3K-Akt, TNF, and TGF-β pathways while suppressing proliferative programs. Functionally, FOSB+JUND remodeled multiple endothelial transport pathways, including induction of ABCB1-mediated Rhodamine 123 efflux, reduced CAV1 expression and albumin uptake and increased transferrin receptor expression and transferrin uptake. These findings identify FOSB+JUND as a combinatorial AP-1 TF complex sufficient to induce functional ABCB1 expression and remodel endothelial transport programs in hPSC-derived CECs. More broadly, this work demonstrates that endothelial transport specialization can be selectively engineered through transcription factors, providing a framework for improving in vitro BBB models and studying BBB transporter regulation.