Notch3 directs differentiation of brain mural cells from human pluripotent stem cell-derived neural crest

Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRβ, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional K_ATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.