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

Abstract

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.

Clinical and Genetic Correlation in Neurocristopathies: Bridging a Precision Medicine Gap

“…During brain morphogenesis, NOTCH signaling is crucial for mural cell (also known as vSMCs and pericytes) differentiation from neural crest-derived stem cells. It has been shown, using hPSC-derived neural crest, that NOTCH signaling is a deciding factor in driving the terminal differentiation of mural cells in the face and the forebrain [92,93]. FOXC1 and PITX2 are two other genes that are implicated in small-vessel disease, with both of them being expressed in NCCs.…”

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“…Mural cells, which include smooth muscle cells and pericytes, are located on the abluminal surface of ECs. Mural cells regulate several functions within the brain, including the control of resting cerebral blood flow, neurovascular coupling, BBB development and maintenance, and neuronal survival [ 16 , 17 , 18 , 19 , 20 ]. Vascular smooth muscle cells can be found within larger arteries and veins.…”