Mural stem cells in normal development and germinal matrix hemorrhage in the preterm human brain

PROJECT ABSTRACT/SUMMARY Brain blood vessel cells play key roles in both physiological and pathological states. Endothelial and mural cells compose the main structural and functional elements of the vasculature. Mural cells, which lie outside of endothelial cells, are critical for the development of the blood brain barrier and regulate blood flow in response to neuronal activity. However, most studies of brain blood vessels use adult mice and therefore little is known about the developing vasculature or human brain blood vessels at any age. These knowledge gaps preferentially affect preterm infants, who are prone to develop hemorrhages specifically located in brain regions called the ganglionic eminences (GE). These hemorrhages confer high risk for permanent brain injury, and we have no treatments. To molecularly define the stages and subtypes of mural cells in the developing human brain, I developed a Fluorescence Activated Cell Sorting (FACS) strategy followed by single cell RNA sequencing (scRNAseq). These data revealed distinct stages of mural cell development from a relatively immature stage at 15GW to the emergence of distinct subtypes at 23 GW. Strikingly, this strategy also uncovered a novel progenitor population that express both mural and neural stem cell markers, suggesting a common neurovascular progenitor that contributes to both angiogenesis and neurogenesis. In the GE, immunohistochemistry with putative neurovascular progenitor markers revealed these cells in a periventricular and perivascular location situated amongst canonical neural stem cells. Consistent with these results, FACS-purified mural cells produce both vascular and neural lineage cells in vitro. In sum, the above experiments lead me to hypothesize that mural cells contribute to both angiogenesis and neurogenesis during development and that a predominance of immature stages of mural cells in the GE contributes to the pathogenesis of hemorrhage. My short-term goals for the proposed K08 are the following: 1. Construct an atlas of human brain mural cells during development using scRNAseq and postmortem human tissue. 2. Develop organoid, mouse transplant, and CRISPR tools to manipulate and interrogate the role of specific signaling pathways of mural cells in human brain development. These results will propel me to an R01. As a physician scientist in Neonatal- Perinatal Medicine, I am uniquely suited to advance our understanding of brain vascular development and the application of this knowledge towards future therapy.