2023 53 B3

Defining the Potential of Mural Progenitor Cells in Human Brain Development

By: Edward Valenzuela

Department: Biology

Faculty Advisors: Dr. Lily Chen, Dr. Christopher Moffatt, Dr. Elizabeth Crouch (UCSF)

Preterm infants born during the second trimester are more susceptible to developing brain hemorrhages and the reasons why the brain vasculature is vulnerable in this developmental period remains unclear. Using FACS, single-cell transcriptomics, and histological and ultrastructural analyses, we find that a group of endothelial and mural cell subtypes make up the brain vasculature during this critical period. To determine their putative developmental trajectories, we performed RNAvelocity analysis on second trimester endothelial and mural cells. These experiments revealed marked stages with distinct endothelial and mural stem cells at the beginning of each trajectory. In mural cells, RNAvelocity revealed smooth muscle cells as the putative stem cells in the lineage. To confirm this finding, we performed FACS and cell culture comparing smooth muscle cell mural cells to other subtypes. We identified caveolin1 and syndecan2 as a putative smooth muscle cell markers for FACS, and syndecan2 appeared to separate distinct populations. Caveolin1 did not label any cells in our experiments. With syndecan2, we then purified high and low expressing cells and cultured them on collagen in smooth muscle cell media or Pericyte Media. After 1-2 weeks in culture, we performed immunocytochemistry against smooth muscle actin, CD248, and Platelet-Derived Growth Factor Beta. To help understand and define the spatiotemporal dynamics of human brain mural stem cells, we performed HiPlex RNAscope using a total of twelve RNA probes. The selected probes included RGS5 and PDGFR-ß for pan-mural cells, CSPG4 and CD248 for classic pericytes, ACTA2 and SDC2 for smooth muscle cells, CLU and SERPING1 for fibroblasts, MKI67 and TMSB15A for mitotic mural cells, and ANXA2 and EMX2 for neural cells. Preliminary results show potential overlap of mural and neural RNA expression but further analysis is required. Together, our results could uncover important mechanisms into vascular development during this critical period of human brain development.