Multiplexed lineage tracing for high-dimensional single-cell analysis of smooth muscle cell differentiation gene control in atherosclerotic plaques

Ischemic heart disease, which occurs when coronary arteries are narrowed and obstruct blood flow to cardiac muscle, is the leading cause of death in the United States and worldwide.1,2 This narrowing is most often caused by atherosclerotic plaque buildup on arterial walls. Throughout atherogenesis, activated vascular smooth muscle cells (VSMCs) have been observed to undergo dedifferentiation and can later differentiate into dangerous phenotypes.8,15 Knockout studies have demonstrated that Klf4, Oct4, Runx2, and TGFb play roles in the dedifferentiation of VSMCs and subsequent differentiation into high-risk phenotypes; however, the precise function of each gene remains unclear. Combinatorial effects are also unknown. We used multiplex lineage tracing to permanently tag gene loci based on unique markers followed by single-cell mass cytometry to understand the heterogeneity of cell types in plaques and the genomic expression histories of those cells. Multiplex lineage tracing was chosen because it allows for combinatorial analysis of all genes involved. This thesis project focused specifically on designing and incorporating the lineage tracing tools into target gene loci. Successful incorporation of these tools allowed for progression of the project and confirmed that our target genes are good candidates for this study. Overall, this long term project is still in its early stages; however, the experiments outlined here confirm that our system design is effective and should continue to be pursued. The long term goal of this study is to provide data to aid in development of gene therapies to control dedifferentiation, differentiation, and/or lysis of heterogeneous atherosclerotic plaques. Furthermore, our findings will hopefully clarify whether treatments that aim to strengthen the fibrous cap to deter lysis or ones that hinder phenotype switching of VSMCs in the first place will be the most effective means of treatment.