Fig. 2

Strategic gating of unlabeled vasculature derived cells from WT mice enriches for SMCs. Further fluorescence activated cell sorting (FACS) analysis of tdTomato (tdT) sorted cells from Cspg4-Cre;Ai9-tdT mice described in Fig. 1 revealed that selectively gating for high side and forward scatter events (A) enriched for the tdT⁺ smooth muscle cell (SMC) populations, where the percent of tdT⁺ cells relative to total gated live cells increased from 3.0% (gating as in Fig. 1) to 60.0% (B). Moreover, 83.2% of these tdT⁺ events represent the SMC populations (relative to 56.0% as in Fig. 1) (C). Popliteal lymphatic vessels (PLVs) and superficial saphenous veins (SSVs) from 8–9-month-old wild-type (WT) mice (n = 3) were harvested, and the gating strategy represented in A was performed and DAPI⁻ live events were selected (13,130 sorted events) for downstream single-cell RNA-sequencing (scRNAseq). The tdT and WT datasets were integrated and a total of 7446 cells (1958 tdT and 5488 WT) were analyzed by minimally supervised shared nearest neighbor (SNN) clustering algorithms in Seurat. Each cluster was labeled by cell type and the total number of cells for each cluster is shown in parentheses (D). A heatmap represents the top 3 differentially expressed genes for all 18 cell clusters (duplicate genes omitted) with each cluster color coated and in the same cluster order (left to right) as the UMAP in (D) (E). A full gene list for each cluster is provided in the Supplementary Materials. Overlay of the two tdT replicates with the WT sample demonstrated remarkable conservation of cell types with limited batch effect (F). UMAPs of the tdT replicates and WT sample are shown side-by-side to further depict the conservation of cell types, and proportional changes in cell populations with the two different sorting strategies for tdT and WT samples (G)