Single-cell RNA sequencing of inguinal and gonadal adipose stromal vascular fractions from wild-type C57BL/6J mice

Endothelial cells (ECs) have been reported to be a source of adipose tissue-derived stromal cells (ADSCs), and ADSCs expressing endothelial markers have been suggested to originate from ECs. To further investigate the lineage relationship between ECs and ADSCs in postnatal mice, we performed single-cell RNA sequencing (scRNA-seq) on ECs and ADSCs isolated from inguinal and gonadal white adipose tissues (IWATs and GWATs) of 12-week-old wild-type C57BL/6J mice, and analyzed the expression of endothelial markers in ADSCs. Overall design: We profiled ECs and ADSCs from mouse IWATs and GWATs using scRNA-seq. Overall, IWATs and GWATs of 12-week-old wild-type mice were collected and digested with a mixture of collagenase, dispase and DNase. Subsequently, the stromal vascular fractions of the samples were subjected to lineage positive cell depletion by fluorescence-activated cell sorting (FACS). Cells positive for B220, Ter119, Gr-1, CD11b, CD3e, CD45, CD71, CD3, CD19 were depleted. Finally, we analyzed ADSCs expressing endothelial markers Pecam1, Cdh5, Tek, and Emcn in the scRNA-seq analysis, and validated these findings with EC lineage tracing models, flow cytometry, and immunostaining analyses.