Pou2f3⁺ Cells Contain Multilineage Potential Progenitors as Revealed by scRNA-seq Analysis

To further study the lineage potential of Pou2f3⁺ cells, we performed single-cell RNA sequencing (scRNA-seq) of lineage-labeled cells isolated from the gastric tissues of Pou2f3-CreERT; Rosa26-tdTomato mice. Lineage-labeled gastric epithelial cells (EPCAM+tdT+) at 21dpi were purified with flow cytometry. We also included an equal mixture of EPCAM⁺ tdT⁻ cells for comparative analysis to define reference epithelial subsets. Three Pou2f3-CreERT; Rosa26-tdTomato transgenic mice were induced with tamoxifen for 2 weeks. TdTomato+ cells were sorted by FACS and pooled from multiple mice. We employed a strategy of mixing TdTomato+ cells with TdTomato−, EpCAM+, CD45− gastric epithelial cells isolated from the same tissues at a 1:1 ratio during sample preparation for scRNA-seq analysis.