Fig 1: DNGR-1-tracing marks a distinct class of ependymal cells, wherein resides latent stem cell ability(A) Spinal cord central canal cryosection from a Clec9aCre/CreRosaLSLtdTomato/LSLtdTomato mouse labeled with anti-CD133 (green). CD133 is expressed apically and marks all ependymal cells (traced and untraced).(B) FACS-sorting of CD133+ DNGR-1-traced and CD133+ non-traced cells.(C) Neurosphere assay of CD133+ DNGR-1-traced and CD133+ non-traced cells from 4 pooled spinal cords. Right, quantification of neurosphere-containing wells and number of neurospheres per well from each fraction. Data are from two independent experiments. Error bars (SEM). Groups were compared using unpaired t test. ****p < 0.0001.(D) Dataset integration and UMAP of CD133+ DNGR-1-traced cells and CD133+ non-traced cells into 3 groups.(E) Heatmap of the top 20 differentially expressed genes in each group from (D).(F) UMAP representation by cellular provenance and quantification of each cellular fraction per group. Orange, CD133+ DNGR-1-traced cells; Green, CD133+ non-traced cells. 97.89% of CD133+ DNGR-1-traced cells were unbiasedly assigned to ependymal cell group (group 2). The remaining CD133+ DNGR-1-traced cells were assigned to groups 1 (1.89%) and 3 (0.23%). Dashed line represents the proportion of CD133+ DNGR-1-traced cells within the UMAP space (15.95%—4,825 of 30,326 cells).(G) UMAP of cells from group 2 (from F).(H) UMAP shown in (G) indicating cellular provenance: CD133+ DNGR-1-traced ependymal cells in salmon and CD133+ non-traced cells in cyan.(I) Percentage of traced and non-traced cells in each of the clusters 0–4 from (G). Dashed line represents the proportion of CD133+ DNGR-1-traced cells within UMAP space (67.9%—4,723 of 6,951 cells).(J) Expression of a selected set of cluster markers from clusters 1 and 3 (from G).(K) Distribution of the top 5 Gene Ontology biological processes defined by the differentially expressed genes in each cluster.Scale bars, 500 µm (C) and 10 µm (A).