Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Overall design: We FACS collected three samples of cells from the dissociated subventricular zone of aSMACreERT2::tdTomato/Sox2-GFP mice. These included one sample of tdTomato/GFP double positive cells, and two samples of tdTomato-negative/GFP-positive cells. 10X Chromium technology was used to prepare single cell barcoded RNA sequencing libraries from each sample. These samples were subsequently sequenced on the Illumina HiSeq 2500 to a total average depth of approx. 50 million reads/sample.