High Autophagic Vesicle Content Identifies a Novel Slow‐cycling Esophageal Basal Cell Population with Stem‐like Properties

Abstract

Esophageal squamous epithelium comprises a basal layer of proliferative cells that differentiate in suprabasal layers before luminal desquamation, facilitating tissue renewal. Controversy exists as to whether the esophageal basal cells are functionally homogenous, with each cell having equal potential to proliferate and differentiate, or heterogenous, with subsets of basal cells harboring differential proliferation and differentiation kinetics. To examine the relationship between the stemness/proliferation axis and autophagy in esophageal basal cells, we stained primary basal cells from wild type mice with the autophagic vesicle (AVs)‐identifying dye Cyto‐ID. AV‐high and AV‐low basal esophageal epithelial cell fractions were sorted by fluorescence‐activated cell sorting (Figure 1A) and stem/progenitor function was assessed by organoid formation assay and passaging. Upon initial plating, organoid formation rate (OFR) was decreased in Cyto‐IDhighbasal cells(0.8%±0.11) as compared to their Cyto‐IDlow counterparts (10.0%±0.06; n=3; p<0.001) (Figure 1B). However, passaging revealed that OFR decreased in Cyto‐IDlow cells (0.36% for passage 2) while increasing in Cyto‐IDhigh cells (14.88% for passage 4) (Figure 1B). Additionally, while Cyto‐IDlowbasal cells maintained spherical morphology upon passaging, structures reminiscent of bud‐like intestinal organoids with enhanced self‐renewal capacity emerged in Cyto‐IDhighcells (Figure 1C). These findings suggest that cells with high AV content may serve as a quiescent stem cell population. To examine the gene expression profiles of Cyto‐IDlowand Cyto‐IDhigh cells, we performed RNA‐Seq and identified >7000 differentially expressed genes (p<0.05, log2 fold‐change= 2) (Figure 1D). Ingenuity pathway analysis revealed reduction in Cell Cycle‐associated genes in Cyto‐IDhigh cells and propidium iodide flow cytometry confirmed impaired cell cycle progression with significant depletion of the S and G2/M fractions in Cyto‐IDhighcells (S: 0.5 %; G2/M: 2.2%) as compared to Cyto‐IDlow cells (S: 3.0%; G2/M: 8.3%, n=3, p=<0.05). Finally, we utilized K5‐CreERT2+/‐; Atg7flox/floxmice with tamoxifen‐inducible autophagy impairment in squamous epithelium to evaluate the functional consequences of autophagy depletion upon esophageal epithelial tissue architecture. Atg7 depletion in vivo resulted in hyperkeratinization, consistent with autophagy limiting cell proliferation of esophageal keratinocytes and supporting a functional role for autophagy in regulating stem cell biology and cell fate determination in the esophagus.These studies identify a novel quiescent/slow‐cycling stem‐like population in esophageal epithelium marked by high AV content. Future studies will define the detailed molecular mechanisms through which autophagy supports stemness in esophageal epithelium and determine the functional contribution of autophagy to the pathogenesis of widely prevalent esophageal diseases.