Identification and characterization of stem cells in mammalian esophageal stratified squamous epithelia

Abstract

Somatic stem cells are essential for the maintenance of tissue homeostasis. Despite its importance, how the esophageal stratified squamous epithelium executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2'-deoxyuridine (BrdU) label-chase in rats in vivo and rat esophageal organoids in vitro together with genome-wide DNA methylation and single-cell RNA sequencing, we identified a slow-cycling/quiescent stem cell population that contained high levels of hemidesmosomes (HDs) and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudotime cell trajectory analysis indicated that tissue cells originated from quiescent basal stem cells in the basal layer. Perturbations of HD component expression and/or Wnt signaling reduced the stem cell population in the basal layer of esophageal keratinocyte organoids, resulting in alterations in the organoid formation rate, size, morphogenesis, and proliferation–differentiation homeostasis. Furthermore, not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined the stem cells of the basal layer. Hence, HDs and Wnt signaling are critical determinants for defining the stem cells of the basal layer required for tissue homeostasis in mammalian esophagi.

Mammalian Esophageal Stratified Tissue Homeostasis is Maintained Distinctively by the Epithelial Pluripotent p63+Sox2+and p63−Sox2+Cell Populations

“…3c). However, these p63 + Sox2 + cells were CK14 high cells, indicating that they represented the basal layer cells with stemness property (Col17A1 high ) and growth property (MKi67 high ), which consistent with our previous study [24]. In contrast, the p63 -Sox2 + population were CK13 high CK14 low/negative cells expressing high levels of differentiation-related genes, such as Sprr1a, suggesting that p63 -Sox2 + cells were suprabasal cells.…”

“…To better understand the dynamics of tissue homeostasis, especially proliferation-differentiation at cellular levels, during formation, development and maturation of D3-organoids, we next performed immunofluorescence analyses using various squamous epithelial cell markers, including the endoderm undifferentiated keratinocyte marker cytokeratin14 (CK14), the endoderm differentiated keratinocyte marker cytokeratin13 (CK13), the stemness pluripotent markers p63 and Sox2, and DNA marker DAPI [24]. Consistent with the results obtained from the daily snapshots of phase-contrast microscopy and H&E-stained organoid cross-section analyses, immunofluorescence results revealed that D3-organoids at Day 2 to Day 3 grew as asymmetric small clumps, in which all cells were stained positively for p63, Sox2 and CK14 but negatively for CK13 (Fig.…”

“…H&E and immunofluorescence analyses were performed on OCT-embedded from primary esophageal epithelial tissues or organoids as previously descried [24]. Briefly, the OCT compound-embedded esophageal tissue or organoids were cut into 5-10 µm sections using a cryosection system.…”

“…cell and organoid cultures from esophageal keratinocytes, transgenic mice, carcinogen-induced esophageal carcinogenesis in rodents and human samples from esophagitis and ESCC patients to seek for solutions [4,[18][19][20][21][22][23]. Recently, we and others demonstrate that the mammalian matured esophageal epithelia display the heterogeneity at the single-cell level [1,[24][25][26]. A small population of cells with high levels of hemidesmosomes (HDs) has stemness potential in the context of tissue homeostasis and aging in the basal layer of stratified epithelium [24,25,27].…”

“…Recently, we and others demonstrate that the mammalian matured esophageal epithelia display the heterogeneity at the single-cell level [1,[24][25][26]. A small population of cells with high levels of hemidesmosomes (HDs) has stemness potential in the context of tissue homeostasis and aging in the basal layer of stratified epithelium [24,25,27]. However, the cells of origin that control proliferations, differentiations and damage-repair responses in esophageal stratified matured squamous epithelia have not been clearly determined.…”