Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Abstract

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescenceactivated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively highthroughput method for identifying and understanding esophageal stem cell niche components in vitro.

Defining the contribution ofTroy-positive progenitor cells to the mouse esophageal epithelium

“…For isolation of esophageal progenitor cells, the muscle layer was removed from the submucosa and the remaining esophagus incubated in 0.5 mg/ml Thermolysin (Sigma) at 37°C for 15 min 47 . After incubation, the submucosa and the epithelium were separated.…”

“…Subsequently, the esophagus was longitudinally cut open and cut into approximately 5 mm pieces. Pieces were incubated for 2-3 hours in 5 mM EDTA in PBS at 37°C and the epithelium peeled away from the underlying tissue 47 . Afterwards, the epithelium was fixed in 4% formaldehyde (FA) in PBS for 30 min.…”

“…Esophagi were dissected and the outer muscle layer mechanically removed and esophageal epithelial cells were isolated as previously described 47 . All epithelial cells were gated on CD324+CD104+ to enrich for basal cells.…”

Regionalized cell and gene signatures govern oesophageal epithelial homeostasis

“…Herein, we employed regionally resolved Smart-seq3 single-cell sequencing 21,22 to characterize the adult mouse oesophageal proximal-distal axis. We capture all major cell populations in the oesophagus, identify and compare proximal-to-distal progenitor-niche networks and molecular gradients and combine in vivo models with organoid co-cultures 23 to functionally demonstrate proof-of-concept cellular interactions and signalling dependencies.…”

“…Herein, we employed regionally resolved Smart-seq3 single-cell sequencing 21,22 to characterize the adult mouse oesophageal proximal-distal axis. We capture all major cell populations in the oesophagus, identify and compare proximal-to-distal progenitor-niche networks and molecular gradients and combine in vivo models with organoid co-cultures 23 to functionally demonstrate proof-of-concept cellular interactions and signalling dependencies. This study provides a comprehensive and importantly, regionally resolved, oesophageal cell atlas, challenging our current understanding of oesophageal tissue homeostasis and providing insights into clinically relevant disease susceptibility.…”