In Vivo Model for Isolating Epithelial Cells of the Anorectal Transition Zone.

Different epithelia line the body and organs and form a continuous lining of cells. The junction of two different types of epithelia represents a special region called transition zone (TZ). TZ are small areas found in numerous places in the body such as between the esophagus and the stomach, in the cervix, in the eye, and between the anal canal and the rectum. These zones are associated with diverse pathologies such as cancers; however, the cellular and molecular mechanisms involved in tumor progression are poorly investigated. We recently characterized the role of anorectal TZ cells during homeostasis and after injury using an in vivo (lineage tracing) approach. To follow TZ cells, we previously developed a mouse model of lineage tracing using cytokeratin 17 (Krt17) as a promoter and GFP as a reporter. Krt17 is expressed by TZ but also by anal glands located below the TZ in the stroma that can interfere with TZ cell population isolation and analysis afterward. In this chapter, we provide a new dissection method to remove specifically anal glands without affecting anorectal TZ cells. This protocol allows the specific dissection and isolation of anal canal, TZ, and rectum epithelia.

Defining Anorectal Transition Zone Heterogeneity Using Single-Cell RNA Sequencing.

Special regions called transition zones (TZs) are found at numerous places in the body. TZs represent the junction between two different types of epithelia and are located between the esophagus and the stomach, in the cervix, in the eye, and between the anal canal and the rectum. TZ is a heterogeneous population, and the detailed characterization of its populations requires an analysis at the single-cell level. In this chapter, we provide a protocol to do single-cell RNA sequencing primary analysis of anal canal, TZ, and rectum epithelia.

A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration.

At numerous locations of the body, transition zones are localized at the crossroad between two types of epithelium and are frequently associated with neoplasia involving both type of tissues. These transition zones contain cells expressing markers of adult stem cells that can be the target of early transformation. The mere fact that transition zone cells can merge different architecture with separate functions implies for a unique plasticity that these cells must display in steady state. However, their roles during tissue regeneration in normal and injured state remain unknown. Here, by using in vivo lineage tracing, single-cell transcriptomics, computational modeling and a three-dimensional organoid culture system of transition zone cells, we identify a population of Krt17+ basal cells with multipotent properties at the squamo-columnar anorectal junction that maintain a squamous epithelium during normal homeostasis and can participate in the repair of a glandular epithelium following tissue injury.