Investigating the plasticity and the differentiation potential of epithelial transition zone cells during normal and perturbed homeostasis – StemTZones

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.

We developed a mouse model to trace TZ Krt17+ cells between the anal canal and the rectum. The model allowed us to show the unipotent property of TZ cells during homeostasis as they maintained only the stratified squamous epithelium of the anal canal. We also successfully developed a 3D organoid culture of anorectal TZ cells that recapitulated the heterogeneity and specific markers of TZ. Next, we challenged those cells with a wounding method called EDTA wound to remove specifically crypt of the rectum. When challenged, a minority of Krt17+ TZ cells were able to change their properties to become multipotent and participate in repair of the simple columnar epithelium constituting the rectum. Using single-cell RNAsequencing and pseudo time analysis, we confirmed our lineage tracing data that is the unipotency of TZ cells during homeostasis and allowed us to define the heterogeneous anorectal TZ cells. We also showed by bioinformatics analysis the emergence of a cell type identified as “hybrid cell” when wounding the region. Hybrid cells showed specific markers at the mRNA and protein level from both squamous stratified and simple columnar cells. Moreover, single-cell RNA sequencing allowed us to highlight the expression of the wound-associated JunB gene present in hybrid cells that we further confirmed is involved in proper differentiation of TZ cells. These findings challenge the idea that each epithelium may be maintained by its own stem cell pool and open novel way for tissue regeneration following rectal injury.

Our epithelia are constantly renewing themselves. To do this, they have their own stem cells, but if these are lacking, those of the transition zone can come to reinforce them. It is like an emergency reservoir that ensures tissue homeostasis - balance. Our discoveries thus open the way to numerous perspectives both in regenerative medicine and in the understanding of the abnormal phenomena governing malignant transformations. We are continuing to characterize another TZ which is the stomach to determine if in another region, keratin 17+ cells have a similar protective role. Finally, if each epithelium surrounding the TZ has its own stem cells, what is the purpose of this transition? Is it simply a barrier separating two different epithelia? To answer these questions, we are trying to eliminate this TZ using genetically modified models to determine if the surrounding epithelia are affected by this deletion or if a phenomenon of tissue adaptation (plasticity) can be envisaged.

Mitoyan L, Chevrier V, Hernandez-Vargas H, Ollivier A, Homayed Z, Pannequin J, Poizat F, De Biasi-Cador C, Charafe-Jauffret E, Ginestier C, and Guasch G. A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration after injury. Nature Communications 2021 12(1):2761.

Alexane Ollivier, Maxime Mahé, Géraldine Guasch Modeling gastrointestinal diseases using organoids to understand healing and regenerative processes. Cells. 2021,10, 1331

Mitoyan L, Gard C, Nin S, Loriod B, Guasch G. Defining anorectal transition zone heterogeneity using single-cell RNA sequencing. Methods Mol Biol. 2022 In press

PhD Louciné Mitoyan «Characterizing the role of epithelial stem cells at transition zones in normal and disease state » defended on October 13 2021

Within adult epithelia, stem cells maintain homeostasis and respond to wound injury, a process critical for the maintenance of the organ. At numerous location of the body, transition zone lies between two types of epithelium, contain cells with stem cell properties, and are frequently associated with neoplasia involving both type of tissues. These junctions between our organs allow us to have a continuous lining of tissues it is thus essential to understand how they participate in the inter-organ relationship and how crucial they are to preserve organ homeostasis and function. StemTZones challenges the idea that each epithelium is maintained by its own stem cell pool. We hypothesize that an unrecognized reservoir of multipotent stem cells, participating to the maintenance of both epithelial types, exists in these transition zones and represent a barrier against metaplasia defined by the replacement of one cell type to another and frequently preceding the dysplasia-cancer progression. Understanding whether transition zone cells represent a new population of cells able to maintain surrounding epithelia is a central issue for many regenerative tissues. More importantly, deciphering the unique and common characteristics of transition zone cells is expected to further our understanding of the frequent tumor susceptibility of these regions.

StemTZones will use novel methods that we recently developed to specifically mark and follow transition zone cells during development by lineage tracing combined with a multidisciplinary approach integrating mouse genetics, bioinformatics, developmental biology, transcriptional profiling and functional experiments in tridimensional culture.

The specific objectives of StemTZones are: 1) To determine the differentiation potential and the fate of transition zone cells from the stomach and the anorectal regions during postnatal grown, adult tissue regeneration and after an injury; 2) To identify the regulatory mechanisms by which transition zone stem cells maintain homeostasis and respond to wound injury; and 3) To define the contribution of transition zone cells to maintain the homeostasis of the surroundings epithelial by selective genetic ablation.