Cambrian Yangshao Liu

Summary: Intestinal epithelial wound healing, which is essential for health, is compromised and represents a therapeutic target in inflammatory bowel disease (IBD). While studies have elucidated important subpopulations of intestinal epithelial cells in repair, these have yet to translate to therapies. Here, in mouse models of acute colitis, we demonstrate a distinct and essential source of wound-healing cells that re-epithelialize the distal colon. Using 3-d imaging, lineage tracing, and single-cell transcriptomics, we show that neighboring skin-like (squamous) cells of the anus rapidly migrate into the injured colon and establish a permanent epithelium of crypt-like morphology. These squamous cells derive from a small unique transition zone, at the boundary of colonic and anal epithelium, that resists colitis. The cells of this zone have a pre-loaded program of colonic differentiation and further upregulate key aspects of colonic epithelium during repair. Thus, heterologous cell-types at tissue junctions represent unique reserve cells capable of repair and plasticity.

Patients with NEUROG3 mutations suffer from diabetes mellitus due to pancreatic endocrinopathy and chronic malabsorptive diarrhea due to enteric endocrinopathy. We have identified a severe truncation mutation in NEUROG3 (P39PfsX38) that results in pancreatic exocrine insufficiency and significantly contributes to chronic malabsorptive diarrhea. We identified this novel phenotype by interrogating induced pluripotent stem cells from the NEUROG3-P39PfsX38 patient’s fibroblasts and an isogenic “wild-type” control cell line generated via CRISPR-Cas9 gene editing. We discovered that NEUROG3- P39PfsX38 lines failed to activate pancreatic progenitor and differentiated lineage markers, suggesting that the mutation may disrupt pancreatic organogenesis and could result in endocrine and exocrine dysfunction. Isogenic corrected cell lines differentiated into all pancreatic lineages. Clinical assessments concluded that the patient has exocrine pancreatic insufficiency. Treatment with pancreatic enzyme replacement therapy improved patient outcomes, including weight gain, fat absorption, and resolution of fat-soluble vitamin deficiency. These results expose a novel role for NEUROG3 in human pancreatic differentiation and illustrate how patient-specific stem cells can be used to interrogate disease etiology and affect patient care.

The development of modern methods to induce optical transparency (“clearing”) in biological tissues has enabled the three-dimensional (3D) reconstruction of intact organs at cellular resolution. New capabilities in visualization of rare cellular events, long-range interactions, and irregular structures will facilitate novel studies in the alimentary tract and gastrointestinal systems. The tubular geometry of the alimentary tract facilitates large-scale cellular reconstruction of cleared tissue without specialized microscopy setups. However, with the rapid pace of development of clearing agents and current relative paucity of research groups in the gastrointestinal field using these techniques, it can be daunting to incorporate tissue clearing into experimental workflows. Here, we give some advice and describe our own experience bringing tissue clearing and whole mount reconstruction into our laboratory’s investigations. We present a brief overview of the chemical concepts that underpin tissue clearing, what sorts of questions whole mount imaging can answer, how to choose a clearing agent, an example of how to clear and image alimentary tissue, and what to do after obtaining the image. This short review will encourage other gastrointestinal researchers to consider how utilizing tissue clearing and creating 3D “maps” of tissue might deepen the impact of their studies.

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we asked whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion led to expanded goblet and tuft cell populations. Sprouty2 loss induced PI3K/Akt signaling, leading to GSK3β inhibition and epithelial interleukin (IL)-33 expression. In vivo, this resulted in increased stromal IL-13+ cells. IL-13 in turn induced tuft and goblet cell expansion in vitro and in vivo. Sprouty2 was downregulated by inflammation; this appeared to be a protective response, as VillinCre;Sprouty2 ^F/F mice were resistant to DSS colitis. In contrast, Sprouty2 was elevated in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.