Cellular Plasticity of Defa4-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury

Abstract

Background & Aims Loss of leucine-rich repeat-containing G-protein–coupled receptor 5–positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. Methods Small intestine and enteroids from Defa4 Cre ; Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function ( Defa4 Cre ;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4 Cre ;Rosa26 reverse tetracycline transactivator–ires Enhanced Green Fluorescent Protein (EGFP) ;TetO NICD ), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function ( Defa4 Cre ;ADAM10 flox/flox ), and Adenomatous polyposis coli (APC) inactivation ( Defa4 Cre ;APC flox/flox ) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. Results Defa4 Cre -expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter–sorted Defa4 Cre -marked cells showed clonogenic enteroid growth. Notch activation in Defa4 Cre -expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4 Cre -expressing cells contributed to regeneration in an ADAM10–Notch–dependent manner. Conclusions Our studies have shown that Defa4 Cre -expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.

Krüppel-like Factor 5 Regulates Stemness, Lineage Specification, and Regeneration of Intestinal Epithelial Stem Cells

“…Recent studies have suggested that activated NOTCH signaling stimulates Paneth cell plasticity during injury-induced regeneration. 42,43 While KLF5 is not expressed in Paneth cells during homeostasis, the majority of cells within regenerative crypts express KLF5, indicating that KLF5 remains a critical player in the regenerative response. Furthermore, this response may be facilitated via KLF5-mediated NOTCH signaling regulation in other precursors.…”

Intestinal Stem Cell Aging: Origins and Interventions

“…Both LGR5 + and Bmi1 + cells can generate intestinal organoids in culture (113). After ablation of LGR5 + cells, Bmi1 + cells can restore the crypt base cell population, providing evidence for significant plasticity of cell identities in the crypt (15), a concept that has been supported by findings that the crypt base can be repopulated by other cell types as well, including differentiated endocrine and Paneth cells (15,16,107,(114)(115)(116)(117). A recent study has also proposed the presence of a distinct revival stem cell population that can repopulate the crypt in conditions of severe damage (118).…”

Plasticity of Paneth cells and their ability to regulate intestinal stem cells

“…In ex vivo study, the plasticity of PCs can be increased, under the Notch pathway was actively or passively activated condition. In vivo studies, Notch target genes, such as Notch intracellular domain (NICD) and Hes1, were activated in the small intestinal PCs of irradiated mice [33]. Forced NICD expression in PCs leads to transient proliferation states, followed by differentiation into other types of intestinal epithelial cells [33,35].…”