The present study revealed four major findings on preserved epithelial clumps in a mouse model of UC. First, the epithelial clumps were detected in the lamina propria in the ulcer region in H&E-stained sections with easy microscopic identification for pathologists; they can be classified into four subgroups according to various morphological characteristics in terms of the number of nuclei and intracellular space in the cytoplasm or intercellular crypt-like structure (Supplemental Fig. 1). Second, most epithelial clumps expressed markers for the epithelial cell adhesion molecules E-cadherin (approximately 80%) and β-catenin (approximately 60%) (Fig. 4G, H). Third, the epithelial clumps heterogeneously expressed the aISC marker Lgr5 and rISC markers Bmi1 and Dll1; a major population of the epithelial clumps expressed Dll1 (50–80%), a part of the population expressed Bmi1 (approximately 20% or less), and a minor population expressed Lgr5 (Fig. 5G-I). Fourth, a population of epithelial clumps expressed the stem cell niche marker Sox9; however, its expression was notably reduced with disease progression (approximately 50% on Day 6 and a low percentage on Day 12) (Fig. 4I). Furthermore, the coexpression profiles of Sox9 and DLLI indicated that a major population of the epithelial clumps was Dll1-single-positive clumps, and a minor population was SOX9low DLL1low or SOX9high DLL1low clumps (Fig. 6C, D). The results of the present study suggest that the epithelial clumps were derived from the crypt base in the villous-crypt axis; the heterogenous cellular properties of the stem cells or their daughter cells in these clumps might be dependent on the degree of resistance to inflammation-mediated epithelial loss.
The intestinal epithelial crypt has a capacity of constant renewal by proliferating stem cells owing to maintenance of homeostasis25. Lgr5+ aISC rapidly proliferates for self-renewal and the generation of all functional epithelial lineages under normal conditions15,26. We detected a small number of Lgr5+ epithelial clumps in the ulcer region; these results might be supported by the evidence that Lgr5+ aISC/CBC cells in the colon crypt are particularly susceptible to mucosal damage in mice with DSS-induced colitis27 and radiation-exposed mice28. The Lgr5high Ki-67+ ISC/CBC cells in the small intestine are active with rapid cell cycling for controlling self-renewal and producing progenitor cells; in contrast, the colon Lgr5low Ki-67− ISC/CBC cells are quiescent with the potential to retain epithelial self-renewal over long periods of time29,30. The negative nuclear expression of Ki-67 in the epithelial clumps might be consistent with the presence of quiescent Lgr5+ ISC/CBC cells in the colon crypt. The epithelial clumps were classified as Lgr5high or Lgr5low; the percentage of Lgr5low epithelial clumps was higher than that of the Lgr5high epithelial clumps (Fig. 5D, G). Several researchers have reported that Lgr5low Ki-67− ISC/CBC cells generate enteroendocrine cells, goblet cells, tuft cells, and deep crypt secretory (DCS) cells and constitute ISC/CBC cells by self-renewal20, 31–33. Lgr5 is directly regulated by Wnt signaling: Lgr5 is targeted to the β-catenin complex, committing to nuclear-translocated β-catenin34. Lgr5high β-cateninnuclear+ cells may lead to premalignant conditions caused by the activation of Wnt signaling35; however, no nuclear translocation of β-catenin was identified in the epithelial clumps. Thus, Lgr5low Ki-67− β-cateninnuclear− epithelial clumps were assumed to be quiescent for the sake of potentially regenerating the crypt and healing the mucosa over long periods of time.
Dll1high cells are called + 5 cells and are categorized as rISC or label-retaining cells (LRC) with quiescent or slow-cycling status; however, after aISC/CBC cell are damaged, the cells can quickly differentiate into secretory lineage cells, that is, tuft cells, enteroendocrine cells, goblet cells, and Paneth cells20. The DCS cells in the colon express the Notch ligand Dll1 and likely serve as a niche for stem cells to regulate their homeostasis36. In addition, transit amplifying (TA) cells are rapidly cycling Dll1+ Ki-67± daughter progenitor cells; they undergo differentiation into an absorptive lineage, that is, enterocytes33,37. Considering that Dll1+ epithelial clumps were the most common type of epithelial clumps according to the present study, we therefore propose that Dll1high epithelial clumps might maintain a potential for reviving stemness, followed by mucosal healing in the ulcer region.
Sox9 belongs to the Sox family of transcription factors, and it is required in the small intestine for the differentiation of the secretory lineage and proliferation of rISC25. Additionally, Sox9 plays a role in negative feedback with respect to the activity of the Wnt-β-catenin pathway; the overexpression of Sox9 inhibits the Wnt-β-catenin pathway, which promotes tumorigenesis, while appropriate expression of Sox9 is required for Wnt-β-catenin pathway activity in rISC21. Roche et al22 proposed that Sox9 expression levels were categorized as high or low according to intensity in ISC: Sox9high rISC is quiescent and slow-cycling under a homeostatic state; however, it can be converted to Sox9low aISC, facilitating epithelial repair, when aISC is lost after damage. Although the role of Sox9 in the colon is not fully understood at present, it is proposed that Sox9 is committed to morphogenesis in the colon epithelium and that it plays a role in the differentiation of the secretory lineage, especially goblet cells38,39. Coexpression analyses of Sox9 and markers of aISC or rISC would confirm the potential of restoring the colon crypt. Although the coexpression analysis of Sox9 and Dlli has not been determined in normal and injured colon crypts to our knowledge, we speculated that SOX9low DLL1high clumps might have the potential to restore the crypts in ulcer sites.
Bmi1+ quiescent ISC (qISC)/LRC or + 4 cells play a crucial role in facilitating regeneration in injured crypts, indicating that they compensate for the absence of Lgr5+ cells in the small intestine28,40; however, they tend to be insensitive to the absence of Lgr5+ cells in the distal small intestine and colon40. Sox9 directly binds to the promoter of Bmi, thus regulating the gene expression of Bmi141. Bmi1 is an epigenetic regulator, which restricts cell proliferation and shows a strong correlation with Sox9 expression42. As damage-sensitive SOX9+ clumps markedly reduced between Day 6 and Day 12, Bmi1+ clumps might exhibit a low potential for mucosal healing in the DSS-induced UC model.
Considering the above results, we propose a model for the formation of epithelial clumps in the colorectum in a DSS-induced mouse model. Inflammatory stimuli impair Lgr5+ aISC/CBC cells and Bmi1+qISC/LRC (or + 4 cells), as these cells are more sensitive than Dll1+ rISC (or TA/+ 5 cells), which are relatively resistant to inflammatory stimuli and which can survive as small clumps of epithelial cells in the ulcer region. Furthermore, the colon crypt base contains Sox9+ DCS cells, instead of Paneth cells; DCS cell-derived clumps can be preserved at an early phase of colitis and subsequently impaired by long-lasting inflammation. The present study indicated that clumps of epithelial cells heterogeneously expressed markers of aISC/CBC cells and their daughter rISC in the colon crypt. The results suggested that the Dll1+ crypt-derived clumps were resistant to DSS-induced colitis; they might have retained the potential to regenerate the crypt and heal the mucosa. Transplantation of primary crypt stem cells and induced human pluripotent stem cells to the colorectum might enhance mucosal healing in patients with UC; however, this approach has some limitations, including interruption with gut microflora and transplant rejection by the immune system8. Induction of regenerative crypts from epithelial clumps with stem cell potential would be an alternative experimental approach to stem cell therapy; therefore, further study is required to observe the morphological changes in epithelial clumps and the relationship between the clumps and regenerative crypts through the process of mucosal healing in the DSS-induced UC model.