TGF-ß promotes fetal gene expression and cell migration velocity in a wound repair model of untransformed intestinal epithelial cells.

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-ß. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-ß. Surprisingly, addition of TGF-ß induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-ß significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.

Organoid-based regenerative medicine for inflammatory bowel disease.

Inflammatory bowel disease (IBD) consists of two major idiopathic gastrointestinal diseases: ulcerative colitis and Crohn's disease. Although a significant advance has been achieved in the treatment of IBD, there remains a particular population of patients that are refractory to the conventional treatments, including the biologic agents. Studies have revealed the importance of "mucosal healing" in improving the prognosis of those difficult-to-treat patients, which indicates the proper and complete regeneration of the damaged intestinal tissue. In this regard, organoid-based regenerative medicine may have the potential to dramatically promote the achievement of mucosal healing in refractory IBD patients, and thereby improve their long-term prognosis as well. So far, studies have shown that hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) may have some beneficial effect on IBD patients through their transplantation or transfusion. Recent advance in stem cell biology has added intestinal stem cells (ISCs) as a new player in this field. It has been shown that ISCs can be grown in vitro as organoids and that those ex-vivo cultured organoids can be employed as donor cells for transplantation studies. Further studies using mice colitis models have shown that ex-vivo cultured organoids can engraft onto the colitic ulcers and reconstruct the crypt-villus structures. Such transplantation of organoids may not only facilitate the regeneration of the refractory ulcers that may persist in IBD patients but may also reduce the risk of developing colitis-associated cancers. Endoscopy-assisted transplantation of organoids may, therefore, become one of the alternative therapies for refractory IBD patients.

Ubiquitin D is Upregulated by Synergy of Notch Signalling and TNF-α in the Inflamed Intestinal Epithelia of IBD Patients.

BACKGROUND AND AIMS: The intestinal epithelium of inflammatory bowel disease [IBD] patients is exposed to various pro-inflammatory cytokines, most notably tumour necrosis factor alpha [TNF-α]. We have previously shown that the Notch signalling pathway is also upregulated in such an epithelium, contributing to intestinal epithelial cell [IEC] proliferation and regeneration. We aimed to reproduce such environment in vitro and explore the gene regulation involved. METHODS: Human IEC cell lines or patient-derived organoids were used to analyse Notch- and TNF-α-dependent gene expression. Immunohistochemistry was performed to analyse expression of ubiquitin D [UBD] in various patient-derived intestinal tissues. RESULTS: In human IEC cell lines, we found that Notch signalling and TNF-α-induced NFκB signalling are reciprocally regulated to promote expression of a specific gene subset. Global gene expression analysis identified UBD to be one of the most highly upregulated genes, due to synergy of Notch and TNF-α. The synergistic expression of UBD was regulated at the transcriptional level, whereas the UBD protein had an extremely short half-life due to post-translational, proteasomal degradation. In uninflamed intestinal tissues from IBD patients, UBD expression was limited to IECs residing at the crypt bottom. In contrast, UBD-expressing IECs were seen throughout the crypt in inflamed tissues, indicating substantial induction by the local inflammatory environment. Analysis using patient-derived organoids consistently confirmed conserved Notch- and TNF-α-dependent expression of UBD. Notably, post-infliximab [IFX] downregulation of UBD reflected favourable outcome in IBD patients. CONCLUSION: We propose that UBD is a novel inflammatory-phase protein expressed in IECs, with a highly rapid responsiveness to anti-TNF-α treatment.