Ex vivogut culture for studying differentiation and migration of small intestinal epithelial cells

Abstract

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health.

Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis

“…Different studies have described how epithelial AMPK can control tight junction protein stabilization [ 29 , 30 ] and maintain epithelial barrier function following injury [ 12 ]. Consistently, altered formation of intestinal paracellular junction, as determined by immunostaining of E-cadherin and β-catenin markers, was reported in an ex vivo gut culture model established from E13.5-day fetal small intestine deleted for AMPKα1 [ 31 ]. Therefore, it is likely that higher permeability in the colon of IEC AMPK KO mice was a predisposing factor for the increased sensitivity to DSS-induced epithelial damage and colitis.…”

“…It is possible that IEC AMPK participates to the migration of cells repopulating the wound as highlighted by recent studies showing the pivotal role of AMPK in the control of cell migration [ 34 , 35 ]. In line, loss of intestinal AMPKα1 impaired epithelial cell migration ex vivo and in vivo [ 11 , 31 ].…”

“…We observed that DSS-induced colitis in IEC AMPK KO mice led to a reduction in the number of colonic goblet cells, with a concomitant striking decrease in the expression of the major mucin-encoding gene Muc2 responsible for mucus synthesis. Interestingly, deficient differentiation has been also demonstrated in an ex vivo gut culture model lacking AMPKα1 and was associated with a reduced prevalence of goblet cells in this epithelium developing model [ 31 ]. Previous studies have reported that cytokines, including IL-22 and IL-23a, produced by cells of the innate immune system enhance colonic restitution and accelerate restoration of goblet cells to promote mucosal healing during acute experimental colitis [ 37 , 38 , 39 ].…”

Intestine Explants in Organ Culture: A Tool to Broaden the Regenerative Studies in Echinoderms

“…Components of the extracellular matrix have been recognized as crucial modulators of different physiological processes [28]. Explants in organ culture from both invertebrate and vertebrate organisms have been used to perform several physiological, biochemical, and biomechanical studies [29][30][31][32]. For example, in cultured gut explants from mice embryos, natural peristalsis, and stem cell differentiation toward Paneth cells, goblet cells, enterocytes, and others were observed.…”

Ex Vivo and In Vitro Studies Revealed Underlying Mechanisms of Immature Intestinal Inflammatory Responses Caused by Aflatoxin M1 Together with Ochratoxin A

“…The intestinal epithelium is an integrated single cell layer that selectively permeates nutrients and specific antigens while constituting an efficient barrier against external contaminants [ 33 ]. The infant intestine has a relatively high endocytic capacity so as to facilitate the transfer of maternal immunoglobulin and specific antigens from breast milk, thus promoting the normal development of intestinal immune system [ 34 ].…”