EGF signaling activates proliferation and blocks apoptosis of mouse and human intestinal stem/progenitor cells in long-term monolayer cell culture

Abstract

The homeostatic renewal of the intestinal epithelium depends on regulation of proliferation and differentiation of stem/ progenitor cells residing in a specific site, called the 'stem cell niche.' Thus, the reconstitution of the microenvironment of the stem cell niche may allow us to maintain intestinal stem/progenitor cells in culture for a longer period. Although epidermal growth factor (EGF) is conventionally used as a supplement of intestinal epithelial cell culture, little has been known regarding a role of EGF signaling in a stem/progenitor cell population. In this study, we attempted to clarify the role of EGF signaling in intestinal stem/progenitor cells, and to establish a culture system in which these cells could be maintained with normal differentiation potential. We first examined the expression pattern of EGF and its receptor, EGFR, and inhibited EGF signaling in mouse intestines. Next, we cultured intestinal cells isolated from mouse and human intestines in the presence of EGF and analyzed the function of EGF signaling in cultured cells. In both embryonic and adult mouse intestines, EGFR and EGF were expressed in immature epithelial cells and adjacent fibroblasts, respectively, and EGF signaling was essential to activate proliferation and inhibit apoptosis of intestinal stem/progenitor cells. Activation of EGF signaling also stimulated proliferation and suppressed apoptosis, both of which are necessary to maintain mouse and human intestinal epithelial cells in culture. Moreover, in these cultured epithelial cells, putative intestinal stem/progenitor cells persisted longer, and gave rise to different types of differentiated intestinal epithelial cells. We conclude that EGF signaling is indispensable for activation of proliferation and inhibition of unexpected cell death, not only in the intestinal stem cell niche, but also in culture of primitive intestinal epithelial cells.

Human Small Intestinal Epithelial Cells Differentiated from Adult Intestinal Stem Cells as a Novel System for Predicting Oral Drug Absorption in Humans

“…The authors demonstrated that ISCs were maintained in culture with continuous proliferation and spontaneous differentiation into four distinct epithelial cell lineages including not only absorptive enterocytes, but also secretory lineages such as enteroendocrine cells, goblet cells, and Paneth cells. Suzuki et al (2010) also found that these differentiated cells formed polarized monolayers with dome-like structures. However, to the best of our knowledge, the utility of this differentiated HIEC for evaluating the permeability of drug candidates to predict absorption in humans has not yet been assessed.…”

“…This systematic process consists of the proliferation of adult intestinal stem cells (ISCs) at the bottom of the crypt, migration into the villi with differentiation/maturation, and eventual apoptosis at the tips of the villi (Yeung et al, 2011). Suzuki et al (2010) recently reported that adult ISCs expressing leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a marker of ISCs, were contained in commercially available primary normal human small intestinal epithelial cells (HIECs). The authors demonstrated that ISCs were maintained in culture with continuous proliferation and spontaneous differentiation into four distinct epithelial cell lineages including not only absorptive enterocytes, but also secretory lineages such as enteroendocrine cells, goblet cells, and Paneth cells.…”

Aldosterone induces myofibroblast EGF secretion to regulate epithelial colonic permeability

“…Since colonic myofibroblasts regulate important functions of epithelial cells via distinct secreted cytokines and autocoids (22), the next step was to study the secretory response of the CCD-18Co cell line to Aldo stimulation. From the many candidates that may mediate the myofibroblast-epithelial interaction, we decided to study TGF-␤ 1 [since it is secreted by primary adult human colonic subepithelial myofibroblasts and enhances barrier function (1) and it is also secreted in response to radiation injury (30)]; VEGF [since it plays a role in epithelial repair and is secreted by CCD-18Co under hormonal regulation (3)]; and EGF [since it is indispensable in the activation of intestinal epithelial cell proliferation (29)]. …”

The intestinal microbiota dysbiosis and Clostridium difficile infection: is there a relationship with inflammatory bowel disease?

“…Moreover, EGFR signaling also activates PI3 kinase, resulting in activation of the downstream effector molecules, such as serine-threonine kinase Akt (AKT) and mammalian target of rapamycin (mTOR) kinase. These molecules induce proliferation and block apoptosis, resulting in cell survival via activating nuclear transcription factors such as NFκB [Suzuki et al 2010]. However in C. difficile infection activation of the EGFR pathway may have dual roles in both tissue repair and proinflammatory response.…”