Transimmortalized mouse intestinal cells (m-ICc12) that maintain a crypt phenotype

Abstract

This study describes the properties of a clone of immortalized cells (m-ICc12 cells) derived from the bases of small intestinal villi from 20-day-old fetuses of L-type pyruvate kinase (L-PK)/ TAg1 transgenic mice. The mice harbor the simian virus 40 large T antigen under the control of the 5' regulatory sequence from the L-PK gene. m-ICc12 cells expressed nuclear large T antigen, had a prolonged life span, and were nontumorigenic when injected into nude mice. They formed confluent monolayers of cuboid cells separated by tight junctions, developed dense, short apical microvilli, and formed domes. They also possessed cytokeratins, villin, aminopeptidase N, dipeptidyl-peptidase IV, and glucoamylase and retained crypt cell features, including intracellular sucrase isomaltase and alpha-L-fucose glycoconjugates accumulation and expression of the polymeric immunoglobulin receptor and the cystic fibrosis transmembrane conductance regulator gene. Thus the m-ICc12 cell line obtained by targeted oncogenesis in transgenic mice maintained in culture several important properties and differentiated functions of intestinal crypt cells.

Ligand-induced differential cross-regulation of Toll-like receptors 2, 4 and 5 in intestinal epithelial cells

“…Like other epithelial cell models, such as Caco-2 and MDCKII cells, mIC cl2 cells form a tight monolayer separated by tight-junctions with a distinct brush-border (apical) and basolateral membrane compartment. They differ, however, from other epithelial cell lines with respect to specific enzymatic functions and the maintenance of functional TLR4 and transport of IgA through pIgRs (Bens et al, 1996;Hornef et al, 2002Hornef et al, , 2003. Our finding that polarized mIC cl2 cells expressed TLR2, TLR4, and TLR5 transcripts and responded to Pam 3 CSK 4 , LPS, and flagellin indicates that the cells carried functional TLR2, TLR4 and TLR5 complexes.…”

“…A key aspect of our work was the use of a mouse intestinal mIC cl2 cell model system, derived from intestinal crypt cells (Bens et al, 1996). Like other epithelial cell models, such as Caco-2 and MDCKII cells, mIC cl2 cells form a tight monolayer separated by tight-junctions with a distinct brush-border (apical) and basolateral membrane compartment.…”

“…The mouse intestinal epithelial cell line mIC cl2 (Bens et al, 1996;Hornef et al, 2002), stably transfected with an NF-B luciferase reporter construct (Hornef et al, 2003), was cultured in DMEM/Ham's F-12 (1:l, v/v), 60 nM sodium selenite, 5 g/ml bovine transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 5 g/ml bovine insulin, 20 mM d-glucose, 10 nM mouse epidermal growth factor, 2% FCS, and 20 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) (pH 7.4). Propagation of the mIC cl2 cell line in IMDM with 10% FCS for several passages to exclude possible effects of the additives present in the complex medium, yielded no noticeable differences in TLR function.…”

“…The mouse intestinal epithelial cell line mIC cl2 (Bens et al, 1996) has been shown to express functional TLR4 (Hornef et al, 2002(Hornef et al, , 2003. Real-time RT-PCR demonstrated that mIC cl2 cells also express TLR2 and TLR5 (Table 1).…”

“…A novel epithelial (mIC cl2 ) cell line derived from intestinal mouse crypts recently has been shown to respond to LPS (Bens et al, 1996;Hornef et al, 2002Hornef et al, , 2003. mIC cl2 Cells form a tight monolayer separated by tightjunctions with a distinct brush-border (apical) and basolateral membrane compartment and have retained specific epithelial features such as dome formation, membrane-associated enzyme activities and expression of pIgRs (Bens et al, 1996). In the present study, we demonstrate that mIC cl2 cells carry a functional TLR2, TLR4 and TLR5 and exploited this system to investigate bacterial ligand driven cross-regulation of the TLR response in intestinal epithelial cells.…”