Nathalie Perreault

Human cell models to study small intestinal functions: Recapitulation of the crypt-villus axis

The intestinal epithelium is continuously and rapidly renewed by a process involving cell generation, migration, and differentiation, from the stem cell population located at the bottom of the crypt to the extrusion of the terminally differentiated cells at the tip of the villus. Because of the lack of normal human intestinal cell models, most of our knowledge about the regulation of human intestinal cell functions has been derived from studies conducted on cell cultures generated from experimental animals and human colon cancers. However, important advances have been achieved over recent years in the generation of normal human intestinal cell models. These models include (a) intestinal cell lines with typical crypt cell proliferative noncommitted characteristics, (b) conditionally immortalized intestinal cell lines that can be induced to differentiate, and (c) primary cultures of differentiated villuslike cells that can be maintained in culture for up to 10 days. Each of these models should help in the investigation of the specific aspects of human intestinal function and regulation. Furthermore, taken together, these models provide an integrated system that allows an in vitro recapitulation of the entire crypt‐villus axis of the normal human small intestine. Microsc. Res. Tech. 49:394–406, 2000.

Expression of Functionally Distinct Variants of the β4A Integrin Subunit in Relation to the Differentiation State in Human Intestinal Cells

Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin α6β4, which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the β4 subunit expression with different antibodies revealed that two forms of β4 exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa β4A subunit, whereas undifferentiated crypt cells express a novel β4A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (β4Actd−). This new form was not found to arise from alternative β4 mRNA splicing. Moreover, we found that these two β4A forms can associate into α6β4A complexes; however, the β4Actd− integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel α6β4Actd− integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.

Uncoordinated, transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes.

The heterogenous expression of brush border membrane hydrolases by the human enterocyte‐like Caco‐2 cell line during morphological and functional differentiation in vitro was investigated at the cellular level. Indirect immunofluorescence revealed that the heretogenous (“mosaic”) expression of sucrase‐isomaltase, lactase, aminopeptidase N, and alkaline phosphatase was, in fact, transient in nature. The labeling indexes for each hydrolase gradually increased during culture at postconfluence in order to reach a maximum (≥90%) after 30 days, concomitant with an upregulation of their respective protein expression levels. In contrast, dipeptidylpeptidase IV labeling remained relatively constant. Backscattered electron imaging analysis in midstage (12 days postconfluence) monolayers demonstrated a lack of correlation between brush border membrane development and expression of each enzyme studied. Moreover, double immunostaining revealed that none of the other four hydrolases correlated directly with sucrase‐isomaltase expression. Finally, immunodetection for the proliferation‐associated antigen Kl‐67 revealed a transient mosaic pattern of proliferation which was inversely related to Caco‐2 cell differentiation. These data indicate that enterocytic differentiation‐related (as well as proliferation‐related) gene expression in Caco‐2 cells is regulated but uncoordinated at the cellular level, suggesting that an overall control mechanism is lacking.

Identification, distribution, and tissular origin of the α5(IV) and α6(IV) collagen chains in the developing human intestine

The basement membrane type IV collagen is a family composed of six genetically distinct but structurally similar polypeptide chains, α1–α6. The α1(IV) and α2(IV) chains are ubiquitous components of all BMs whereas the other four have a restricted tissue distribution. In the present study, we have analyzed the expression, distribution, and cellular origin of the α5(IV) and α6(IV) chains in the developing and adult human small intestine and in well‐characterized in vitro models by indirect immunofluorescence, Western blot, and RT‐PCR. We have found that in the fetal small intestine, α5(IV) and α6(IV) are present in the epithelial BM and, in contrast to α1(IV) and α2(IV), are produced by both epithelial and mesenchymal cells. A distinct tissular origin for the α1/α2(IV) and α5/α6(IV) chains suggests that α5(IV) and α6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of α5(IV)/α6(IV) chain expression occurs in the adult intestine. Indeed, as compared with the fetal intestine, α6(IV) chain production is maintained while the expression of the α5(IV) chain is substantially reduced. Altered expression of the α5(IV) chain was also observed in the differentiating enterocytic‐like Caco‐2/15 cells, suggesting that in the intestinal model, the α5(IV) chain is subject to a regulated expression. Taken together, these observations indicate that the human intestinal epithelial BM contains up to four type IV collagen chains: the classical α1(IV)/α2(IV) chains, which originate from mesenchymal cells, and the α5(IV)/α6(IV) chains, which are of both epithelial and mesenchymal origin and have their expression regulated throughout development. Dev. Dyn. 1998; 212:437–447.

Regulated expression of the integrin α9β1 in the epithelium of the developing human gut and in intestinal cell lines: Relation with cell proliferation

The integrin α9β1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the α9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The α9β1 integrin was also detected in the intestinal cell lines HIEC‐6 and Caco‐2/15. The presence of α9β1 in HIEC‐6 was found to be consistent with their proliferative crypt‐like status. In Caco‐2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco‐2/15 cells in a proliferative state, resulted in higher levels of α9 as compared to control cells. Taken together, these observations suggest a relation between integrin α9β1 expression and proliferation in human intestinal cells. J. Cell. Biochem. 71:536–545, 1998.

Expression of the α-5(IV) collagen chain in the fetal human small intestine

Abstract Background/Aims: The basement membrane type IV collagen is a family composed of at least five genetically distinct but structurally similar polypeptide chains, α1–α5. The α1(IV) and α2(IV) chains are ubiq-uitous components of basement membranes, whereas the α3(IV), α4(IV), and α5(IV) chains have a restricted tissue distribution. The aim of this study was to analyze the presence of these minor type IV collagen chains in the small intestinal mucosa. Methods: The expression of type IV collagen chains in the developing and adult human small intestine was determined by indirect immunofluorescence with monoclonal and polyclonal antibodies. Western blotting and Northern hybridization analysis were also used to additionally investigate the expression of theα1(IV) and α5(IV) chains. Results: The α3–α5(IV) chains were absent from the adult epithelium, but, surprisingly, the α5(IV) chain was consistently detected in the fetal mucosa. Its expression was confirmed by Western blotting, complementary DNA polymerase chain-reaction amplification, and Northern hybridization analysis. Conclusions: The α5(IV) chain of collagen is expressed in the fetal but not adult human intestinal epithelium. Its position at the basolateral domain of epithelial cells suggests a potential role for this molecule during development.

Relationship between tenascin and α-smooth muscle actin expression in the developing human small intestinal mucosa

The expression of tenascin (Tn) and α-smooth muscle actin (α-SMA) was analyzed in the developing and adult human small intestine by means of double immunofluorescent staining with specific antibodies. By 7 weeks of gestation, the gut anlage has a simple tubular shape and is formed of a stratified undifferentiated epithelium surrounded by a poorly organized mesenchyme. Both Tn and α-SMA were found exclusively at the periphery of the tissue, corresponding to the presumptive muscularis propria. By 9 weeks, villus rudiments had formed but Tn and α-SMA remained restricted to the muscularis propria. Tn was first detected in the mesenchyme at 11 weeks. By 13 weeks, a preferential distribution of Tn in the subepithelial region of the mesenchyme was readily observed while α-SMA was still absent. From this stage to 20 weeks, Tn gradually concentrated in this region that, as determined by α-SMA detection, corresponded to the future muscularis mucosa area. As shown by double staining of Tn and α-SMA, deposition of Tn also preceded the appearance of the other α-SMA-expressing cells in the mucosa. These observations suggest that Tn could have a role in the differentiation of intestinal contractile cells.