Expression Profiling of Stationary and Migratory Intestinal Epithelial Cells After <i>in vitro</i> Wounding: Restitution is Accompanied by Cell Differentiation

Abstract

Rapid repair by cell migration, a process called “restitution”, is essential for wound healing of mucous epithelia. Here, an established in vitro model for restitution, i.e., migration of the non-transformed intestinal epithelial cell line IEC-18 after scratch wounding, was investigated. This cell line is also known for its retained differentiation potential. The aim of this study was to test by expression profiling whether the differentiation state is altered during restitution in vitro. Using a sensitive RT-PCR method a systematic analysis of separated stationary and migratory cells was performed 48 h after in vitro wounding. Most characteristically, the differentiation state was changed in migratory cells when compared with stationary cells. For example, migratory cells lost markers of terminal differentiation and changed to a phenotype that assists the process of restitution by up-regulating the expression of genes such as plasminogen activator inhibitor-1, transforming growth factor α, heparin-binding EGF-like growth factor, α-smooth muscle actin, ornithine decarboxylase, and glyceraldehyde-3-phosphate dehydrogenase. However, there were no unequivocal signs of epithelial-mesenchymal transition (EMT) found in migratory cells.

TFF1 is Differentially Expressed in Stationary and Migratory Rat Gastric Epithelial Cells (RGM-1) afterin VitroWounding: Influence of TFF1 RNA Interference on Cell Migration

“…After being photographed at 24 h, the cells were sorted for RT-PCR analysis by detaching first the “stationary cells” as described previously in detail [49]. RNA isolation was performed using 450 µL lysis buffer from the ISOLATE I (or II) RNA mini Kit (Bioline GmbH, Germany) according to the manufacturer's instructions.…”

“…Then, all remaining stationary cells as well as the migratory cells close to the stationary cells were removed using a 4-5 mm fragment of double edge stainless-steel razor blades as previously described [49,52]. The remaining cells were rinsed gently with 37°C serum free medium (∼ 5 times), so that only a population of pioneer cells were left over (i.e., about 10 rows of cells directly behind the migratory front).…”

“…In the past, we have developed a sensitive method for analyzing the gene expression of separated stationary and migratory cells after in vitro wounding [49]. Here, we apply this method to analyze systematically RGM-1 cells.…”

IL-1β stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a

“…1). Stimulated myofibroblasts have previously been shown to play a role in wound healing through expression of TGF-␤ (22), but a link between myofibroblasts and the changes in differentiation observed at the wound edge (38) has not been demonstrated. We wished to examine whether stimulated myofibroblasts induced changes in the state of epithelial cell differentiation during the model wound regeneration.…”

“…Wound healing initiates a sequence of cellular changes that begin with dedifferentiation and migration (38) and progress to proliferation and differentiation, in a way that is reminiscent of normal crypt-to-villus epithelial maturation. The Wnt signaling pathway has emerged as one of the key regulators of the self-renewing capacity of the intestine (15).…”