Establishment of esophageal-like non-keratinized stratified epithelium using normal human bronchial epithelial cells

Abstract

rent experimental models of esophageal epithelium in vitro suffer from either poor differentiation or complicated culture systems. We have established a model to study stratified squamous epithelium in vitro, which is very similar to esophageal epithelium in vivo. A stratified squamous multilayer epithelium was formed by seeding primary normal human bronchial epithelial (NHBE) cells onto collagen-and fibronectin-coated trans-well inserts and then cultivating the cells under air-liquid interface (ALI) conditions in the presence of growth factors and low levels of all-trans-retinoic acid. Trans-epithelial electrical resistance (TEER) measurements revealed the presence of a tight barrier, previously only achievable with esophageal biopsies mounted in Ussing chambers. Molecular markers for desmosomes, cornified envelope, tight junctions, and mature esophageal epithelium were upregulated in the differentiating culture in parallel with functional properties, such as decreased permeability and acid resistance and restoration. Acid exposure resulted in a decrease in TEER, but following 1-h recovery the TEER values were fully restored. Treatment with all-trans-retinoic acid decreased TEER and inhibited the recovery after acid challenge. PPAR-delta agonist treatment increased TEER, and this temporary increase in TEER was consistent with an increase in involucrin mRNA. Global gene expression analysis showed that ALI-differentiated NHBE cells had expression profiles more similar to epithelial biopsies from the esophageal tissue of healthy volunteers than to any other cell line. With respect to morphology, molecular markers, barrier properties, and acid resistance, this model presents a new way to investigate barrier properties and the possible effects of different agents on human esophagus-like epithelium. esophageal epithelium; acid; barrier function; air-liquid interface ONE OF THE PRIMARY FUNCTIONS of the esophageal epithelium is to protect the underlying tissue against mechanical and chemical insult. The epithelium of the distal esophagus also needs to withstand reflux from the stomach, which contains acid, bile, and proteases. Failure of the epithelium to protect the underlying tissue from these attacks results in erosion, esophagitis, and painful symptoms. The esophagus is covered by a nonkeratinized stratified squamous epithelium. The keratinocytes in a non-keratinized squamous epithelium can be assigned to three layers with distinct features, namely the basal, intermediate, and superficial layers (1), of which the epithelial barrier properties reside in the upper intermediate and superficial layers.Different cell lines and primary cells are available to use as esophageal epithelial cell models. Het-1A, an immortalized normal human esophageal cell line (28), grows as a monolayer. Kyse-140 and Kyse-510 are esophageal carcinoma cell lines that also grow as monolayers. The TR146 cell line originates from human buccal epithelium (24) and has been shown to form a stratified epithelium (four to seven cell layers) and have ...

Acid modulates the squamous epithelial barrier function by modulating the localization of claudins in the superficial layers

“…It has been recently reported that TEER markedly increases between days 5 and 7 of ALI-cultured NHBE cells. 9 In the present study, on day 5 of the ALI culture, claudin-1 and claudin-4 were detected at the cell-cell contact area in the suprabasal layers in a continuous linear pattern but occludin and ZO-1 were not. On day 7 of the ALI culture, occludin and ZO-1 were detected as dots and whisker-like lines, and were co-localized at the junctions of the superficial layers, while claudin-1 and claudin-4 were also detected as dots and whisker-like lines in the superficial layers, and as a continuous linear pattern in the suprabasal layers.…”

“…We recently established an esophageal-like stratified epithelial cell layer under ALI conditions using NHBE cells. 9,15 Although the cells were not originally from the esophagus, these cells created stratified epithelial layers, expressed cytokeratins like the esophagus, and the TJ expression pattern and gene expression pattern were similar to human esophagus. 9 Here, the stratified epithelial cell layers acquired more than 1500 O .…”

“…9,15 Although the cells were not originally from the esophagus, these cells created stratified epithelial layers, expressed cytokeratins like the esophagus, and the TJ expression pattern and gene expression pattern were similar to human esophagus. 9 Here, the stratified epithelial cell layers acquired more than 1500 O . cm 2 resistance, which is similar to the resistance obtained from esophageal biopsy specimens using an Ussing chamber.…”

“…The medium was changed every 2 days until the cells reached confluence. ALI culture, which has previously been shown to induce multilayering and differentiation to squamous epithelial cells, 9 was introduced by removing the apical medium and decreasing the outer medium to the level of the epithelial cells. The medium was switched to ALI medium, which consists of BEGM and DMEM (1:1) with ATRA (0.3 nM).…”

“…Optimizing culture conditions, such as selecting appropriate media, is known to be critical for stratified epithelial cell culture to regulate differentiation and to form suitable epithelial barriers with TJs. 9 After characterizing the localization of TJ proteins in stratified epithelial cell layers, we examined the influence of acid on the barrier function of the cell layers, and on the expression and distribution of TJ proteins.…”