Bile Acid-Mediated Transdifferentiation of Stem Cells From the Deep Esophageal Glands
CitationFass, Ofer. 2017. Bile Acid-Mediated Transdifferentiation of Stem Cells From the Deep Esophageal Glands. Doctoral dissertation, Harvard Medical School.
AbstractBackground: Barrett’s esophagus (BE) is a precancerous condition characterized by replacement of the normal stratified squamous epithelium by metaplastic intestine-like columnar mucosa as a result of chronic damage from acid reflux. The pathogenesis of BE is poorly understood and the cells giving rise to the metaplastic tissue remain largely unknown. Previous research from our lab suggested a role for myoepithelial cells within the submucosal gland in the initiation of metaplastic progression in response to deoxycholic acid (DCA), a secondary bile acid and major constituent of bile acid reflux in humans.
Aims: Given the increasing body of evidence for the role of bile acids in the metaplastic transformation observed in BE, we aim to assess whether DCA alters cells from the deep esophageal gland towards columnar epithelial and glandular phenotypes, similar to that observed in BE.
Methods: Isolated myoepithelial cells from pig esophageal glands (iPEMC), were exposed to DCA (100uM, pH 7.4) for 72 hours and harvested for flow cytometry and quantitative polymerase chain reaction (qPCR). Flow cytometry was performed on untreated (negative control) and DCA-treated iPEMC to quantify percent of cells expressing antibody markers characteristic of a squamous epithelial phenotype (cytokeratin 5, CK5), glandular epithelial phenotype (cytokeratin 7, CK7), or myoepithelial phenotype (smooth muscle actin, SMA). qPCR was performed to assess for relative expression of various stem cell markers, namely transformation-related protein 63 (TP63), NANOG, and LGR5, in both untreated and DCA-treated iPEMC. Relative gene expression of phenotypic markers, specifically CK5, CK7, and SMA, were also measured.
Results: Flow cytometry demonstrated an increase in the number of CK5-expressing cells after DCAtreatment from 26% (untreated control cells) to 42% (p < 0.0001). The percentage of CK7 expressing cells also increased from 2% to 40% (p = .001153). SMA similarly increased from 40% to 73% following DCA exposure (p < 0.0001). qPCR results were non-significant, but exhibited increased CK7 and decreased expression of the stem cell markers TP63, NANOG, and LGR5 following DCA exposure, which may reflect a differentiation into columnar epithelial cells and loss of stemness.
Conclusions: The results from this study suggest that DCA, a major component of bile acid reflux and potential mediator of cell injury, also promotes the differentiation of iPEMC in vitro, as demonstrated by an increased expression of CK7, suggesting transdifferentiation into a glandular phenotype. Differentiation was further demonstrated by the loss of stem cell marker expression. An increase in expression of CK7 and SMA suggests that myoepithelial cells are able to differentiate into additional cell types, supporting their pluripotency and hypothetical role as adult progenitor cells of the esophageal submucosal gland. The effect of DCA, inducing cellular injury and promoting the initial steps of glandular differentiation in this cell culture model, offers a preliminary potential mechanism for the origin of esophageal metaplasia seen in BE.
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