Induction of ER stress identifies potential esophageal stem cell
markers
S.L. Rosekrans,1,2 J. Heijmans,1,2 E.J. Westerlund,1 C. Puylaert, 1
V. Muncan,1 G.R. van den Brink1,2
1
Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71 1105BK
Amsterdam, the Netherlands
2
Department of Gastroenterology & Hepatology, Academic Medical Center, Amsterdam, the Netherlands
The esophageal squamous epithelium is a rapidly renewing tissue. It has recently been shown
that the esophageal epithelium is hierarchically organized with long lived stem cells and
rapidly proliferating cells that maintain cellular renewal, both being localized in the basal
layer. To date, no specific markers have been identified that distinguish stem cells from non
stem cell proliferating cells. In the intestinal epithelium, long lived stem cells and transcripts
that mark stemness are rapidly depleted upon induction of endoplasmic reticulum (ER) stress
(unpublished data, Heijmans, Rosekrans, van den Brink). We hypothesize that esophageal
stem cell markers exhibit a similar sensitivity to ER stress. We aim to identify stem cell genes
that mark a hierarchically distinct population of cells in the basal layer cells by analysis of a
gene signature that is lost upon induction of ER stress.
Sensitivity of esophageal precursor cells to ER stress was examined in vivo and in vitro. For
proof of principle experiments in vivo, we chemically induced ER stress in mice, by injections
with thapsigargin. For in vitro experiments, ER stress was induced in vitro in TE7 and OE21
esophageal squamous cell carcinoma (SCC) cell lines using SubAB, a cytotoxin that induces
ER stress by depleting the major ER chaperone GRP78. From in vitro experiments, we next
performed gene arrays to identify those transcripts that were significantly down regulated in
both cell lines upon induction of ER stress. Next, we localized mRNA of all these genes in
wild type mouse esophagus by Dig labeled in situ hybridization.
Induction of ER stress in mice resulted in rapid depletion of esophageal precursor cells and
accelerated differentiation, thus showing that in analogy to the intestine, esophageal stemness
is lost. Using RNA micro-arrays, we identified a gene signature of 47 genes that were lost in
both individual cell lines upon induction of ER stress. Of these genes, we found 29 genes to
be restricted to the basal layer of the mouse esophagus. Out of these 29, nine genes show
expression in only a small proportion of the basal cells, potentially marking stem cells.
Conclusion: ER stress depletes esophageal precursor cells. Our in vitro screen combined with
in situ hybridization identified nine genes that are specifically expressed in a subset of
proliferating genes, thereby potentially marking esophageal stem cells.