Identification and genetic manipulation of human and mouse oesophageal stem cells
Authors
Youngtae Jeong, Horace Rhee, Shanique Martin, Daniel Klass, Yuan Lin, Le Xuan Truong Nguyen, Weiguo Feng, Maximilian Diehn
Institution
Stanford University
Country
United States
Year
2015
Journal
Gut Online First
Abstract
Objective Human oesophageal stem cell research is
hampered by the lack of an optimal assay system to study
self-renewal and differentiation. We aimed to identify and
characterise human and mouse oesophageal stem/
progenitor cells by establishing 3-dimensional organotypic
sphere culture systems for both species.
Design Primary oesophageal epithelial cells were freshly
isolated and fluorescence-activated cell sorting (FACS)-
sorted from human and mouse oesophagus and 3-
dimensional organotypic sphere culture systems were
developed. The self-renewing potential and differentiation
status of novel subpopulations were assessed by sphereforming
ability, cell cycle analysis, immunostaining, qPCR
and RNA-Seq.
Results Primary human and mouse oesophageal
epithelial cells clonally formed esophagospheres
consisting of stratified squamous epithelium. Sphereforming
cells could self-renew and form esophagospheres
for over 43 passages in vitro and generated stratified
squamous epithelium when transplanted under the
kidney capsule of immunodeficient mice. Sphere-forming
cells were 10–15-fold enriched among human
CD49fhiCD24low cells and murine
CD49f+CD24lowCD71low cells compared with the most
differentiated cells. Genetic elimination of p63 in mouse
and human oesophageal cells dramatically decreased
esophagosphere formation and basal gene expression
while increasing suprabasal gene expression.
Conclusions We developed clonogenic and organotypic
culture systems for the quantitative analyses of human
and mouse oesophageal stem/progenitor cells and
identified novel cell surface marker combinations that
enrich for these cells. Using this system, we demonstrate
that elimination of p63 inhibits self-renewal of human
oesophageal stem/progenitor cells. We anticipate that
these esophagosphere culture systems will facilitate
studies of oesophageal stem cell biology and may prove
useful for ex vivo expansion of human oesophageal stem
cells.