title: Identification and genetic manipulation of human and mouse oesophageal stem cells.
abstract: 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.
  
  
  
  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 sphere-forming ability, cell cycle analysis, immunostaining, qPCR and RNA-Seq.
  
  
  
  Primary human and mouse oesophageal epithelial cells clonally formed esophagospheres consisting of stratified squamous epithelium. Sphere-forming 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 CD49f(hi)CD24(low) cells and murine CD49f(+)CD24(low)CD71(low) 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.
  
  
  
  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.
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Youngtae
authors.initials: Y
authors.lastname: Jeong
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Horace
authors.initials: H
authors.lastname: Rhee
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Shanique
authors.initials: S
authors.lastname: Martin
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Klass
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Le Xuan Truong
authors.initials: le XT
authors.lastname: Nguyen
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Feng
authors.affiliation: Stanford Cancer Institute and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Diehn
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics; D051927:CD24 Antigen / Q000032:analysis / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D000066673:Cell Self Renewal / Q000235:genetics*; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D004848:Epithelium / Q000254:growth & development*; D004947:Esophagus / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D039503:Integrin alpha6 / Q000032:analysis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000235:genetics; D061251:Primary Cell Culture / Q000379:methods; D011990:Receptors, Transferrin / Q000032:analysis / Q000235:genetics; D018874:Spheroids, Cellular / Q000166:cytology* / Q000637:transplantation; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D015703:Antigens, CD; D051927:CD24 Antigen; C068126:CD71 antigen; D039503:Integrin alpha6; D010750:Phosphoproteins; D011990:Receptors, Transferrin; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C486419:Trp63 protein, mouse; D025521:Tumor Suppressor Proteins
keywords: EPITHELIAL CELLS; OESOPHAGEAL PHYSIOLOGY; STEM CELLS