title: Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.
abstract: OBJECTIVE
  Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
  
  
  METHODS
  We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.
  
  
  RESULTS
  CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.
  
  
  CONCLUSIONS
  We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
authors: Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
authors: Fengchao
authors: F
authors: Wang
authors: David
authors: D
authors: Scoville
authors: Xi C
authors: XC
authors: He
authors: Maxime M
authors: MM
authors: Mahe
authors: Andrew
authors: A
authors: Box
authors: John M
authors: JM
authors: Perry
authors: Nicholas R
authors: NR
authors: Smith
authors: Nan Ye
authors: NY
authors: Lei
authors: Paige S
authors: PS
authors: Davies
authors: Megan K
authors: MK
authors: Fuller
authors: Jeffrey S
authors: JS
authors: Haug
authors: Melainia
authors: M
authors: McClain
authors: Adam D
authors: AD
authors: Gracz
authors: Sheng
authors: S
authors: Ding
authors: Matthias
authors: M
authors: Stelzner
authors: James C Y
authors: JC
authors: Dunn
authors: Scott T
authors: ST
authors: Magness
authors: Melissa H
authors: MH
authors: Wong
authors: Martin G
authors: MG
authors: Martin
authors: Michael
authors: M
authors: Helmrath
authors: Linheng
authors: L
authors: Li
mesh_terms: D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000378:metabolism; D053687:Adult Stem Cells / Q000378:metabolism*; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D051927:CD24 Antigen / Q000378:metabolism; D018929:Cell Culture Techniques; D003106:Colon / Q000166:cytology; D003114:Colony-Forming Units Assay; D000091342:Endoplasmic Reticulum Chaperone BiP; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology*; D007421:Intestine, Small / Q000166:cytology; D051379:Mice; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism
chemical_list: D020410:Activated-Leukocyte Cell Adhesion Molecule; D000954:Antigens, Surface; D051927:CD24 Antigen; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018960:Hyaluronan Receptors; D019009:Proto-Oncogene Proteins c-kit
keywords: CBC; CFE; CoSC; Differentiation; FACS; Flow Cytometry Analysis; GRP; IEC; IHC; ISC; PC; Paneth cell; Single-Cell Sorting; Stemness; colonic stem cell; colony-forming efficiency; crypt base columnar; fluorescence-activated cell sorting; green fluorescent protein; immunohistochemistry; intestinal epithelial cell; intestinal stem cell; qRT-PCR; quantitative reverse-transcription polymerase chain reaction
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