Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression.
OBJECTIVE
The surface epithelium of the colon is being replaced constantly with cells derived from the stem cells of the crypt. Although the location of the stem cells is known, there are no markers for these cells. This study tested the hypothesis that colonic stem cells might be isolated and cultured on the basis of specific integrin expression patterns in normal human colonic epithelium.


METHODS
Integrin expression in normal human colonic mucosa was determined by using indirect immunofluorescence. Crypt cells were then isolated as single cells from normal colon tissues and the expression pattern of integrins was analyzed by flow cytometry. Based on the specific expression of integrin beta1 in colonic crypts, the cells were sorted by using a flow cytometer, and colony assays in soft agar were performed to evaluate the clonogenicity of the sorted cells.


RESULTS
By immunofluorescence, the cells located in the lower one third of crypts expressed higher levels of beta1-integrin than the cells in the remainder of the crypt. When isolated crypt cells were stained with the beta1-integrin antibody and examined in a flow cytometer, there were 2 peaks of fluorescence. Sorting of crypt cells based on staining with anti-beta1 integrin antibody produced a cell population with a significantly enhanced ability to form colonies.


CONCLUSIONS
beta1-integrin is a candidate surface marker for the proliferative zone of the human colonic crypt. Our in vitro culture system for the clonal growth of a single colonic crypt cell suspension could facilitate the identification of other candidate stem cell markers.
Fujimoto
Koji
K
Department of Surgery, Vanderbilt University Medical Center and Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232, USA.
Beauchamp
R Daniel
RD
Whitehead
Robert H
RH
D002448:Cell Adhesion / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells / Q000166:cytology; D003106:Colon / Q000166:cytology* / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000502:physiology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D016023:Integrins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology; D014018:Tissue Distribution
D016326:Extracellular Matrix Proteins; D019012:Integrin beta1; D016023:Integrins; D008565:Membrane Proteins; D020033:Protein Isoforms