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Profiling of normal human colonic epithelial cells according to their PTK7 surface abundance

ABSTRACT: Human mucosa was collected from two different individuals undergoing colectomy. After treatment with EDTA, colonic crypts were isolated and further dis-aggregated using Dispase. Next, cells were stained using antibodies directed against the extracellular domain of EpCAM, PTK7, CD11, CD31, and CD45. Cells were analyzed and sorted via flow cytometry (BD Aria). After excluding non-epithelial cells (Epcam. CD11, CD31, and CD45 negative), the EpCAM positive fraction was divided into fractions expressing either high, medium, low, or negative levels of PTK7. Sorted cells were transferred to Trizol for RNA extraction and RNA was purified using the Qiagen RNA Mini Kit. Colonic crypts were isolated from normal human mucosa derived from individuals undergoing colectomy. Single cell fractions from these crypts were sorted and isolated RNA processed and hybridized to Affymetrix PrimeView Arrays

ORGANISM(S): Homo sapiens

SUBMITTER: Antonio Berenguer-LLergo 

PROVIDER: E-GEOD-68340 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were  ...[more]

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