title: Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived.
abstract: In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca(2+) (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3-5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1-3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3-4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Dame
authors.affiliation: Department of Internal Medicine, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Henry D
authors.initials: HD
authors.lastname: Appelman
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Kelly D
authors.initials: KD
authors.lastname: Copley
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Shannon D
authors.initials: SD
authors.lastname: McClintock
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Muhammad Nadeem
authors.initials: MN
authors.lastname: Aslam
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Durga
authors.initials: D
authors.lastname: Attili
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
authors.forename: B Joseph
authors.initials: BJ
authors.lastname: Elmunzer
authors.affiliation: 1] Department of Internal Medicine, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA [2] Medical Service and Geriatrics Research Education and Clinical Center, VA Medical Center, Ann Arbor, MI, USA.
authors.forename: Dean E
authors.initials: DE
authors.lastname: Brenner
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: James
authors.initials: J
authors.lastname: Varani
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
authors.forename: D Kim
authors.initials: DK
authors.lastname: Turgeon
mesh_terms: D000236:Adenoma / Q000503:physiopathology*; D015415:Biomarkers / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003094:Collagen; D003106:Colon / Q000166:cytology*; D003110:Colonic Neoplasms / Q000503:physiopathology*; D004338:Drug Combinations; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology*; D053554:Keratin-20 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D007797:Laminin; D018613:Microscopy, Confocal; D008954:Models, Biological; D055262:Mucin-2 / Q000378:metabolism; D011509:Proteoglycans; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*
chemical_list: D015415:Biomarkers; D015820:Cadherins; D004338:Drug Combinations; D053554:Keratin-20; D019394:Ki-67 Antigen; C116884:LGR5 protein, human; D007797:Laminin; C524625:MUC2 protein, human; D055262:Mucin-2; D011509:Proteoglycans; D043562:Receptors, G-Protein-Coupled; C059485:matrigel; D003094:Collagen; D002118:Calcium