title: PKH26 staining defines distinct subsets of normal human colon epithelial cells at different maturation stages.
abstract: OBJECTIVE
  Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon.
  Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells.
  
  
  CONCLUSIONS
  Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Pastò
authors.forename: Maddalena
authors.initials: M
authors.lastname: Marchesi
authors.forename: Adamo
authors.initials: A
authors.lastname: Diamantini
authors.forename: Chiara
authors.initials: C
authors.lastname: Frasson
authors.forename: Matteo
authors.initials: M
authors.lastname: Curtarello
authors.forename: Claudia
authors.initials: C
authors.lastname: Lago
authors.forename: Giorgia
authors.initials: G
authors.lastname: Pilotto
authors.forename: Anna Rosita
authors.initials: AR
authors.lastname: Parenti
authors.forename: Giovanni
authors.initials: G
authors.lastname: Esposito
authors.forename: Marco
authors.initials: M
authors.lastname: Agostini
authors.forename: Donato
authors.initials: D
authors.lastname: Nitti
authors.forename: Alberto
authors.initials: A
authors.lastname: Amadori
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D003106:Colon / Q000166:cytology*; D005786:Gene Expression Regulation; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000378:metabolism; D009930:Organic Chemicals / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D015415:Biomarkers; D009930:Organic Chemicals; C070080:PKH 26