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Background and Aim  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.    Methodology and Major Findings  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.

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages