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

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: Dissociat...

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Autores principales: Pastò, Anna, Marchesi, Maddalena, Diamantini, Adamo, Frasson, Chiara, Curtarello, Matteo, Lago, Claudia, Pilotto, Giorgia, Parenti, Anna Rosita, Esposito, Giovanni, Agostini, Marco, Nitti, Donato, Amadori, Alberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425557/
https://www.ncbi.nlm.nih.gov/pubmed/22927961
http://dx.doi.org/10.1371/journal.pone.0043379
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author Pastò, Anna
Marchesi, Maddalena
Diamantini, Adamo
Frasson, Chiara
Curtarello, Matteo
Lago, Claudia
Pilotto, Giorgia
Parenti, Anna Rosita
Esposito, Giovanni
Agostini, Marco
Nitti, Donato
Amadori, Alberto
author_facet Pastò, Anna
Marchesi, Maddalena
Diamantini, Adamo
Frasson, Chiara
Curtarello, Matteo
Lago, Claudia
Pilotto, Giorgia
Parenti, Anna Rosita
Esposito, Giovanni
Agostini, Marco
Nitti, Donato
Amadori, Alberto
author_sort Pastò, Anna
collection PubMed
description 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.
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spelling pubmed-34255572012-08-27 PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages Pastò, Anna Marchesi, Maddalena Diamantini, Adamo Frasson, Chiara Curtarello, Matteo Lago, Claudia Pilotto, Giorgia Parenti, Anna Rosita Esposito, Giovanni Agostini, Marco Nitti, Donato Amadori, Alberto PLoS One Research Article 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. Public Library of Science 2012-08-22 /pmc/articles/PMC3425557/ /pubmed/22927961 http://dx.doi.org/10.1371/journal.pone.0043379 Text en © 2012 Pastò et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Pastò, Anna
Marchesi, Maddalena
Diamantini, Adamo
Frasson, Chiara
Curtarello, Matteo
Lago, Claudia
Pilotto, Giorgia
Parenti, Anna Rosita
Esposito, Giovanni
Agostini, Marco
Nitti, Donato
Amadori, Alberto
PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title_full PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title_fullStr PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title_full_unstemmed PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title_short PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages
title_sort pkh26 staining defines distinct subsets of normal human colon epithelial cells at different maturation stages
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425557/
https://www.ncbi.nlm.nih.gov/pubmed/22927961
http://dx.doi.org/10.1371/journal.pone.0043379
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