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

et al. (2012) PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages. PLoS ONE 7(8): e43379. doi:10.1371/journal.pone.0043379 PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages Anna Pasto` 0 Maddalena Marchesi 0 Adamo Diamantini 0 Chiara Frasson 0 Matteo Curtarello 0 Claudia Lago 0 Giorgia Pilotto 0 Anna Rosita Parenti 0 Giovanni Esposito 0 Marco Agostini 0 Donato Nitti 0 Alberto Amadori 0 Shree Ram Singh, National Cancer Institute, United States of America 0 1 Department of Surgery, Oncology and Gastroenterology, University of Padova , Padova , Italy , 2 Hemato-Oncology Laboratory, Department of Pediatrics, University of Padova , Padova , Italy , 3 IRCCS Istituto Oncologico Veneto , Padova , Italy , 4 Department of Diagnostic Sciences and Special Therapies, University of Padova , Padova , Italy 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 PKHpos population survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/ Lgr5+ cells were only found within PKHhigh cells, whereas Msi-1+/Lgr52 cells were also observed in the PKHlow 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 PKHlow and PKHneg cells, a small PKHhigh 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: PKHhigh/Lgr5+/Msi-1+/CK202, PKHhigh/Lgr52/Msi-1+/CK20+, PKHlow/Lgr52/Msi-1+/Ck202, and PKHlow/ Lgr52/Msi-12/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. - Funding: This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), Ministry of Health, Ministry of Education 60% and PRIN, Regione Veneto. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Normal tissues are organized in a hierarchical fashion, where rare somatic cells endowed with stem-like properties give origin to a population of differentiated cells forming the bulk of tissue. The skin and the gastrointestinal tract undergo a tremendous turnover of the epithelial component, which entails the existence of a selfrenewing population able to face the continuous replacement of the dying epithelial cells [1,2]. These self-maintaining cells, called stem cells, are characterized by 4 fundamental properties: longevity, multipotency, quiescence, and asymmetric cell division. These two latter features, however, have been recently a matter of debate, as poor cell cycling and invariant asymmetric division may not represent obligatory properties of stem cells [36]. In the small intestine, the mucosa is organized into crypts and villi, populated by different types of cells that maintain their position along the intestinal architecture; in colon instead only crypts are found. While in the small intestine the location of stem cells is still debated [79], the situation is apparently less complicated at the colon level, where Paneth cells are not found [10], and stem cells may be identified at the very bottom of the crypt [11]. Surprisingly, despite the anatomy of the colon crypt being uniquely suited to study adult stem cells within their niche, only a few attempts have been made to isolate by laser microdissection the cells present in the different segments of the intestinal crypt, and to exploit their molecular profile as a gold standard [12]. Most information on the physiology of intestinal stem cells has been obtained in the mouse, as obvious limitations preclude transplantation or genetic marking approaches in humans. Very recently, Sato et al. [13] and Jung et al. [14] first succeeded in isolating and expanding in vitro normal human colonic stem cells by an experimental protocol which entailed the use of a complex combination of several growth factors [13] and a positive selection strategy based on the expression of the Ephrin type-B receptor 2 (EphB2) [14]. Here we derived in vitro spheroids from normal human colonic mucosa not by surface marker-driven selection, but by simply taking advantage of their slow proliferation rate in the absence of serum. In these spheroids, according to the phenotypic/molecular expression of several putative stemness markers including Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) [15], Musashi-1 (Msi-1) [16], B-lymphoma Mo-MLV insertion region 1 (Bmi-1) [17], EphB2 [18], Epithelial cell adhesion molecule (EpCAM) [19] and Aldehyde dehydrogenase 1 (ALDH1) [20], we could identify several discrete cell populations, in which mRNA expression profiles of specific genes closely mirrored the transcriptomic properties of epithelial cells isolated from different colon crypt portions by microdissection, as representative of different maturation stages. Materials and Methods Tissue Specimens and Cell Isolation Following informed consent, 80 histologically normal and 5 tumoral human colonic mucosa samples were obtained from colon cancer-bearing patients undergoing colectomy. Immediately after resection, the tissues were washed in cold phosphate-buffered saline (PBS) containing Penicillin/Streptomycin, gentamicin (1 ml/ ml) and amphotericin (1.25 mg/ml). Morphologically normal colon mucosa samples and tumor specimens were divided in two parts: one fragment was snap-frozen in liquid nitrogen, and stored at 280uC until use, while the other was p (...truncated)