Table 1.
Sequences of the primers used in qRT-PCR experiments.
Figure 1.
Kinetics of PKH26 and In vitro Spheroid Formation.
(A) Cells obtained from human normal colon samples were cultured in vitro as detailed in Materials and Methods, and stained with PKH26. Cytofluorimetric analysis of PKH staining over 6 weeks of culture showed the progressive selection of a cell population expressing the dye at high intensity. At the 3rd week of culture, within the PKHpos subset it was possible to distinguish two major peaks (PKHhigh and PKHlow). One representative experiment is shown. (B) Three weeks after PKH26 staining, PKHpos and PKHneg cells were FACS-sorted and cultured under serum-free conditions as detailed in Materials and Methods. While PKHneg cells rapidly died in culture (left panel), PKHpos cells began to form spheroid-like structures, which progressively grew over time in culture (right panels; magnification 20×, scale bars: 100 µm). (C) Confocal microscopy analysis of spheroids showed cells with different intensity of PKH26 staining at 3 weeks of culture; magnification 20×. (D) Spheroids obtained after 3-week culture from PKHpos cells isolated from tumor [T] and pre-cancerous [PC] human colon samples were morphologically comparable to those obtained from normal [N] colon tissue. Scale bars: 100 µm. (E) Kinetics of cell survival in culture; in the absence of serum, the number of living cells decreased until about 5% of input cells at the end of the culture period. Results obtained in 4 consecutive experiments are shown. (F) CK18 staining of a human colon mucosa section (10× magnification). (G) Kinetics of CK18 expression in cultured colon cells; data are expressed as mean values ± SD of six different experiments.
Table 2.
Extreme limiting dilution analysis of spheroid-forming cell precursors in normal colon epithelial cell populations.
Figure 2.
Expression of Stemness-Associated Markers in Cultured Colon Cells.
(A) Immunohistochemical analysis of Msi-1 and Lgr5 expression in human normal colon biopsies; Msi-1+ and Lgr5+ cells are localized at the crypt base, where bona fide stem-like cells home; magnification is indicated in the boxes. (B) Cytofluorimetric analysis of Msi-1 and Lgr5 expression by PKH26-labelled cells expressing different intensities of PKH26 staining (PKHneg, PKHlow and PKHhigh; upper panel) at the 3rd week of culture. Msi-1+ cells were found within both PKHhigh and PKHlow cell populations (middle panels), whereas Lgr5+ cells were confined to the PKHhigh subset (lower panels). One representative experiment out of three consecutive is shown. (C) Cytofluorimetric analysis of Msi-1 and Lgr5 expression by PKHhigh cells over 4 weeks of culture. Data are expressed as percent mean values (± SD) of 5 consecutive experiments. (D) Kinetics of Msi-1 and Lgr5 expression by PKHhigh cells at the 2nd, 3rd and 4th week of culture. All Lgr5+ cells also co-expressed Msi-1, whereas a small fraction of Msi-1+ cells did not express Lgr5. One representative experiment out of 4 is shown. (E) qRT-PCR analysis of expression of Bmi-1, EphB2, EpCAM and ALDH1 in FACS-isolated Msi-1+/Lgr5− and Msi-1+/Lgr5+ cell subsets at the 3rd week of culture. Data were calculated as mean values ± SD as detailed in Materials and Methods, normalized to the housekeeping gene β2-microglobulin and relative to the reference sample (Msi-1+/Lgr5− cells). * p<0.05.
Figure 3.
Expression of Msi-1 and CK20 in Cultured Colon Cells.
(A) Immunohistochemical analysis of CK20 showed labeling of the epithelial cells along the crypt wall and at the mucosal surface, but not at the crypt bottom. (B) Cytofluorimetric analysis of Msi-1 and CK20 expression by PKHneg, PKHlow and PKHhigh populations at the 3rd week of culture; a small subset of PKHhigh cells co-expressed both markers. One representative experiment out of 4 consecutive is shown. (C) Confocal microscopy analysis of CK20 and Msi-1 expression in PKHpos cells. The cells co-expressing both markers are indicated by an arrow. Magnification 40×. (D) Immunofluorescence analysis of Msi-1 and CK20 expression in frozen colon sections. Double-positive cells are evident at the very crypt base. (E) qRT-PCR analysis of Muc-1 and Muc-2 expression on FACS-sorted Msi-1+/CK20− and Msi-1+/CK20+ subsets at the 3rd week of culture. Data are calculated as mean values ± SD as detailed in Materials and Methods, normalized to the housekeeping gene β2-microglobulin and relative to the reference sample (Msi-1+/CK20− cells). * p<0.05.
Figure 4.
Comparison by qRT-PCR of Stemness and Differentiation Marker Expression in Cultured Colon Cells and Microdissected Colon Crypt Populations.
mRNA levels of Lgr5, Msi-1 and CK20 genes were evaluated by qRT-PCR on FACS-sorted PKHhigh, PKHlow and PKHneg cells at the 3rd week of culture (Panel A) and on Differentiated (Diff), TA and CBC cells (Panel C) isolated by laser microdissection from colon crypts (as shown by the picture reported in Panel B). Data are shown as mean values ± SD calculated as described in detail in Materials and Methods, normalized to the housekeeping gene β2-microglobulin and relative to the reference sample (PKHlow for sorted cultured cells, and TA for cells isolated by microdissection). * p<0.05.
Figure 5.
In vitro Differentiation of Cultured PKHpos Cells.
(A) After 3 weeks of culture the spheroids obtained from PKH-stained cells maintained in serum-free medium in non-adherent plates (upper panel) were dissociated, and PKHpos cells were FACS-sorted and cultured in the presence of 10% FCS in adherent plates or included into Matrigel. In both conditions, the cells changed their morphology (lower panel), and in the presence of Matrigel they formed branched structures surrounding a “hole”. (B) Cytofluorimetric analysis of Msi-1 and Lgr5 expression in PKHpos cells cultured for 3 weeks in serum-free conditions (Spheroids) and after 10 days in differentiating conditions (Differentiated). (C) WB analysis of Msi-1 and Muc-1 proteins in Spheroids and Differentiated cell lysates. α-tubulin was used as a control for protein contents. (D) qRT-PCR analysis of CK20, Muc-1 and Muc-2 expression in Spheroids and Differentiated cells. Data are calculated as mean values ± SD as detailed in Materials and Methods, normalized to the housekeeping gene β2-microglobulin and relative to the reference sample (spheroid cells). *p<0.05. (E) Confocal microscopy analysis of differentiated cells. In the presence of serum and adhesion conditions, the cells presented a fragmented PKH26 dye pattern along the cellular membrane, completely loosing the expression of Msi-1 while acquiring Muc-1 (sample # 1) and CK20 (samples #2) expression.