The human colon epithelial cancer cell line Caco2 was obtained from American Type Culture Collection (ATCC, Manassas, VA). Caco2 cells at passage 10 were infected with a Lentivirus reporter vectors that contain the mouse Melk promoter driving enhancer green fluorescent protein (MELK-GFP) (11
) or a control PGK promoter driven H2B-GFP vector. Individual clones were isolated and two of the clones were used for further experiments. Caco2 cells were cultured as suggested by the supplier. Caco2 cells were cultured, 2 days (sub-confluent), 3 days (confluent) and 14 days (post-confluent) to generate differentiated cells. The melanoma cell line WM115, provided by Boris Fichtman and Zeev Ronai (Burnham Institute for Medical Research, La Jolla, CA), was cultured in RPMI 1640 supplemented with 10% fetal bovine serum. The H9 hES cells were provided by Brandon Nelson and Mark Mercola, (Burnham Institute for Medical Research La Jolla, CA). They were cultivated with mouse embryo fibroblast feeder cell conditioned medium supplemented with bFGF as described (12
). The undifferentiated state of the hES cells was routinely monitored by staining for Oct4 and other hES markers.
Single cell suspensions were applied to glass slides with a Shandon Cytospin 3 centrifuge at 500 rpm for 5 minutes. The cells were fixed for 5 minutes in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) at room temperature, washed twice in PBS then blocked with 1.5% normal goat serum for 1 hour at room temperature. The cells were incubated with a 1:10 dilution of the primary antibody (anti CD133-PE, AC133, Miltenyi Biotec, Auburn, CA) at 37°C for 1 hour and subsequently washed twice with 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO) in PBS and twice with PBS. The primary antibody was detected with Alexa 568-conjugated goat anti mouse IgG 1:100 (Invitrogen, Carlsbad, CA) and nuclei were stained with DAPI. The stained cells were mounted in Vectashield mounting medium (VECTOR, Burlingame, CA).
Flow cytometry analysis
AC133 reaction was identified by direct immunofluorescent staining using the AC133 mouse monoclonal antibody direct conjugated with phycoerythrin (PE). All cells were stained according to manufacturer's recommendations. In brief, 2×105 live cells were suspended in 100μl of buffer (0.5% FCS and 2mM EDTA) and stained for 10 minutes at 4°C with 10μl of the AC133 antibody (1:11). Cells were analyzed for PE and GFP expression by flow cytometry on a FACSort cytometer (Becton Dickinson, San Jose, CA). 10,000 events were acquired and analyzed using FlowJo software.
Cell cycle analysis
DNA in MELK-GFP expressing Caco2 cells was stained using Hoechst 33342 (Invitrogen, Carlsbad CA) while WM115 and hES cells were stained with Draq5 (Biostatus Ltd, Leicestershire, UK). For the Hoechst 33342 staining 2-3×105 cells, previously stained for AC133, were washed 1× in wash buffer (0.5% FCS and 2mM EDTA in PBS). Cells were resuspended in 250μl of culture media (DMEM) and Hoechst 33342 was added to a final concentration of 15ug/ml. Cells were incubated at 37°C for 90 minutes. For the Draq5 staining 1×105 cells, previously stained for AC133, were washed 1× in wash buffer. The cells were resuspended in DMEM and Draq5 at a final concentration of 10μM. Cells stained with Hoechst 33342 were analyzed on a FACSDiVa flow cytometer (Becton Dickinson) and cells stained with Draq5 were analyzed on a FACSort cytometer (Becton Dickinson). All FACS data was analyzed using FlowJo software.
For sorting cells expressing AC133, the cells were removed from the culture dish with 0.05% trypsin and 0.02% EDTA (Invitrogen, Carlsbad, CA), washed in PBS containing 1% FCS, stained as described above and resuspended at 106 cells per ml in the same buffer. The cells were filtered through a 35μM nylon filter prior to FACSort. Sorting was performed on a FACSDiVa flow cytometer (Becton Dickinson). Side and forward scatter profiles and propidium iodide (PI) staining were used to eliminate cell doublets and dead cells. The top ten percent of the AC133 reactive cells and the AC133 negative cells were collected. An aliquot was removed at the end of the sort and reanalyzed to evaluate purity.
Colony formation assay
Caco2 cells sorted on AC133 reactivity were cultured in a 24-well plates at concentrations of 100, 300, 1000 and 5000 cells per well, in triplicates. After 7 days the cells were fixed in methanol, stained with giemsa stain and counted with a dissection microscope at 10× magnification.
AC133 high and negative sorted cells were plated at a concentration of 50, 500 and 5000 cells per well in duplicate. Cells were cultured for 1, 2 or 3 days. The cells were washed 2× in PBS before they were frozen at -70°C in the 96-well plate. The CyQUANT Cell Proliferation Assay Kit (Invitrogen, Carlsbad, CA) was used according to manufacturer's instruction.
Total RNA from AC133 high and negative sorted Caco2 and WM115 cells was extracted using the TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's protocol. Two samples each of two cell lines were analyzed as biological duplicates. Labeled cRNA was prepared from 500 ng RNA using the Illumina® RNA Amplification Kit from Ambion (Austin, TX, USA). The Biotin labeled cRNA (750 ng) was hybridized 18 hr at 58°C to the HumanRef-8 v2 Expression BeadChip (>22,000 gene transcripts; Illumina, San Diego, CA, USA) according to the manufacturer's instructions. BeadChips were scanned with an Illumina BeadArray Reader and hybridization efficiency was monitored using BeadStudio software (Illumina). BeadStudio software was used to normalize and to quality control the data. To identify statistically significant changes the data were evaluated by GeneSpring software. A volcano plot was used to identify genes with changed a least 2 fold and had reproducibility p-values of 0.05 or less. The list of genes passing these thresholds was compared to publically available data using the Nextbio search engine. Complete primary data are available though the Gene Expression Omnibus support by the National Center for Biotechnology Information as GEO accession number (GSE11757).