Case selection: A total of 52 samples of liver tissues were obtained from the hepatobiliary disease file of the Division of Pathology, Kanazawa University Hospital in Japan between 2005 and 2009. This study consisted of three cases of normal liver, five cases of chronic viral hepatitis or liver cirrhosis, 33 cases of HCC, six cases of intrahepatic cholangiocarcinoma, and five cases of combined hepatocellular and cholangiocarcinoma (combined carcinoma). All cases used in this study were surgically resected cases. Normal liver tissues used in this study were background liver tissues of metastatic colon cancers. Age, sex and clinicopathological characteristics are shown in Table 1.
Expression of CD133 (mRNA level): Total RNA was extracted from the frozen section of all 47 cases using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Total RNA was dissolved in 50 μL of distilled water that contained 0.1% diethylpyrocarbonate, and quantitated using a spectrophotometer at OD260. Isolated RNA was used for the subsequent reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of CD133 mRNA was examined by nested RT-PCR using two sets of primers. The oligonucleotide sequences, numbers of cycles, and annealing temperatures of these primers are shown in Table 2. After PCR, 5-μL aliquots of the products were subjected to 1.5% or 2.0% agarose gel electrophoresis and stained with ethidium bromide.
Immunostaining of CD133, cytokeratin 19 (CK19) and hepatocyte paraffin-1 (HepPar-1): Frozen sections of 52 samples of non-neoplastic and neoplastic liver tissues were used for immunostaining. Immunostaining for CD133, CK19 and HepPar-1 was performed using a mouse monoclonal antibody against human CD133 (clone AC133; Miltenyi Biotec, Auburn, CA, USA), a mouse monoclonal antibody against human CK19 (Dako Cytomation, Glostrup, Denmark), and a mouse monoclonal antibody against human HepPar-1 (Dako Cytomation).
Serial sections were used in each case to examine the co-localization of CD133, CK19 and HepPar-1 expression. Sliced frozen sections were fixed with acetone for 20 min. After blocking endogenous peroxidases, the sections were incubated in protein block solution (Dako Cytomation) for 20 min and incubated at 4°C with each primary antibody. These sections were incubated for 1 h at room temperature with goat anti-mouse immunoglobulins, which were conjugated to peroxidase-labeled polymer (Envision+; Dako Cytomation). 3,3'-Diaminobenzidine tetrahydrochloride was used as the chromogen, followed by light counterstaining with hematoxylin. Negative controls were evaluated by substituting the primary antibody with similarly diluted non-immunized mouse serum.
Cell culture: Three human HCC cell lines (HuH7, PLC5 and HepG2) and two human cholangiocarcinoma cell lines (CCKS1 and HuCCT1) were used in this study. HuH7, PLC5 and HepG2 were obtained from the Hearth Science Research Bank (Osaka, Japan). HuCCT-1 was obtained from the Cell Resource Center for Biochemical Research, Tohoku University, Sendai, Japan. CCKS1 was established in our laboratory. HuH7 and PLC5 were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen Corp., Carlsbad, CA, USA), and HepG2 was maintained in minimum essential medium (Invitrogen Corp.) with 1% nonessential amino acids (Specialty Media, Phillipsburg, NJ, USA). CCKS1 and HuCCT1 were cultured in RPMI-1640 medium (Invitrogen Corp.) Each medium was supplemented with 10% fetal bovine serum (Invitrogen Corp.) and 1% antibiotic-antimycotic (Invitrogen Corp.).
Dual fluorescent immunostaining of CD133/CK19 and CD133/alpha-fetoprotein (AFP): Cell lines were cultured on Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL, USA) for fluorescent immunostaining. After culturing for 2 d, the specimens were fixed in 4% paraformaldehyde for 10 min at 4°C. After incubation in protein block solution (Dako Cytomation) for 10 min, the specimens were incubated with antibodies against CD133 and CK19, or antibodies against CD133 and AFP for 1 h at room temperature. The antibodies used were as follows: CD133, mouse monoclonal, clone AC133, Miltenyi Biotec; CK19, goat polyclonal, clone G-14, Santa Cruz Biotechnology (Santa Cruz, CA, USA); and AFP, a rabbit polyclonal, Dako Cytomation. The reaction product was visualized with fluorescent goat anti-mouse and anti-rabbit IgG antibodies (1:500, Molecular Probes Inc., Eugene, OR, USA). Specimens were counterstained with DAPI (Molecular Probes Inc.), and fluorescent signals were observed using a fluorescence microscope (Olympus, Tokyo, Japan).
Fluorescence-activated cell sorting (FACS) with reference to CD133 expression: HuH7 and HuCCT1 cells were used for FACS. Cultured cells were harvested after treatment with 0.25% of trypsin-EDTA solution (Sigma Chemical Co., St Louis, MO, USA) for 20 min, and washed three times in Hanks’ Balanced Salt Solution (Invitrogen Corp.). Cultured cells were stained live in a staining solution containing bovine serum albumin, insulin, and phycoerythrin (PE)-conjugated monoclonal antibody to CD133 (clone AC133; Miltenyi Biotec) for 30 min at 4°C. As negative controls, cultured cells were incubated similarly with non-immunized mouse immunoglobulin. Samples were analyzed and sorted by JSAN (Bay Bioscience, Kobe, Japan). Cell debris and cell aggregates were gated out electronically. For the positive population, only the top 5%-10% of the most brightly stained cells were selected. For the negative population, only the bottom 5%-10% of the most dimly stained cells were selected. Then, 1.0 × 105 cells were sorted from the positive or negative population at the most specific mode. Sorted cells were plated on culture dishes for subculture. After sorting, CD133+ and CD133- cells were cultured separately. After 4-wk culture, cultured cells were sorted again into CD133+ and CD133- cells using flow cytometry to evaluate how the CD133+ cell ratios were altered in each subpopulation. After subculturing for 3 or 4 wk, cultured cells were sorted again into CD133+ and CD133- cells to evaluate how the CD133+ or CD133- populations changed during subculture. The percentages of CD133+ cells were calculated in a total of 1000-5000 cells in each group.
RNA expression in culture cells: Total RNA was extracted from five types of cultured cells using an RNeasy Mini Kit (Qiagen). Total RNA was similarly extracted from CD133+ and CD133- cells. RT-PCR was performed for CD133, hepatocyte makers (AFP and albumin), biliary markers (CK19 and CK7 and β-actin. The oligonucleotide sequences, numbers of cycles and annealing temperatures of these primers are shown in Table 2. After PCR, 5-μL aliquots of the products were subjected to 1.5% or 2.0% agarose gel electrophoresis and stained with ethidium bromide.
Real-time RT-PCR: The alterations of CD133 expression levels were examined in non-sorted or sorted (CD133+ or CD133-) cultured cells time-dependently (days 0, 7, 14, 21 and 28) after the passage or sorting. Real-time analysis was performed using premade CD133 and β-actin-specific primers and probes with the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Warrington, UK). RT-PCR was done with the TaqMan Universal PCR Master Mix (PE Applied Biosystems) using 2 μL cDNA in a 25-μL final reaction mixture. Cycling conditions were as follows: incubation at 50°C for 2 min, 10 min at 95°C, and 50 cycles of 15 s at 95°C and 1 min at 60°C. CD133 was normalized (ΔCt) to β-actin from the Ct value of CD133. Each experiment was performed in triplicate, and the mean adopted.
Cell proliferation assay of CD133+ and CD133-cells: CD133+ and CD133- cells were plated on a Lab-Tek II chamber slide (Nalge Nunc International), and cultured for 7 d before the cell proliferation assay. Cell proliferation was assayed using BrdU. Cultured cells were incubated on slides with BrdU solution (10 mmol/L) at 37°C for 30 min. After fixing with 70% ethanol (50 mmol/L glycine buffer solution, pH 2.0) for more than 20 min, the slides were incubated with anti-BrdU solution at 37°C for 30 min. After additional incubation with IgG fluorochrome solution for 30 min, positive signals were detected by a fluorescence microscope (Olympus).
Relationship between side population (SP) and CD133+ cells: SP is currently estimated as one of the most reliable stem cell phenotypes[19,20]. The relationship between SP and CD133+ cells was examined by FACS. After detaching and washing, the cultured cells were then incubated at 37°C for 90 min with 20 μg/mL Hoechst 33342 (Sigma Chemical Co.), PE-conjugated monoclonal antibody to CD133 (clone AC133; Miltenyi Biotec), bovine serum albumin, in the presence or absence of 100 μmol/L verapamil (Sigma Chemical Co.). After incubation, 1 μg/mL propidium iodide (Sigma Chemical Co.) was added and the cells were filtered through a 40-μm cell strainer (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. The relationship between SP and CD133 expression was analyzed by JSAN (Bay Bioscience). Hoechst 33342 was excited with a UV laser at 350 nm and fluorescence emission was measured with 405/BP30 (Hoechst blue) and 570/BP20 (Hoechst red) optical filters. Propidium iodide labeling was measured through a 630/BP30 filter for the discrimination of dead cells. Next, HuH7 cells were sorted into SP/CD133+, SP/CD133-, non-SP/CD133+, and non-SP/CD133-. After 4 wk subculturing, each population was analyzed again with regard to CD133 expression by FACS.