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  • br Previously we successfully isolated established CSCs CICs from human


    Previously, we successfully isolated established CSCs/CICs from human endometrial carcinoma as sphere-cultured SB 203580 (Hashimoto et al., 2017). The sphere-cultured cells showed higher tumor-initiating ability than that of serum-cultured cells, indicating that sphere-cultured cells are enriched with CSCs/CICs. Moreover, single cell transcriptome analysis revealed heterogeneity in serum-cultured cells (Hashimoto et al., 2017). We thus analyzed primary sphere-cultured cells at single cell levels and found functional heterogeneity in the cells. These find-ings indicate that CSCs/CICs isolated as sphere-forming cells are com-posed of heterogenic cells including higher tumorigenic clones and treatment-resistant cells. This report is the first report on heterogeneity of CSCs/CICs, and our model is a reasonable model for analyzing the heterogeneity of CSCs/CICs.
    2. Materials and methods
    2.1. Ethics statement
    Mice were maintained and experimented on in accordance with the guidelines of and after approval by the Committee of Sapporo Medical University School of Medicine, Animal Experimentation Center under permit number 08-006. Any animal found unhealthy or sick was promptly euthanized. All the studies were approved by the Institutional Review Board (IRB) of Sapporo Medical University Hospital. Written informed consent was obtained from all patients according to the guidelines of the Declaration of Helsinki.
    2.2. Isolation of primary cancer cells from clinical tumors
    This study was approved by the IRB of Sapporo Medical University Hospital. Written informed consent was obtained from all patients ac-cording to the guidelines of the Declaration of Helsinki. Solid tumors were cut into fragments, washed in phosphate buffered saline (PBS), and centrifuged at 2000 rpm for 10 min. Then cell aggregates were incubated at 37 °C for about 30 min with 2 mg Liberase™ research grade (Roche, Basel, Switzerland) in DMEM/F12 (ThermoFisher Scientific, MA, USA) until they had been separated into single cells. The cells obtained by these procedures were cultured as described below.
    2.3. Cell culture conditions and establishment of S clone cells and LL clone cells
    We cultured cells from a primary tumor in two conditions. One condition was culture using an attachment plate in DMEM/F12 medium containing 10% FBS and 1% penicillin and streptomycin (serum cul-ture). The other condition was a culture using an ultra-low attachment flask (Corning Inc., Corning, NY, USA) in serum-free DMEM/F12 medium supplemented with N-2 supplement (Wako, Osaka, Japan), 
    20 ng/ml recombinant human epithelial growth factor (ThermoFisher Scientific), 10 ng/ml human basic fibroblast growth factor (Sigma-Aldrich, MO, USA), 4 μg/ml heparin (AY pharma, Tokyo, Japan) and 1% penicillin and streptomycin (sphere culture).
    Single cell cloning was performed to establish clone cells from sphere cultured cells by single cell sorting using FACS Aria II™ (Becton, Dickinson and Campany, NJ, USA) or limiting dilution. Clone cells were classified into sphere clone (S clone) cells and leukemia-like clone (LL clone) cells.
    2.4. Reverse transcription polymerase chain reaction analysis (RT-PCR)
    RT-PCR was performed to check the expression of candidate genes acquired by SAGE-Seq. The thermal cycling conditions were 94 °C for 2 min followed by 40 cycles of 15 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C. Primers for GAPDH (glyceraldehyde-3-phosphate dehy-drogenase), used as an internal control, were 5’-ACCACAGTCCATGCC ATCAC-3′ and 5’-TCCACCACCCTGTTGCTGTA-3′ with an expected PCR product size of 452 bp. Other primer information is summarized in Supplementary Table S1.
    2.5. Quantitative real-time PCR analysis (qRT-PCR)