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Cancer stem cells (CSCs) are considered to play important roles in tumor initiation, progression, metastasis, and recurrence.However, separation and long-term culture of CSCs remain technical problems, limiting the investigations either on regulation network involving in stemness maintenance of CSCs or treatment strategies targeting to CSCs.In the previous study, we have established a universal culture system for liver cancer stem cells, by which a list of CSCs strains from liver cancer cell lines or primary liver cancer tissue have been successfully obtained and their characteristics of CSCs are maintained across long-term passages.