Isolation, Culture And Identification Of Human Hepatic Cancer Stem Cells From Hepatocellular Carcinom Tissues

Objective To isolate and culture human hepatic cancer stem cells from tissues of hepatocellular carcinoma and identify their biological characteristics.Methods Hepatocellular carcinoma tissues from patients with liver cancer after surgery were collected. Hepatic cancer cells were isolated by digestion and cultured in the serum free medium, and the survival of tumor spheres were observed in ultra low adhesion 24-well plates. Besides, the proliferation of hepatic cancer cells was analysed by MTT assay, and hepatic cancer cells were sorted by flow cytometry based on CD90 and CD133, and devided into four groups CD90+CD133-, CD90-CD133+,CD90+CD133+and CD90-CD133- respectively.Then, the four group cells were subcutaneously engrafted into nude mice, and tumor were resected and subjected to HE staining.Results Tumor spheres could be found in the serum free culture condition and the hepatic cancer cells kept in an increased state in continued 72 hours, moreover, the ratio of expression of CD90 and CD133 was 7.2%、8.8% respectively. The CD90+,CD133+,CD90- and CD133- cells were scucessfully sorted by flow cytometry. Cell transplantation assays indicated CD90+CD133-,CD90-CD133+ and CD90+CD133+ groups could formed tumors, while CD90-CD133- was not, moreover, the tumor of CD90+CD133+ group emerged firstly, there was no tumor observed in CD90-CD133- group during the whole process. HE staining showed the Pathological features of tumor were similar with that of human.Conclusion We succesfully established the method to isolate and culture human hepatic cancer stem cells, which could formed tumor spheres and have ability of self-renewal. Some of the cells had the ability to form tumor spheres, self-renewal. Some of the cells expressed CD90+ or CD133+, and could formed tumors under the skin of nude mice. Importantly, the CD90+CD133+ cells possess the properties of stem cells, which would be hepatic cancer cells.