Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs.
The cancer stem cells (CSCs) from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified. The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension, and mixed homogeneously into 1.2% alginate gel. Single-cell alginate gel was cultured with serum-free DMEM/F12 medium. Epirubicin (0.8 microg/mL) was added to the medium to enrich CSCs. After cultured conventionally for 7 to 10 days, most of cells suspended in alginate gel were killed by epirubicin. But few cells survived and some single-cell cloning spheres formed. Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells with properties of self-renewal and multi-potential differentiation. Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo. The results showed that some cells positive for Oct3/4 (TRITC) and Nanog (TRITC) were found in single-cell cloning spheres, and most of positive cells were concentrated in the core of sphere. Cells from spheres could form osteosarcoma in the body of mice. It was concluded that cells from single-cell cloning spheres had the properties of the expression of parts of stem cell genes (Oct3/4 and Nanog), resisting anti-cancer drugs, and tumorigenicity in vivo. To sum up, it is believed that cells obtained from osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs are cancer stem cells.
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. zhousong_tjmu@163.com
Song
S
Zhou
Feng
F
Li
Jun
J
Xiao
Wei
W
Xiong
Zhong
Z
Fang
Wenjian
W
Chen
Pengyan
P
Niu
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001859:Bone Neoplasms / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D015251:Epirubicin / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D014411:Neoplastic Stem Cells / Q000473:pathology* / Q000637:transplantation; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D012516:Osteosarcoma / Q000473:pathology*
D000970:Antineoplastic Agents; D016895:Culture Media, Serum-Free; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D015251:Epirubicin