The cultivation and identification of tumor stem cells from neuroblastoma derived tumor spheres.
OBJECTIVE
Since the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells.


METHODS
Bone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed.


RESULTS
Neuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 10(4) sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology.


CONCLUSIONS
Neuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.
Department of Hematology/Oncology, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, P. R. China.
Qiu-Xia
QX
Liu
Jing-Yan
JY
Tang
Jiao-Yang
JY
Cai
Min-Zhi
MZ
Yin
Ben-Shang
BS
Li
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects*; D002648:Child; D016895:Culture Media, Serum-Free; D006801:Humans; D015474:Isotretinoin / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D018874:Spheroids, Cellular / Q000473:pathology*; D023041:Xenograft Model Antitumor Assays
C069077:BMI1 protein, human; D016895:Culture Media, Serum-Free; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1; D015474:Isotretinoin