Skip to main content
SpringerLink
Log in

Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs

  • Published:
Journal of Huazhong University of Science and Technology [Medical Sciences] Aims and scope Submit manuscript

Summary

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 μg/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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Gatta G, Capocaccia R, Stiller C, et al. Childhood cancer survival trends in Europe: a EUROCARE Working Group study. J Clin Oncol, 2005,23(16):3742–3751

    Article  PubMed  Google Scholar 

  2. Meyers PA, Schwartz CL, Krailo M, et al. Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol, 2005,23(9):2004–2011

    Article  CAS  PubMed  Google Scholar 

  3. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells. Nature, 2001,414(6859):105–111

    Article  CAS  PubMed  Google Scholar 

  4. Marx J. Mutant stem cells may seed cancer. Science, 2003,301(5638):1308–1310

    Article  CAS  PubMed  Google Scholar 

  5. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA, 2003,100:3983–3988

    Article  CAS  PubMed  Google Scholar 

  6. Galli R, Binda E, Orfanelli U, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res, 2004,64(19): 7011–7021

    Article  CAS  PubMed  Google Scholar 

  7. Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res, 2005,65(23):10 946–10 951

    Article  CAS  Google Scholar 

  8. Kim CF, Jackson EL, Woolfenden AE, et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell, 121(6):823–835

  9. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature, 2007,445(7123):111–115

    Article  CAS  PubMed  Google Scholar 

  10. Yin S, Li J, Hu C, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer, 2007,120(7):1444–1450

    Article  CAS  PubMed  Google Scholar 

  11. Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology, 2008,47(3):919–928

    Article  CAS  PubMed  Google Scholar 

  12. Woodward WA, Sulman EP. Cancer stem cells: markers or biomarkers? Cancer Metastasis Rev. 2008, 27(3):459–470

    Article  CAS  PubMed  Google Scholar 

  13. Phillips TM, McBride WH, Pajonk F. The response of CD24 (/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst, 2006,98(24):1777–1785

    Article  PubMed  Google Scholar 

  14. Inagaki A, Soeda A, Oka N, et al. Long-term maintenance of brain tumor stem cell properties under at non-adherent and adherent culture conditions. Biochem Biophys Res Commun, 2007,361(3):586–592

    Article  CAS  PubMed  Google Scholar 

  15. Sutherland RM. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science, 1988,240(4849):177–184

    Article  CAS  PubMed  Google Scholar 

  16. Kelm JM, Kramer BP, Gonzalez-Nicolini V, et al. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng, 2003,83(2):173–180

    Article  CAS  PubMed  Google Scholar 

  17. Fischbach C, Chen R, Matsumoto T, et al. Engineering tumors with 3D scaffolds. Nat Methods, 2007,4(10):855–860

    Article  CAS  PubMed  Google Scholar 

  18. Jeon O, Bouhadir KH, Mansour JM, et al. Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties. Biomaterials, 2009,30(14):2724–2734

    Article  CAS  PubMed  Google Scholar 

  19. de Vos P, Faas MM, Strand B, et al. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials, 2006,27(32):5603–5617

    Article  PubMed  CAS  Google Scholar 

  20. West ER, Xu M, Woodruff TK, et al. Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials, 2007,28(30):4439–4448

    Article  CAS  PubMed  Google Scholar 

  21. Markusen JF, Mason C, Hull DA, et al. Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. Tissue Eng, 2006,12(4):821–830

    Article  CAS  PubMed  Google Scholar 

  22. Sagar J, Chaib B, Sales K, et al. Role of stem cells in cancer therapy and cancer stem cells: a review. Cancer Cell Int, 2007,7:9

    Article  PubMed  CAS  Google Scholar 

  23. Toda S, Watanabe K, Yokoi F, et al. A new organotypic culture of thyroid tissue maintains three-dimensional follicles with C cells for a long term. Biochem Biophys Res Commun, 2002,294(4):906–911

    Article  CAS  PubMed  Google Scholar 

  24. Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct-4. Cell, 1998,95(3): 379–391

    Article  CAS  PubMed  Google Scholar 

  25. Hoei-Hansen CE, Almstrup K, Nielsen JE, et al. Stem cell pluripotency factor Nanog is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours. Histopathology, 2005,47(1):48–56

    Article  CAS  PubMed  Google Scholar 

  26. Yuan X, Curtin J, Xiong Y, et al. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene, 2004,23(58):9392–9400

    Article  CAS  PubMed  Google Scholar 

  27. Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res, 2003,63(18):5821–5828

    CAS  PubMed  Google Scholar 

  28. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA, 2004,101(3):781–786

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Song Zhou  (周 松), Feng Li  (李 锋), Jun Xiao  (肖 骏), Wei Xiong  (熊 伟), Zhong Fang  (方 忠), Wenjian Chen  (陈文坚) & Pengyan Niu  (牛鹏彦)

Authors
  1. Song Zhou  (周 松)
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Li  (李 锋)
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Xiao  (肖 骏)
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Xiong  (熊 伟)
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhong Fang  (方 忠)
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenjian Chen  (陈文坚)
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pengyan Niu  (牛鹏彦)
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Feng Li  (李 锋).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhou, S., Li, F., Xiao, J. et al. Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 30, 81–84 (2010). https://doi.org/10.1007/s11596-010-0114-4

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11596-010-0114-4

Key words

Search

Navigation