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Journal of Neuro-Oncology

, Volume 104, Issue 2, pp 509–522 | Cite as

Optimization of glioblastoma multiforme stem cell isolation, transfection, and transduction

  • Demirkan B. Gürsel
  • Robel T. Beyene
  • Christoph Hofstetter
  • Jeffry P. Greenfield
  • Mark M. Souweidane
  • Michael Kaplitt
  • Margarita Arango-Lievano
  • Brian Howard
  • John A. Boockvar
Laboratory Investigation - Human/Animal Tissue

Abstract

It has been postulated that brain tumor stem cells (TSCs) may be the population of cells responsible for the maintenance and recurrence of glioblastoma multiforme (GBM). The purpose of this study was to optimize a reproducible protocol for generating TSCs for their subsequent transfection or transduction. Patient GBMs were enzymatically and mechanically dissociated and tumor spheres were resuspended in appropriate media and analyzed to ensure they met stem cell criteria. These cells were then transfected with a plasmid or transduced with a viral vector to introduce a previously absent gene and then allowed to form tumor spheres. Tumor spheres were generated from patient GBMs without contamination. These cells met stringent criteria as stem cells, including multipotentiality and self-renewal. High efficiency transfection and transduction of tumor spheres was possible, even at the core of the sphere. This allowed for the introduction of new genes to the TSCs, as evidenced by fluorescent microscopy and Western blot analysis. This study is a guide to optimize the generation of patient derived GBM tumor spheres without RBC and dead cell contamination. GBM TSCs within tumor spheres can easily be transfected with plasmids or transduced with a virus. This is important from a therapeutic perspective if gene replacement is to be successful in replacing genes lost in GBM progression or to knock down or silence genes that are over-expressed in malignant brain tumors.

Keywords

Neurosphere Glioblastoma multiforme Neural stem cells Differentiation Transfection Transduction 

Notes

Acknowledgments

We would like to the acknowledge Lisa Gursel for her thorough reading of this manuscript and helpful suggestions made therein.

References

  1. 1.
    Bleau AM, Howard BM, Taylor LA et al (2008) New strategy for the analysis of phenotypic marker antigens in brain tumor-derived neurospheres in mice and humans. Neurosurg Focus 24:E27CrossRefGoogle Scholar
  2. 2.
    Pollard SM, Yoshikawa K, Clarke ID et al (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4:568–580PubMedCrossRefGoogle Scholar
  3. 3.
    Sanai N, Alvarez-Buylla A, Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353:811–822PubMedCrossRefGoogle Scholar
  4. 4.
    Wang JC (2007) Evaluating therapeutic efficacy against cancer stem cells: new challenges posed by a new paradigm. Cell Stem Cell 1:497–501CrossRefGoogle Scholar
  5. 5.
    Lee J, Kotliarova S, Kotliarov Y et al (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391–403PubMedCrossRefGoogle Scholar
  6. 6.
    Galli R, Binda E, Orfanelli U et al (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021PubMedCrossRefGoogle Scholar
  7. 7.
    Windrem MS, Roy NS, Wang J et al (2002) Progenitor cells derived from the adult and human subcortical white matter disperse and differentiate as oligodendrocytes within demyelinated lesions of the rat brain. J Neurosci Res 69:966–975PubMedCrossRefGoogle Scholar
  8. 8.
    Ahmed S (2009) The culture of neural stem cells. J Cell Biochem 106:1–6PubMedCrossRefGoogle Scholar
  9. 9.
    Chong YK, Toh TB, Zaiden N et al (2009) Cryopreservation of human tumor neurospheres. Stem Cells 27:29–39PubMedCrossRefGoogle Scholar
  10. 10.
    Qiang L, Yang Y, Ma YJ et al (2009) Isolation and characterization of cancer stem like cells in human glioblastoma cell lines. Cancer Lett 279:13–21PubMedCrossRefGoogle Scholar
  11. 11.
    Howard BM, Gursel DB, Bleau AM et al (2010) EGFR signaling is differentially activated in patient-derived glioblastoma stem cells. J Exp Ther Oncol 8:247–260PubMedGoogle Scholar
  12. 12.
    Gunther HS, Schmidt NO, Phillips HS et al (2008) Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 27:2897–2909PubMedCrossRefGoogle Scholar
  13. 13.
    Suslov ON, Kukekov VG, Ignatova TN et al (2002) Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sci USA 99:14506–14511PubMedCrossRefGoogle Scholar
  14. 14.
    Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710PubMedCrossRefGoogle Scholar
  15. 15.
    Barami K (2008) Relationship of neural stem cells with their vascular niche: implications in the malignant progression of gliomas. J Clin Neurosci 15:1193–1197PubMedCrossRefGoogle Scholar
  16. 16.
    Bez A, Corsini E, Curti D et al (2003) Neurosphere and neurosphere forming cells: morphological and ultrastructural characterization. Brain Res 993:18–29PubMedCrossRefGoogle Scholar
  17. 17.
    Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401PubMedCrossRefGoogle Scholar
  18. 18.
    Clarke MF, Dick JE, Dirks PB et al (2006) Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344PubMedCrossRefGoogle Scholar
  19. 19.
    Rich JN, Eyler CE (2008) Cancer stem cells in brain tumor biology. Cold Spring Harb Symp Quant Biol 73:411–420PubMedCrossRefGoogle Scholar
  20. 20.
    Varghese M, Olstorn H, Sandberg C et al (2008) A comparison between stem cells from the adult human brain and from brain tumors. Neurosurgery 63:1022–1034PubMedCrossRefGoogle Scholar
  21. 21.
    Hemmati HD, Nakano I, Lazareff JA et al (2003) Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci USA 100:15178–15183PubMedCrossRefGoogle Scholar
  22. 22.
    Doetsch F (2007) Glial identity in neural stem cells. Nat Neurosci 6:1127–1134CrossRefGoogle Scholar
  23. 23.
    Messam CA, Hou J, Major EO (2000) Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody. Exp Neurol 161:585–596PubMedCrossRefGoogle Scholar
  24. 24.
    Beier D, Hau P, Proescholdt M et al (2007) CD133(+) and CD133(−) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67:4010–4015PubMedCrossRefGoogle Scholar
  25. 25.
    Ogden AT, Waziri AE, Lochhead RA et al (2008) Identification of A2B5 + CD133- tumor-initiating cells in adult human gliomas. Neurosurgery 62:505–514 discussion 514–5PubMedCrossRefGoogle Scholar
  26. 26.
    Liu G, Yuan X, Zeng Z et al (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:67PubMedCrossRefGoogle Scholar
  27. 27.
    Salmaggi A, Boiardi A, Gelati M et al (2006) Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 54:850–860PubMedCrossRefGoogle Scholar
  28. 28.
    Wakimoto H, Kesari S, Farrell CJ et al (2009) Human glioblastoma-derived cancer stem cells: establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors. Cancer Res 69:3472–3481PubMedCrossRefGoogle Scholar
  29. 29.
    Fidler IJ, Hart IR (1982) Biological diversity in metastatic neoplasms: origins and implications. Science 217:998–1003PubMedCrossRefGoogle Scholar
  30. 30.
    Fidler IJ, Kripke ML (1977) Metastasis results from preexisting variant cells within a malignant tumor. Science 197:893–895PubMedCrossRefGoogle Scholar
  31. 31.
    Heppner GH (1984) Tumor heterogeneity. Cancer Res 44:2259–2265PubMedGoogle Scholar
  32. 32.
    Holland EC (2001) Progenitor cells and glioma formation. Curr Opin Neurol 14:683–688PubMedCrossRefGoogle Scholar
  33. 33.
    Li Z, Wang H, Eyler CE et al (2009) Turning cancer stem cells inside out: an exploration of glioma stem cell signaling pathways. J Biol Chem 284:16705–16709PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Demirkan B. Gürsel
    • 1
    • 2
    • 3
  • Robel T. Beyene
    • 1
    • 2
    • 3
  • Christoph Hofstetter
    • 1
    • 2
    • 3
  • Jeffry P. Greenfield
    • 3
  • Mark M. Souweidane
    • 3
  • Michael Kaplitt
    • 3
  • Margarita Arango-Lievano
    • 3
  • Brian Howard
    • 1
    • 2
    • 3
  • John A. Boockvar
    • 1
    • 2
    • 3
  1. 1.Laboratory for Translational Brain Tumor and Stem Cell ResearchWeill Cornell Medical College, Cornell UniversityNew YorkUSA
  2. 2.Weill Cornell Brain Tumor CenterWeill Cornell Medical College, Cornell UniversityNew YorkUSA
  3. 3.Department of Neurological Surgery, Weill Cornell Brain Tumor CenterWeill Medical College, Cornell UniversityNew YorkUSA

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