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*

*Corresponding author for this work

Research output: Contribution to journalArticle

14 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)509-522
Number of pages14
JournalJournal of Neuro-Oncology
Volume104
Issue number2
DOIs
StatePublished - Sep 1 2011

Keywords

  • Differentiation
  • Glioblastoma multiforme
  • Neural stem cells
  • Neurosphere
  • Transduction
  • Transfection

ASJC Scopus subject areas

  • Oncology
  • Neurology
  • Clinical Neurology
  • Cancer Research

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  • Cite this

    Gürsel, D. B., Beyene, R. T., Hofstetter, C., Greenfield, J. P., Souweidane, M. M., Kaplitt, M., Arango-Lievano, M., Howard, B., & Boockvar, J. A. (2011). Optimization of glioblastoma multiforme stem cell isolation, transfection, and transduction. Journal of Neuro-Oncology, 104(2), 509-522. https://doi.org/10.1007/s11060-011-0528-2