title: Optimization of glioblastoma multiforme stem cell isolation, transfection, and transduction.
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.
authors.affiliation: Laboratory for Translational Brain Tumor and Stem Cell Research, Weill Cornell Medical College, Cornell University, New York, NY 10021, USA.
authors.forename: Demirkan B
authors.initials: DB
authors.lastname: Gürsel
authors.forename: Robel T
authors.initials: RT
authors.lastname: Beyene
authors.forename: Christoph
authors.initials: C
authors.lastname: Hofstetter
authors.forename: Jeffry P
authors.initials: JP
authors.lastname: Greenfield
authors.forename: Mark M
authors.initials: MM
authors.lastname: Souweidane
authors.forename: Michael
authors.initials: M
authors.lastname: Kaplitt
authors.forename: Margarita
authors.initials: M
authors.lastname: Arango-Lievano
authors.forename: Brian
authors.initials: B
authors.lastname: Howard
authors.forename: John A
authors.initials: JA
authors.lastname: Boockvar
mesh_terms: D015153:Blotting, Western; D001932:Brain Neoplasms; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D005909:Glioblastoma; D006801:Humans; D008856:Microscopy, Fluorescence; D014411:Neoplastic Stem Cells / Q000166:cytology*; D014161:Transduction, Genetic; D014162:Transfection