Isolation and Culturing of Glioma Cancer Stem Cells

Sang‐Soo Kim1, Kathleen F. Pirollo1, Esther H. Chang1

1 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.10
DOI:  10.1002/0471143030.cb2310s67
Online Posting Date:  June, 2015
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In many human cancers including malignant glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for tumor initiation, metastasis and resistance to conventional anti‐cancer therapies. Therefore, a CSC‐targeted drug delivery strategy to eliminate CSCs is a desirable approach for developing a more effective therapeutic. Moreover, isolated CSCs will provide an invaluable tool for studying the underlying cellular mechanisms of tumor development and provide insight into therapeutic options for successful eradication of CSCs. This unit describes a method for the isolation and culture of CSCs from human GBM tumor tissue. © 2015 by John Wiley & Sons, Inc.

Keywords: glioblastoma multiforme; cancer stem cells; culture; neurosphere; tumorsphere

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of CSCs from Brain Tumor
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of CSCs from Brain Tumor

  • Fresh GBM tumor tissue specimen
  • Stem cell medium (see recipe)
  • Hank's balanced salt solution (HBSS; Mediatech)
  • Enzyme digestive mix (see recipe)
  • Trypan blue (Life Technologies)
  • FACS buffer (see recipe)
  • Human Fc block (BD Biosciences)
  • Ice
  • Anti‐human SSEA‐1 (Stemgent)
  • Anti‐human Nestin (Abcam)
  • Anti‐human Nanog (Cell Signaling Technology)
  • Anti‐human Msi1 (BD Pharmingen)
  • Anti‐human CD133 (Miltenyi Biotec)
  • Anti‐human Nestin (Abcam)
  • Anti‐human CD71 (TfR) (BioLegend)
  • Fixing buffer (see recipe)
  • 10× Phosphate‐buffered saline (PBS; Mediatech)
  • 4% paraformaldehyde solution (see recipe)
  • Triton X‐100 (Sigma)
  • Bovine serum albumin (Sigma)
  • PBS‐T (see recipe)
  • ProLong Gold Antifade Mountant with DAPI (Life Technology)
  • DAPI
  • Nail polish
  • Vertical laminar flow hood certified for Level II
  • 35‐mm tissue culture dishes (Corning)
  • No. 10 curved scalpel blades (Fine Science Tools)
  • 5‐ and 10‐ml disposable pipets (Denville)
  • Pipet‐aid (Drummond Scientific)
  • Rotator multimix (VWR Scientific)
  • 70‐μm cell strainer (Fisher Scientific)
  • 15‐ and 50‐ml conical centrifuge tubes (Corning)
  • 5‐ml syringes (BD)
  • Benchtop centrifuge (Eppendorf)
  • Hemacytometer (Hausser Scientific)
  • 5‐ml polystyrene round‐bottom tubes (BD Falcon)
  • BD FACSAria flow cytometer (BD Biosciences)
  • 100‐mm ultra‐low attachment culture dishes (Corning)
  • 37°C CO 2 incubator with humidity and gas control
  • Pasture pipets (Fisher Scientific)
  • LabTek 8‐well chamber slide (Nunc)
  • 1‐ml pipets
  • Glass coverslips (Fisher Scientific)
  • IX71 inverted epifluorescent microscope (Olympus)
  • LSM 510 META laser scanning microscopes (Zeiss)
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Literature Cited

Literature Cited
  Brescia, P., Richichi, C., and Pelicci, G. 2012. Current strategies for identification of glioma stem cells: Adequate or unsatisfactory? J. Oncol. 2012:376894.
  Chen, J., McKay, R.M., and Parada, L.F. 2012. Malignant glioma: Lessons from genomics, mouse models, and stem cells. Cell 149:36‐47.
  Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., Visvader, J., Weissman, I.L., and Wahl, G.M. 2006. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 66:9339‐9344.
  Hutton, S.R. and Pevny, L.H. 2008. Isolation, culture, and differentiation of progenitor cells from the central nervous system. CSH Protoc. 2008:pdb.prot5077.
  Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, N.M., Pastorino, S., Purow, B.W., Christopher, N., Zhang, W., Park, J.K., and Fine, H.A. 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 9:391‐403.
  Reynolds, B.A. and Weiss, S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707‐1710.
  Sakariassen, P.Ø., Immervoll, H., and Chekenya, M. 2007. Cancer stem cells as mediators of treatment resistance in brain tumors: Status and controversies. Neoplasia 9:882‐892.
  Tropepe, V., Sibilia, M., Ciruna, B.G., Rossant, J., Wagner, E.F., and van der Kooy, D. 1999. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev. Biol. 208:166‐188.
  Tunici, P., Bissola, L., Lualdi, E., Pollo, B., Cajola, L., Broggi, G., Sozzi, G., and Finocchiaro, G. 2004. Genetic alterations and in vivo tumorigenicity of neurospheres derived from an adult glioblastoma. Mol. Cancer 3:25.
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