Methods for Analysis of Brain Tumor Stem Cell and Neural Stem Cell Self-Renewal

  • Ichiro Nakano
  • Harley I. Kornblum
Part of the Methods in Molecular Biology book series (MIMB, volume 568)


Neural stem cells (NSC) self-renew and are multipotent, producing neurons and glia. Recent studies have shown that brain tumors (BT) contain cells that, like NSC, self-renew and are multipotent, producing the different types of cells found within the brain tumors. These brain tumor stem cells are a kind of cancer stem cell, competent to form tumors that mimic the parent tumor in experimental animals. Studies from our laboratory and others have demonstrated that brain tumor stem cells and NSC share similar mechanisms and pathways for proliferation. For example, we have identified that one of the AMPK/snf1 kinases, maternal embryonic leucine zipper kinase (MELK), is highly expressed in NSC and malignant brain tumors, as well as in brain tumor stem cell-enriched cell cultures. Analysis of transgenic MELK-reporter mice indicated that MELK is expressed in NSC in vivo, and our in vitro studies demonstrated that MELK is required for NSC self-renewal. We have also found that MELK is required for proliferation of putative BT stem cells. Utilizing our studies with MELK as an example, this chapter describes methods to culture NSC and BT stem cells, and to analyze the pathways, which regulate self-renewal of those cells.

Key words

MELK Brain tumor stem cells Cancer stem cells Signaling pathway Proliferation Self-renewal Proliferation RNA interference Cell culture 



This work was supported by NIMH grant MH065756 and the Miriam and Sheldon Adelson Program in Neural Repair Research. IN is supported by the ISCBM-CIRM fellowship and the Khan Family Foundation.


  1. 1.
    Alvarez-Buylla, A., and Lim, D. A. (2004) For the long run: maintaining germinal niches in the adult brain. Neuron 41, 683–6.PubMedCrossRefGoogle Scholar
  2. 2.
    Nakano, I., and Kornblum, H. I. (2006) Brain tumor stem cells. Pediatr Res 59, 54R–8R.PubMedCrossRefGoogle Scholar
  3. 3.
    Reya, T., Morrison, S. J., Clarke, M. F., and Weissman, I. L. (2001) Stem cells, cancer, and cancer stem cells. Nature 414, 105–11.PubMedCrossRefGoogle Scholar
  4. 4.
    Dirks, P. B. (2006) Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444, 687–8.PubMedCrossRefGoogle Scholar
  5. 5.
    Bao, S., Wu, Q., McLendon, R. E., Hao, Y., Shi, Q., Hjelmeland, A. B., Dewhirst, M. W., Bigner, D. D., and Rich, J. N. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–60.PubMedCrossRefGoogle Scholar
  6. 6.
    Bao, S., Wu, Q., Sathornsumetee, S., Hao, Y., Li, Z., Hjelmeland, A. B., Shi, Q., McLendon, R. E., Bigner, D. D., and Rich, J. N. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Cancer Res 66, 7843–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D., and Dirks, P. B. (2004) Identification of human brain tumour initiating cells. Nature 432, 396–401.PubMedCrossRefGoogle Scholar
  8. 8.
    Hemmati, H. D., Nakano, I., Lazareff, J. A., Masterman-Smith, M., Geschwind, D. H., Bronner-Fraser, M., and Kornblum, H. I. (2003) Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A 100, 15178–83.PubMedCrossRefGoogle Scholar
  9. 9.
    Phillips, H. S., Kharbanda, S., Chen, R., Forrest, W. F., Soriano, R. H., Wu, T. D., Misra, A., Nigro, J. M., Colman, H., Soroceanu, L., Williams, P. M., Modrusan, Z., Feuerstein, B. G., and Aldape, K. (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9, 157–73.PubMedCrossRefGoogle Scholar
  10. 10.
    Nakano, I., Paucar, A. A., Bajpai, R., Dougherty, J. D., Zewail, A., Kelly, T. K., Kim, K. J., Ou, J., Groszer, M., Imura, T., Freije, W. A., Nelson, S. F., Sofroniew, M. V., Wu, H., Liu, X., Terskikh, A. V., Geschwind, D. H., and Kornblum, H. I. (2005) Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation. J Cell Biol 170, 413–27.PubMedCrossRefGoogle Scholar
  11. 11.
    Nakano, I., Masterman-Smith, M., Saigusa, K., Paucar, A., Horvath, S., Watanabe, M., Negro, A., Bajpai, R., Howes, A., Lelievre, V., Washek, J. A., Lazareff, J. A., Freije, W. A., Liau, L. M., Gilbertson, R. J., Cloughesy, T., Geschwind, D. H., Nelson, S. F., Mischel, P. S., Terskikh, A., and Kornblum, H. I. (2007) Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells. J Neursci Res 86, 48–60.CrossRefGoogle Scholar
  12. 12.
    Reynolds, B. A., Tetzlaff, W., and Weiss, S. (1992) A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12, 4565–74.PubMedGoogle Scholar
  13. 13.
    Lie, D. C., Song, H., Colamarino, S. A., Ming, G. L., and Gage, F. H. (2004) Neurogenesis in the adult brain: new strategies for central nervous system diseases. Annu Rev Pharmacol Toxicol 44, 399–421.PubMedCrossRefGoogle Scholar
  14. 14.
    Gage, F. H. (2000) Mammalian neural stem cells. Science 287, 1433–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Kornblum, H., and Geschwind, D. (2001) Molecular markers in CNS stem cell research: hitting a moving target. Restor Neurol Neurosci 18, 89–94.PubMedGoogle Scholar
  16. 16.
    Kornblum, H. I., and Geschwind, D. H. (2001) Molecular markers in CNS stem cell research: hitting a moving target. Nat Rev Neurosci 2, 843–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Estivill-Torrus, G., Pearson, H., van Heyningen, V., Price, D. J., and Rashbass, P. (2002) Pax6 is required to regulate the cell cycle and the rate of progression from symmetrical to asymmetrical division in mammalian cortical progenitors. Development 129, 455–66.PubMedGoogle Scholar
  18. 18.
    Molofsky, A. V., He, S., Bydon, M., Morrison, S. J., and Pardal, R. (2005) Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev 19, 1432–7.PubMedCrossRefGoogle Scholar
  19. 19.
    Molofsky, A. V., Pardal, R., Iwashita, T., Park, I. K., Clarke, M. F., and Morrison, S. J. (2003) Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–7.PubMedCrossRefGoogle Scholar
  20. 20.
    Lee, J. P., Jeyakumar, M., Gonzalez, R., Takahashi, H., Lee, P. J., Baek, R. C., Clark, D., Rose, H., Fu, G., Clarke, J., McKercher, S., Meerloo, J., Muller, F. J., Park, K. I., Butters, T. D., Dwek, R. A., Schwartz, P., Tong, G., Wenger, D., Lipton, S. A., Seyfried, T. N., Platt, F. M., and Snyder, E. Y. (2007) Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med 13, 439–47.PubMedCrossRefGoogle Scholar
  21. 21.
    Ge, W., He, F., Kim, K. J., Blanchi, B., Coskun, V., Nguyen, L., Wu, X., Zhao, J., Heng, J. I., Martinowich, K., Tao, J., Wu, H., Castro, D., Sobeih, M. M., Corfas, G., Gleeson, J. G., Greenberg, M. E., Guillemot, F., and Sun, Y. E. (2006) Coupling of cell migration with neurogenesis by proneural bHLH factors. Proc Natl Acad Sci U S A 103, 1319–24.PubMedCrossRefGoogle Scholar
  22. 22.
    Nakano, I., Dougherty, J. D., Kim, K. J., Klement, I., Geschwind, D. H., and Kornblum, H. I. (2007) Phosphoserine phosphatase is expressed in the neural stem cell niche and regulates neural stem and progenitor cell proliferation. Stem Cells 25, 1975–84PubMedCrossRefGoogle Scholar
  23. 23.
    Capela, A., and Temple, S. (2006) LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1. Dev Biol 291, 300–13.PubMedCrossRefGoogle Scholar
  24. 24.
    Groszer, M., Erickson, R., Scripture-Adams, D. D., Dougherty, J. D., Le Belle, J., Zack, J. A., Geschwind, D. H., Liu, X., Kornblum, H. I., and Wu, H. (2006) PTEN negatively regulates neural stem cell self-renewal by modulating G0-G1 cell cycle entry. Proc Natl Acad Sci U S A 103, 111–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Groszer, M., Erickson, R., Scripture-Adams, D. D., Lesche, R., Trumpp, A., Zack, J. A., Kornblum, H. I., Liu, X., and Wu, H. (2001) Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 294, 2186–9.PubMedCrossRefGoogle Scholar
  26. 26.
    Chow, L. M., and Baker, S. J. (2006) PTEN function in normal and neoplastic growth. Cancer Lett 241, 184–96.PubMedCrossRefGoogle Scholar
  27. 27.
    Ohgaki, H. (2005) Genetic pathways to glioblastomas. Neuropathology 25, 1–7.PubMedCrossRefGoogle Scholar
  28. 28.
    Mischel, P. S., and Cloughesy, T. F. (2003) Targeted molecular therapy of GBM. Brain Pathol 13, 52–61.PubMedCrossRefGoogle Scholar
  29. 29.
    Li, Q., Ford, M. C., Lavik, E. B., and Madri, J. A. (2006) Modeling the neurovascular niche: VEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells: an in vitro study. J Neurosci Res 84, 1656–68.PubMedCrossRefGoogle Scholar
  30. 30.
    Jung, K. H., Chu, K., Lee, S. T., Kim, S. J., Sinn, D. I., Kim, S. U., Kim, M., and Roh, J. K. (2006) Granulocyte colony-stimulating factor stimulates neurogenesis via vascular endothelial growth factor with STAT activation. Brain Res 1073–1074, 190–201.PubMedCrossRefGoogle Scholar
  31. 31.
    Jackson, E. L., Garcia-Verdugo, J. M., Gil-Perotin, S., Roy, M., Quinones-Hinojosa, A., VandenBerg, S., and Alvarez-Buylla, A. (2006) PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling. Neuron 51, 187–99.PubMedCrossRefGoogle Scholar
  32. 32.
    Easterday, M. C., Dougherty, J. D., Jackson, R. L., Ou, J., Nakano, I., Paucar, A. A., Roobini, B., Dianati, M., Irvin, D. K., Weissman, I. L., Terskikh, A. V., Geschwind, D. H., and Kornblum, H. I. (2003) Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations. Dev Biol 264, 309–22.PubMedCrossRefGoogle Scholar
  33. 33.
    Terskikh, A. V., Easterday, M. C., Li, L., Hood, L., Kornblum, H. I., Geschwind, D. H., and Weissman, I. L. (2001) From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc Natl Acad Sci U S A 98, 7934–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Geschwind, D. H., Ou, J., Easterday, M. C., Dougherty, J. D., Jackson, R. L., Chen, Z., Antoine, H., Terskikh, A., Weissman, I. L., Nelson, S. F., and Kornblum, H. I. (2001) A genetic analysis of neural progenitor differentiation. Neuron 29, 325–39.PubMedCrossRefGoogle Scholar
  35. 35.
    Dougherty, J. D., Garcia, A. D., Nakano, I., Livingstone, M., Norris, B., Polakiewicz, R., Wexler, E. M., Sofroniew, M. V., Kornblum, H. I., and Geschwind, D. H. (2005) PBK/TOPK, a proliferating neural progenitor-specific mitogen-activated protein kinase kinase. J Neurosci 25, 10773–85.PubMedCrossRefGoogle Scholar
  36. 36.
    Wong, M. L., Kaye, A. H., and Hovens, C. M. (2007) Targeting malignant glioma survival signalling to improve clinical outcomes. J Clin Neurosci 14, 301–8.PubMedCrossRefGoogle Scholar
  37. 37.
    Salhia, B., Tran, N. L., Symons, M., Winkles, J. A., Rutka, J. T., and Berens, M. E. (2006) Molecular pathways triggering glioma cell invasion. Expert Rev Mol Diagn 6, 613–26.PubMedCrossRefGoogle Scholar
  38. 38.
    Tomera, J. F. (2000) Glioma: Novel considerations and treatment modalities. Drugs Today (Barc) 36, 355–67.Google Scholar
  39. 39.
    Ware, M. L., Berger, M. S., and Binder, D. K. (2003) Molecular biology of glioma tumorigenesis. Histol Histopathol 18, 207–16.PubMedGoogle Scholar
  40. 40.
    Fox, I. J., Paucar, A. A., Nakano, I., Mottahedeh, J., Dougherty, J. D., and Kornblum, H. I. (2004) Developmental expression of glial fibrillary acidic protein mRNA in mouse forebrain germinal zones – implications for stem cell biology. Brain Res Dev Brain Res 153, 121–5.PubMedCrossRefGoogle Scholar
  41. 41.
    Mellinghoff, I. K., Wang, M. Y., Vivanco, I., Haas-Kogan, D. A., Zhu, S., Dia, E. Q., Lu, K. V., Yoshimoto, K., Huang, J. H., Chute, D. J., Riggs, B. L., Horvath, S., Liau, L. M., Cavenee, W. K., Rao, P. N., Beroukhim, R., Peck, T. C., Lee, J. C., Sellers, W. R., Stokoe, D., Prados, M., Cloughesy, T. F., Sawyers, C. L., and Mischel, P. S. (2005) Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 353, 2012–24.PubMedCrossRefGoogle Scholar
  42. 42.
    Horvath, S., Zhang, B., Carlson, M., Lu, K. V., Zhu, S., Felciano, R. M., Laurance, M. F., Zhao, W., Qi, S., Chen, Z., Lee, Y., Scheck, A. C., Liau, L. M., Wu, H., Geschwind, D. H., Febbo, P. G., Kornblum, H. I., Cloughesy, T. F., Nelson, S. F., and Mischel, P. S. (2006) Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target. Proc Natl Acad Sci U S A 103, 17402–7.PubMedCrossRefGoogle Scholar
  43. 43.
    Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., and Dirks, P. B. (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821–8.PubMedGoogle Scholar
  44. 44.
    Torroglosa, A., Murillo-Carretero, M., Romero-Grimaldi, C., Matarredona, E. R., Campos-Caro, A., and Estrada, C. (2007) Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway. Stem Cells 25, 88–97.PubMedCrossRefGoogle Scholar
  45. 45.
    Imura, T., Nakano, I., Kornblum, H. I., and Sofroniew, M. V. (2006) Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes. Glia 53, 277–93.PubMedCrossRefGoogle Scholar
  46. 46.
    Cheng, J. C., Horwitz, E. M., Karsten, S. L., Shoemaker, L., Kornblum, H. I., Malik, P., and Sakamoto, K. M. (2007) Report on the workshop “New Technologies in Stem Cell Research,” Society for Pediatric Research, San Francisco, California, April 29, 2006. Stem Cells 25, 1070–88.PubMedCrossRefGoogle Scholar
  47. 47.
    Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X., Fan, G., and Greenberg, M. E. (2001) Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104, 365–76.PubMedCrossRefGoogle Scholar
  48. 48.
    Wachs, F. P., Couillard-Despres, S., Engelhardt, M., Wilhelm, D., Ploetz, S., Vroemen, M., Kaesbauer, J., Uyanik, G., Klucken, J., Karl, C., Tebbing, J., Svendsen, C., Weidner, N., Kuhn, H. G., Winkler, J., and Aigner, L. (2003) High efficacy of clonal growth and expansion of adult neural stem cells. Lab Invest 83, 949–62.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ichiro Nakano
    • 1
  • Harley I. Kornblum
    • 2
  1. 1.Departments of Neurosurgery and PediatricsDavid Geffen School of Medicine at UCLALos AngelesUSA
  2. 2.Pediatrics, Pharmacology, and PsychiatryDavid Geffen School of Medicine at UCLALos AngelesUSA

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