Stem Cell Reviews and Reports

, Volume 7, Issue 4, pp 815–835 | Cite as

Identity, Fate and Potential of Cells Grown as Neurospheres: Species Matters

  • Carolin Steffenhagen
  • Sabrina Kraus
  • Franz-Xaver Dechant
  • Mahesh Kandasamy
  • Bernadette Lehner
  • Anne-Maria Poehler
  • Tanja Furtner
  • Florian A. Siebzehnrubl
  • Sebastien Couillard-Despres
  • Olaf Strauss
  • Ludwig Aigner
  • Francisco J. Rivera


It is commonly accepted that adult neurogenesis and gliogenesis follow the same principles through the mammalian class. However, it has been reported that neurogenesis might differ between species, even from the same order, like in rodents. Currently, it is not known if neural stem/progenitor cells (NSPCs) from various species differ in their cell identity and potential. NSPCs can be expanded ex vivo as neurospheres (NSph), a model widely used to study neurogenesis in vitro. Here we demonstrate that rat (r) and mouse (m) NSph display different cell identities, differentiation fate, electrophysiological function and tumorigenic potential. Adult rNSph consist mainly of oligodendroglial progenitors (OPCs), which after repeated passaging proliferate independent of mitogens, whereas adult mNSph show astroglial precursor-like characteristics and retain their mitogen dependency. Most of the cells in rNSph express OPC markers and spontaneously differentiate into oligodendrocytes after growth factor withdrawal. Electrophysiological analysis confirmed OPC characteristics. mNSph have different electrophysiological properties, they express astrocyte precursor markers and spontaneously differentiate primarily into astrocytes. Furthermore, rNSph have the potential to differentiate into oligodendrocytes and astrocytes, whereas mNSph are restricted to the astrocytic lineage. The phenotypic differences between rNSph and mNSph were not due to a distinct response to species specific derived growth factors and are probably not caused by autocrine mechanisms. Our findings suggest that NSph derived from adult rat and mouse brains display different cell identities. Thus, results urge for caution when data derived from NSph are extrapolated to other species or to the in vivo situation, especially when aimed towards the clinical use of human NSph.


Adult neural stem cells Cell phenotype Differentiation potential Cell fate Glial progenitor cells 



The authors would like to thank the following funding agencies for their support: the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell grant to C.S. and A.-M.P.), the Germany Federal Ministry of Education and Research (BMBF grants #01GG0706, #01GN0979; #0312134; #01GN0505; NGFNplus Brain Tumor Network. Subproject 7 #01GS0887), Alexander von Humboldt Foundation (Georg Forster Program to F.J.R.), Deutsche Forschungsgesellschaft (DFG grant #AI31/3-1, #AI31/4-1) and by the state of Salzburg. We disclose any conflict of interest.


The authors indicate no potential conflicts of interest.


  1. 1.
    Gage, F. H., Ray, J., & Fisher, L. J. (1995). Isolation, characterization, and use of stem cells from the CNS. Annual Review of Neuroscience, 18, 159–192.PubMedCrossRefGoogle Scholar
  2. 2.
    Palmer, T. D., Markakis, E. A., Willhoite, A. R., Safar, F., & Gage, F. H. (1999). Fibroblast Growth Factor-2 Activates a Latent Neurogenic Program in Neural Stem Cells from Diverse Regions of the Adult CNS. The Journal of Neuroscience, 19, 8487–8497.PubMedGoogle Scholar
  3. 3.
    Palmer, T. D., Ray, J., & Gage, F. H. (1995). FGF-2-Responsive Neuronal Progenitors Reside in Proliferative and Quiescent Regions of the Adult Rodent Brain. Molecular and Cellular Neuroscience, 6, 474–486.PubMedCrossRefGoogle Scholar
  4. 4.
    Reynolds, B. A., & Weiss, S. (1992). Generation of Neurons and Astrocytes from Isolated Cells of the Adult Mammalian Central-Nervous-System. Science, 255, 1707–1710.PubMedCrossRefGoogle Scholar
  5. 5.
    Wachs, F. P., Couillard-Despres, S., Engelhardt, M., et al. (2003). High efficacy of clonal growth and expansion of adult neural stem cells. Laboratory Investigation, 83, 949–962.PubMedCrossRefGoogle Scholar
  6. 6.
    Reynolds, B. A., & Rietze, R. L. (2005). Neural stem cells and neurospheres–re-evaluating the relationship. Natural Methods, 2, 333–336.CrossRefGoogle Scholar
  7. 7.
    Gritti, A., Parati, E. A., Cova, L., et al. (1996). Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. The Journal of Neuroscience, 16, 1091–1100.PubMedGoogle Scholar
  8. 8.
    Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M., & McKay, R. D. (1996). Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes & Development, 10, 3129–3140.CrossRefGoogle Scholar
  9. 9.
    Takahashi, J., Palmer, T. D., & Gage, F. H. (1999). Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. Journal of Neurobiology, 38, 65–81.PubMedCrossRefGoogle Scholar
  10. 10.
    Gross, R. E., Mehler, M. F., Mabie, P. C., Zang, Z., Santschi, L., & Kessler, J. A. (1996). Bone Morphogenetic Proteins Promote Astroglial Lineage Commitment by Mammalian Subventricular Zone Progenitor Cells. Neuron, 17, 595–606.PubMedCrossRefGoogle Scholar
  11. 11.
    Nakashima, K., Yanagisawa, M., Arakawa, H., & Taga, T. (1999). Astrocyte differentiation mediated by LIF in cooperation with BMP2. FEBS Letters, 457, 43–46.PubMedCrossRefGoogle Scholar
  12. 12.
    Hsieh, J., Aimone, J. B., Kaspar, B. K., Kuwabara, T., Nakashima, K., & Gage, F. H. (2004). IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes. The Journal of Cell Biology, 164, 111–122.PubMedCrossRefGoogle Scholar
  13. 13.
    Rivera, F. J., Couillard-Despres, S., Pedre, X., et al. (2006). Mesenchymal Stem Cells Instruct Oligodendrogenic Fate Decision on Adult Neural Stem Cells. Stem Cells, 24, 2209–2219.PubMedCrossRefGoogle Scholar
  14. 14.
    Gage, F. H. (2000). Mammalian Neural Stem Cells. Science, 287, 1433–1438.PubMedCrossRefGoogle Scholar
  15. 15.
    Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., et al. (1998). Neurogenesis in the adult human hippocampus. Natural Medicines, 4, 1313–1317.CrossRefGoogle Scholar
  16. 16.
    Snyder, J. S., Choe, J. S., Clifford, M. A., et al. (2009). Adult-born hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice. The Journal of Neuroscience, 29, 14484–14495.PubMedCrossRefGoogle Scholar
  17. 17.
    Gil-Mohapel, J., Simpson, J. M., Titterness, A. K., & Christie, B. R. (2010). Characterization of the neurogenesis quiescent zone in the rodent brain: Effects of age and exercise. The European Journal of Neuroscience, 31, 797–807.PubMedCrossRefGoogle Scholar
  18. 18.
    Melvin, N. R., Spanswick, S. C., Lehmann, H., & Sutherland, R. J. (2007). Differential neurogenesis in the adult rat dentate gyrus: An identifiable zone that consistently lacks neurogenesis. The European Journal of Neuroscience, 25, 1023–1029.PubMedCrossRefGoogle Scholar
  19. 19.
    Dziembowska, M., Tham, T. N., Lau, P., Vitry, S., Lazarini, F., & Dubois-Dalcq, M. (2005). A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors. Glia, 50, 258–269.PubMedCrossRefGoogle Scholar
  20. 20.
    Gong, X., He, X., Qi, L., Zuo, H., & Xie, Z. (2006). Stromal cell derived factor-1 acutely promotes neural progenitor cell proliferation in vitro by a mechanism involving the ERK1/2 and PI-3 K signal pathways. Cell Biology International, 30, 466–471.PubMedCrossRefGoogle Scholar
  21. 21.
    Bauer, S., & Patterson, P. H. (2006). Leukemia inhibitory factor promotes neural stem cell self-renewal in the adult brain. The Journal of Neuroscience, 26, 12089–12099.PubMedCrossRefGoogle Scholar
  22. 22.
    Dictus, C., Tronnier, V., Unterberg, A., & Herold-Mende, C. (2007). Comparative analysis of in vitro conditions for rat adult neural progenitor cells. Journal of Neuroscience Methods, 161, 250–258.PubMedCrossRefGoogle Scholar
  23. 23.
    Ray, J., & Gage, F. H. (2006). Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells. Molecular and Cellular Neurosciences, 31, 560–573.PubMedCrossRefGoogle Scholar
  24. 24.
    Ginis, I., & Rao, M. S. (2003). Toward cell replacement therapy: Promises and caveats. Experimental Neurology, 184, 61–77.PubMedCrossRefGoogle Scholar
  25. 25.
    Ginis, I., Luo, Y., Miura, T., et al. (2004). Differences between human and mouse embryonic stem cells. Developmental Biology, 269, 360–380.PubMedCrossRefGoogle Scholar
  26. 26.
    Rao, M. (2004). Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Developmental Biology, 275, 269–286.PubMedCrossRefGoogle Scholar
  27. 27.
    Theise, N. D. (2005). On experimental design and discourse in plasticity research. Stem Cell Reviews, 1, 9–13.PubMedCrossRefGoogle Scholar
  28. 28.
    Glover, J. C., Boulland, J. L., Halasi, G., & Kasumacic, N. (2009). Chimeric animal models in human stem cell biology. ILAR Journal, 51, 62–73.PubMedGoogle Scholar
  29. 29.
    Endl, E., Steinbach, P., Knuchel, R., & Hofstadter, F. (1997). Analysis of cell cycle-related Ki-67 and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling technique. Cytometry, 29, 233–241.PubMedCrossRefGoogle Scholar
  30. 30.
    Amit, M., Carpenter, M. K., Inokuma, M. S., et al. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology, 227, 271–278.PubMedCrossRefGoogle Scholar
  31. 31.
    Cheng, J., Dutra, A., Takesono, A., Garrett-Beal, L., & Schwartzberg, P. L. (2004). Improved generation of C57BL/6 J mouse embryonic stem cells in a defined serum-free media. Genesis, 39, 100–104.PubMedCrossRefGoogle Scholar
  32. 32.
    Smith, A. G., Heath, J. K., Donaldson, D. D., et al. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature, 336, 688–690.PubMedCrossRefGoogle Scholar
  33. 33.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods, 25, 402–408.PubMedCrossRefGoogle Scholar
  34. 34.
    Raff, M. C. (1984). Williams BPa, Miller RH. The in vitro differentiation of a bipotential glial progenitor cell. The EMBO Journal, 3, 1857–1864.PubMedGoogle Scholar
  35. 35.
    Ahlgren, S. C., Wallace, H., Bishop, J., Neophytou, C., & Raff, M. C. (1997). Effects of Thyroid Hormone on Embryonic Oligodendrocyte Precursor Cell Developmentin Vivoandin Vitro. Molecular and Cellular Neuroscience, 9, 420–432.PubMedCrossRefGoogle Scholar
  36. 36.
    Calver, A. R., Hall, A. C., Yu, W.-P., et al. (1998). Oligodendrocyte Population Dynamics and the Role of PDGF In Vivo. Neuron, 20, 869–882.PubMedCrossRefGoogle Scholar
  37. 37.
    Davies, J. E., & Miller, R. H. (2001). Local Sonic Hedgehog Signaling Regulates Oligodendrocyte Precursor Appearance in Multiple Ventricular Zone Domains in the Chick Metencephalon. Developmental Biology, 233, 513–525.PubMedCrossRefGoogle Scholar
  38. 38.
    Fruttiger, M., Karlsson, L., Hall, A. C., et al. (1999). Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice. Development, 126, 457–467.PubMedGoogle Scholar
  39. 39.
    Koper, J., Hoeben, R., Hochstenbach, F., van Golde, L., & Lopes-Cardozo, M. (1986). Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures. Biochimica et Biophysica Acta, 887, 327–334.PubMedCrossRefGoogle Scholar
  40. 40.
    Samanta, J., & Kessler, J. A. (2004). Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation. Development, 131, 4131–4142.PubMedCrossRefGoogle Scholar
  41. 41.
    Zhu, G., Mehler, M. F., & Zhao, J. (1999). Yu Yung S, Kessler JA. Sonic Hedgehog and BMP2 Exert Opposing Actions on Proliferation and Differentiation of Embryonic Neural Progenitor Cells. Developmental Biology, 215, 118–129.PubMedCrossRefGoogle Scholar
  42. 42.
    Siebzehnrubl, F. A., Jeske, I., Muller, D., et al. (2009). Spontaneous in vitro transformation of adult neural precursors into stem-like cancer cells. Brain Pathology, 19, 399–408.PubMedCrossRefGoogle Scholar
  43. 43.
    Dascal, N., & Lotan, I. (1991). Activation of protein kinase C alters voltage dependence of a Na + channel. Neuron, 6, 165–175.PubMedCrossRefGoogle Scholar
  44. 44.
    Jia, Z., Jia, Y., Liu, B., et al. (2008). Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms. Pflugers Arch European Journal of Physiology, 456, 857–866.CrossRefGoogle Scholar
  45. 45.
    Filippov, V., Kronenberg, G., Pivneva, T., et al. (2003). Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Molecular and Cellular Neurosciences, 23, 373–382.PubMedCrossRefGoogle Scholar
  46. 46.
    Kronenberg, G., Wang, L. P., Geraerts, M., et al. (2007). Local origin and activity-dependent generation of nestin-expressing protoplasmic astrocytes in CA1. Brain Structure & Function, 212, 19–35.CrossRefGoogle Scholar
  47. 47.
    Matthias, K., Kirchhoff, F., Seifert, G., et al. (2003). Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. The Journal of Neuroscience, 23, 1750–1758.PubMedGoogle Scholar
  48. 48.
    Sontheimer, H., Perouansky, M., Hoppe, D., Lux, H. D., Grantyn, R., & Kettenmann, H. (1989). Glial cells of the oligodendrocyte lineage express proton-activated Na + channels. Journal of Neuroscience Research, 24, 496–500.PubMedCrossRefGoogle Scholar
  49. 49.
    Brown, J. P., Couillard-Despres, S., Cooper-Kuhn, C. M., Winkler, J., Aigner, L., & Kuhn, H. G. (2003). Transient expression of doublecortin during adult neurogenesis. The Journal of Comparative Neurology, 467, 1–10.PubMedCrossRefGoogle Scholar
  50. 50.
    Couillard-Despres, S., Winner, B., Schaubeck, S., et al. (2005). Doublecortin expression levels in adult brain reflect neurogenesis. The European Journal of Neuroscience, 21, 1–14.PubMedCrossRefGoogle Scholar
  51. 51.
    Tamura, Y., Kataoka, Y., Cui, Y., Takamori, Y., Watanabe, Y., & Yamada, H. (2007). Multi-directional differentiation of doublecortin- and NG2-immunopositive progenitor cells in the adult rat neocortex in vivo. The European Journal of Neuroscience, 25, 3489–3498.PubMedCrossRefGoogle Scholar
  52. 52.
    Guo, F., Ma, J., McCauley, E., Bannerman, P., & Pleasure, D. (2009). Early postnatal proteolipid promoter-expressing progenitors produce multilineage cells in vivo. The Journal of Neuroscience, 29, 7256–7270.PubMedCrossRefGoogle Scholar
  53. 53.
    Singh, S. K., Hawkins, C., Clarke, I. D., et al. (2004). Identification of human brain tumour initiating cells. Nature, 432, 396–401.PubMedCrossRefGoogle Scholar
  54. 54.
    Sutter, R., Shakhova, O., Bhagat, H., et al. (2010). Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas. Oncogene, 29, 1845–1856.PubMedCrossRefGoogle Scholar
  55. 55.
    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 Research, 67, 4010–4015.PubMedCrossRefGoogle Scholar
  56. 56.
    Lindberg, N., Kastemar, M., Olofsson, T., Smits, A., & Uhrbom, L. (2009). Oligodendrocyte progenitor cells can act as cell of origin for experimental glioma. Oncogene, 28, 2266–2275.PubMedCrossRefGoogle Scholar
  57. 57.
    Djojosubroto, M., Bollotte, F., Wirapati, P., Radtke, F., Stamenkovic, I., & Arsenijevic, Y. (2009). Chromosomal number aberrations and transformation in adult mouse retinal stem cells in vitro. Investigative Ophthalmology Visual Science, 50, 5975–5987.PubMedCrossRefGoogle Scholar
  58. 58.
    McBurney, M. W. (1976). Clonal lines of teratocarcinoma cells in vitro: Differentiation and cytogenetic characteristics. Journal of Cellular Physiology, 89, 441–455.PubMedCrossRefGoogle Scholar
  59. 59.
    Park, J., Yoshida, I., Tada, T., Takagi, N., Takahashi, Y., & Kanagawa, H. (1998). Trisomy 8 does not affect differentiative potential in a murine parthenogenetic embryonic stem cell line. The Japanese Journal of Veterinary Research, 46, 29–35.PubMedGoogle Scholar
  60. 60.
    Ray, J., & Gage, F. H. (2006). Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells. Molecular and Cellular Neuroscience, 31, 560–573.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Carolin Steffenhagen
    • 1
    • 2
  • Sabrina Kraus
    • 3
  • Franz-Xaver Dechant
    • 2
  • Mahesh Kandasamy
    • 1
  • Bernadette Lehner
    • 2
  • Anne-Maria Poehler
    • 4
  • Tanja Furtner
    • 1
  • Florian A. Siebzehnrubl
    • 5
  • Sebastien Couillard-Despres
    • 1
  • Olaf Strauss
    • 3
  • Ludwig Aigner
    • 1
  • Francisco J. Rivera
    • 1
  1. 1.Institute of Molecular Regenerative MedicineParacelsus Medical University SalzburgSalzburgAustria
  2. 2.Department of NeurologyUniversity of RegensburgRegensburgGermany
  3. 3.Department of OphthalmologyUniversity of RegensburgRegensburgGermany
  4. 4.Division of Molecular NeurologyUniversity Hospital of ErlangenErlangenGermany
  5. 5.Department of Neuropathology. Franz-Penzoldt-CenterUniversity of Erlangen-NurembergNurembergGermany

Personalised recommendations