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Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology

Nature Methods volume 3, pages 801–806 (2006)Cite this article

Abstract

For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.

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Figure 1: Characterization of typical neurospheres isolated from the postnatal mouse forebrain and expanded in bFGF and EGF.
Figure 2: Frequent, rapid and multiple 'coalescence' of secondary neurospheres.
Figure 3: Chimeric neurospheres ultimately predominate in cocultures of spheres from EGFP+ and β-gal+ mice.
Figure 4: Cocultured human and mouse neurospheres display chimerism.

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References

  1. Reynolds, B.A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).

    Article  CAS  Google Scholar 

  2. Reynolds, B.A., Tetzlaff, W. & Weiss, S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12, 4565–4574 (1992).

    Article  CAS  Google Scholar 

  3. Reynolds, B.A. & Weiss, S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175, 1–13 (1996).

    Article  CAS  Google Scholar 

  4. Craig, C.G. et al. In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J. Neurosci. 16, 2649–2658 (1996).

    Article  CAS  Google Scholar 

  5. Represa, A., Shimazaki, T., Simmonds, M. & Weiss, S. EGF-responsive neural stem cells are a transient population in the developing mouse spinal cord. Eur. J. Neurosci. 14, 452–462 (2001).

    Article  CAS  Google Scholar 

  6. Chojnacki, A. & Weiss, S. Isolation of a novel platelet-derived growth factor-responsive precursor from the embryonic ventral forebrain. J. Neurosci. 24, 10888–10899 (2004).

    Article  CAS  Google Scholar 

  7. Tropepe, V. et al. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev. Biol. 208, 166–188 (1999).

    Article  CAS  Google Scholar 

  8. Tropepe, V. et al. Retinal stem cells in the adult mammalian eye. Science 287, 2032–2036 (2000).

    Article  CAS  Google Scholar 

  9. Seaberg, R.M. & van der Kooy, D. Adult neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J. Neurosci. 22, 1784–1793 (2002).

    Article  CAS  Google Scholar 

  10. Ramirez-Castillejo, C. et al. Pigment epithelium-derived factor is a niche signal for neural stem cell renewal. Nat. Neurosci. 9, 331–339 (2006).

    Article  CAS  Google Scholar 

  11. Hitoshi, S. et al. Primitive neural stem cells from the mammalian epiblast differentiate to definitive neural stem cells under the control of Notch signalling. Genes Dev. 18, 1806–1811 (2004).

    Article  CAS  Google Scholar 

  12. Kippin, T.E., Martens, D.J. & van der Kooy, D. p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity. Genes Dev. 19, 756–767 (2005).

    Article  CAS  Google Scholar 

  13. Kippin, T.E., Kapur, S. & van der Kooy, D. Dopamine specifically inhibits forebrain neural stem cell proliferation, suggesting a novel effect of antipsychotic drugs. J. Neurosci. 25, 5815–5823 (2005).

    Article  CAS  Google Scholar 

  14. Kukekov, V.G., Laywell, E.D., Thomas, L.B. & Steindler, D.A. A nestin-negativ precursor cell from the adult mouse brain gives rise to neurons and glia. Glia 21, 399–407 (1997).

    Article  CAS  Google Scholar 

  15. Suslov, O.N., Kukekov, V.G., Ignatova, T.N. & Steindler, D.A. Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc. Natl. Acad. Sci. USA 99, 14506–14511 (2002).

    Article  CAS  Google Scholar 

  16. Imura, T., Kornblum, H.I. & Sofroniew, M.V. The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP. J. Neurosci. 23, 2824–2832 (2003).

    Article  CAS  Google Scholar 

  17. Kim, M. & Morshead, C.M. Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis. J. Neurosci. 23, 10703–10709 (2003).

    Article  CAS  Google Scholar 

  18. Morshead, C.M., Garcia, A.D., Sofroniew, M.V. & van der Kooy, D. The ablation of glial fibrillary acidic protein-positive cells from the adult central nervous system results in the loss of forebrain neural stem cells but not retinal stem cells. Eur. J. Neurosci. 18, 76–84 (2003).

    Article  Google Scholar 

  19. Molofsky, A.V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003).

    Article  CAS  Google Scholar 

  20. Nunes, M.C. et al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat. Med. 9, 439–447 (2003).

    Article  CAS  Google Scholar 

  21. Zhang, S.C., Lipsitz, D. & Duncan, I.D. Self-renewing canine oligodendroglial progenitor expanded as oligospheres. J. Neurosci. Res. 54, 181–190 (1998).

    Article  CAS  Google Scholar 

  22. Seaberg, R.M. et al. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat. Biotechnol. 22, 1115–1124 (2004).

    Article  CAS  Google Scholar 

  23. Toma, J.G. et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol. 3, 778–784 (2001).

    Article  CAS  Google Scholar 

  24. Matsuda, M. et al. Serotonin regulates mammary gland development via an autocrine-paracrine loop. Dev. Cell 6, 193–203 (2004).

    Article  CAS  Google Scholar 

  25. Hemmati, H.D. et al. Cancerous stem cells can arise from pediatric brain tumors. Proc. Natl. Acad. Sci. USA 100, 15178–15183 (2003).

    Article  CAS  Google Scholar 

  26. Snyder, E.Y. et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68, 33–51 (1992).

    Article  CAS  Google Scholar 

  27. Parker, M.A. et al. Expression profile of an operationally-defined neural stem cell clone. Exp. Neurol. 194, 320–332 (2005).

    Article  CAS  Google Scholar 

  28. Gritti, A. et al. Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J. Neurosci. 22, 437–445 (2002).

    Article  CAS  Google Scholar 

  29. Lobo, M.V. et al. Cellular characterization of epidermal growth factor-expanded free-floating neurospheres. J. Histochem. Cytochem. 51, 89–103 (2003).

    Article  Google Scholar 

  30. Morshead, C.M., Benveniste, P., Iscove, N.N. & van der Kooy, D. Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nat. Med. 8, 268–273 (2002).

    Article  CAS  Google Scholar 

  31. Wilson, H.V. On some phenomena of coalescence and regeneration in sponges. J. Exp. Zool. 5, 245–258 (1907).

    Article  Google Scholar 

  32. Studer, L., Tabar, V. & McKay, R.D.G. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat. Neurosci. 1, 290–295 (1998).

    Article  CAS  Google Scholar 

  33. Garcia, A.D., Doan, N.B., Imura, T., Bush, T.G. & Sofroniew, M.V. GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat. Neurosci. 7, 1233–1241 (2004).

    Article  CAS  Google Scholar 

  34. Reynolds, B.A. & Rietze, R.L. Neural stem cells and neurospheres – re-evaluating the relationship. Nat. Methods 2, 333–336 (2005).

    Article  CAS  Google Scholar 

  35. Gritti, A. et al. Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J. Neurosci. 19, 3287–3297 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the contributions of M. Ditter, C.E. Hagemeyer, A. Papazoglou, A. Klein and A. Fahrner. We thank W. Wünsch for his help digitizing the videos. We also thank J. Loring for technical help with Supplementary Video 6 and A. Eroshkin for discussion on the mathematical model in the Supplementary Note. This work was supported by the Deutsche Forschungsgemeinschaft (Si874/2-1), the US National Institutes of Health, the American Parkinson's Disease Association, Children's Neurobiological Solutions, the March of Dimes, Margot Anderson Brain Restoration Foundation and Project ALS.

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Authors and Affiliations

  1. Institute of Anatomy and Cell Biology, University of Freiburg, Albertstr. 17, Freiburg, D-79104, Germany

    Ilyas Singec & Michael Frotscher

  2. Department of Neuropathology, Neurocenter, University of Freiburg, Breisacher Str. 64, Freiburg, D-79106, Germany

    Ilyas Singec, Rolf Knoth, Ralf P Meyer & Benedikt Volk

  3. Department of Stereotactic Neurosurgery, Neurocenter, University of Freiburg, Breisacher Str. 64, Freiburg, D-79106, Germany

    Jaroslaw Maciaczyk & Guido Nikkhah

  4. Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    Ilyas Singec & Evan Y Snyder

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Supplementary information

Supplementary Fig. 1

Beating cellular specializations of neurosphere cells. (PDF 102 kb)

Supplementary Note

Mathematical analysis of proliferative cells in secondary neurospheres. (PDF 131 kb)

Supplementary Video 1

Representative time-lapse video microscopy demonstrating frequent, rapid, and multiple “coalescence” of secondary neurospheres. Note that neurospheres are capable of active locomotion, apparently propelled by tiny beating cellular processes. The movie shows a ∼2.5 hr segment of the 10-hr time-lapse recording excerpted as well in Fig. 2. The video recorder captured images every 3.5 seconds. This neurosphere culture was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of 50 cells/μl and 104 cells/cm2 (standard “medium density”), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO2 at the time of this videomicrograph. For illustrative purposes, this movie presents an “overview” of a “medium” cell density culture in a 6-well plate. Numerous time-lapse video-microscopic recordings confirmed merging of spheres grown at even “clonal” cell density (Supplementary Video 2 online). The beating cellular processes are better appreciated at higher power in Supplementary Video 3 online. (MOV 1971 kb)

Supplementary Video 2

Time-lapse video-microscopy demonstrating, at higher magnification, neurosphere locomotion and “coalescence” despite plating at “clonal” density. Cellular processes (see also Supplemental Fig. 1 and Supplemental Video 3) propel neurospheres over considerable distances increasing the probability of coalescence events in even low density cultures. The movie shows a time-lapse recording of ∼2 hr. This neurosphere culture (24-well plate) was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of only 5 cells/μl and 103 cells/cm2 (“clonal” density), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO2 at the time of this videomicrograph. (MOV 676 kb)

Supplementary Video 3

Time-lapse video microscopy of human fetal cortex-derived neurospheres at higher magnification. Note the tiny beating cellular processes covering the surface of two merging human neurospheres. These beating structures were abundant on human and rodent neurospheres irrespective of their origin (e.g. cortex, striatum, spinal cord) and seem to propel them in suspension culture. (MOV 9235 kb)

Supplementary Video 4

Time-lapse video microscopy of human neurospheres grown in 96-well plates. Neurospheres are motile and merge in 96-well plates similar to the findings obtained in 6-well (Video 1) and 24-well (Video 2) plates. In this experiment with 96-well plates, the initial plating density was 10 cells/μl or 2000 cells/well (personal communication, B. Reynolds), but nevertheless still generated polyclonal spheres. Note that movie speed here is 5 times faster than in the other Videos. (MOV 6887 kb)

Supplementary Video 5

Time-lapse video microscopy of mouse cell culture showing neurosphere fusion and interaction of motile spheres with single cells attached to uncoated plastic dish. This example shows, besides the merging of two neurospheres, that single cells can attach to or detach from a motile neurosphere compromising their clonal boundaries even at “clonal” plating density (5 cells/μl). (MOV 2174 kb)

Supplementary Video 6

Time-lapse video microscopy of neurosphere-like cells that have become adherent. Note that adherent spheres, too, migrate and coalesce. Only if this concentrated culture dish had begun as a single isolated cell in a single isolated miniwell would an assumption of clonal boundaries and identity be justified and legitimate. In this experiment mouse forebrain stem cells (20 cells/μl) were plated into 24-well plates with glass coverslips coated with poly-L-lysine and laminin. The videomicrograph was taken on day 7. (MOV 5136 kb)

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Singec, I., Knoth, R., Meyer, R. et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 3, 801–806 (2006). https://doi.org/10.1038/nmeth926

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