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The neurosphere assay, a method under scrutiny

Published online by Cambridge University Press:  24 June 2014

Loic P. Deleyrolle*
Affiliation:
Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia
Rodney L. Rietze
Affiliation:
Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia
Brent A. Reynolds
Affiliation:
Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia
*
Loic P. Deleyrolle, Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia. Tel: +07 33466300; Fax: +07 33466301; E-mail: [email protected]

Abstract

Objectives:

The aim of this review is to provide an overview of the fundamental features of the neurosphere assay (NSA), which was initially described in 1992, and has since been used not only to detect the presence of stem cells in embryonic and adult mammalian neural tissues, but also to study their characteristics in vitro. Implicit in this review is a detailed examination of the limitations of the NSA, and how this assay is most accurately and appropriately used. Finally we will point out criteria that should be challenged to design alternative ways to overcome the limits of this assay.

Methods:

NSA is used to isolate putative neural stem cells (NSCs) from the central nervous system (CNS) and to demonstrate the critical stem cell attributes of proliferation, extensive self-renewal and the ability to give rise to a large number of differentiated and functional progeny. Nevertheless, the capability of neural progenitor cells to form neurospheres precludes its utilisation to accurately quantify bona fide stem cell frequency based simply on neurosphere numbers. New culture conditions are needed to be able to distinguish the activity of progenitor cells from stem cells.

Conclusion:

A commonly used, and arguably misused, methodology, the NSA has provided a wealth of information on precursor activity of cells derived from the embryonic through to the aged CNS. Importantly, the NSA has contributed to the demise of the ‘no new neurogenesis’ dogma, and the beginning of a new era of CNS regenerative medicine. Nevertheless, the interpretations arising from the utilisation of the NSA need to take into consideration its limits, so as not to be used beyond its specificity and sensitivity.

Type
Review article
Copyright
Copyright © 2007 Blackwell Munksgaard

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References

Evans, GS, Potten, CS. Stem cells and the elixir of life. Bioessays 1991;13:135138. CrossRefGoogle ScholarPubMed
Potten, CS, Loeffler, M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990;110:10011020. Google ScholarPubMed
Reynolds, BA, Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:17071710. CrossRefGoogle ScholarPubMed
Hall, PA, Watt, FM. Stem cells: the generation and maintenance of cellular diversity. Development 1989;106:619633. Google ScholarPubMed
Gritti, A, Cova, L, Parati, EA, Galli, R, Vescovi, AL. Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS. Neurosci Lett 1995;185:151154. CrossRefGoogle Scholar
Gritti, A, Frolichsthal-Schoeller, P, Galli, Ret 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 1999;19:32873297. Google ScholarPubMed
Gritti, A, Parati, EA, Cova, Let al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 1996;16:10911100. Google ScholarPubMed
Reynolds, BA, Weiss, S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996;175:113. CrossRefGoogle ScholarPubMed
Weiss, S, Dunne, C, Hewson, Jet al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 1996;16:75997609. Google ScholarPubMed
Weiss, S, Reynolds, BA, Vescovi, ALet al. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci 1996;19:387393. CrossRefGoogle Scholar
Gross, CG. Neurogenesis in the adult brain: death of a dogma. Nat Rev Neurosci 2000;1:6773. CrossRefGoogle ScholarPubMed
Hitoshi, S, Tropepe, V, Ekker, M, Van der Kooy, D. Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain. Development 2002;129:233244. Google Scholar
Lu, F, Wong, CS. A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation. Radiat Res 2005;163:6371. CrossRefGoogle ScholarPubMed
Yang, Z, Levison, SW. Hypoxia/ischemia expands the regenerative capacity of progenitors in the perinatal subventricular zone. Neuroscience 2006;139:555564. CrossRefGoogle ScholarPubMed
Morshead, CM, Reynolds, BA, Craig, CGet al. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 1994;13:10711082. CrossRefGoogle ScholarPubMed
Maslov, AY, Barone, TA, Plunkett, RJ, Pruitt, SC. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci 2004;24:17261733. CrossRefGoogle Scholar
Marshall, GP II, Scott, EW, Zheng, T, Laywell, ED, Steindler, DA. Ionizing radiation enhances the engraftment of transplanted in vitro-derived multipotent astrocytic stem cells. Stem Cells 2005;23:12761285. CrossRefGoogle ScholarPubMed
Ivanova, NB, Dimos, JT, Schaniel, Cet al. A stem cell molecular signature. Science 2002;298:601604. CrossRefGoogle ScholarPubMed
Ramalho-Santos, M, Yoon, S, Matsuzaki, Y, Mulligan, RC, Melton, DA. “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597600. CrossRefGoogle ScholarPubMed
Enwere, E, Shingo, T, Gregg, Cet al. Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J Neurosci 2004;24:83548365. CrossRefGoogle ScholarPubMed
Kohyama, J, Tokunaga, A, Fujita, Yet al. Visualization of spatiotemporal activation of Notch signaling: live monitoring and significance in neural development. Dev Biol 2005;286:311325. CrossRefGoogle ScholarPubMed
Pitman, M, Emery, B, Binder, Met al. LIF receptor signaling modulates neural stem cell renewal. Mol Cell Neurosci 2004;27:255266. CrossRefGoogle ScholarPubMed
Deleyrolle, L, Marchal-Victorion, S, Dromard, Cet al. Exogenous and fibroblast growth factor 2/epidermal growth factor-regulated endogenous cytokines regulate neural precursor cell growth and differentiation. Stem Cells 2006;24:748762. CrossRefGoogle ScholarPubMed
Gritti, A, Bonfanti, L, Doetsch, Fet al. Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J Neurosci 2002;22:437445. Google ScholarPubMed
Aguirre, AA, Chittajallu, R, Belachew, S, Gallo, V. NG2-expressing cells in the subventricular zone are type C-like cells and contribute to interneuron generation in the postnatal hippocampus. J Cell Biol 2004;165:575589. CrossRefGoogle ScholarPubMed
Doetsch, F, Caille, I, Lim, DA, Garcia-Verdugo, JM, Alvarez-Buylla, A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999;97:703716. CrossRefGoogle ScholarPubMed
Doetsch, F, Petreanu, L, Caille, I, Garcia-Verdugo, JM, Alvarez-Buylla, A. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 2002;36:10211034. CrossRefGoogle ScholarPubMed
Palmer, TD, Markakis, EA, Willhoite, AR, Safar, F, Gage, FH. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 1999;19:84878497. Google ScholarPubMed
Zhao, M, Momma, S, Delfani, Ket al. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 2003;100:79257930. CrossRefGoogle ScholarPubMed
Doetsch, F. A niche for adult neural stem cells. Curr Opin Genet Dev 2003;13:543550. CrossRefGoogle ScholarPubMed
Sanai, N, Tramontin, AD, Quinones-Hinojosa, Aet al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 2004;427:740744. CrossRefGoogle ScholarPubMed
Vescovi, AL, Parati, EA, Gritti, Aet al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 1999;156:7183. CrossRefGoogle ScholarPubMed
Svendsen, CN, Caldwell, MA, Ostenfeld, T. Human neural stem cells: isolation, expansion and transplantation. Brain Pathol 1999;9:499513. CrossRefGoogle ScholarPubMed
Uchida, N, Buck, DW, He, Det al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2000;97:1472014725. CrossRefGoogle ScholarPubMed
Pincus, DW, Harrison-Restelli, C, Barry, Jet al. In vitro neurogenesis by adult human epileptic temporal neocortex. Clin Neurosurg 1997;44:1725. Google ScholarPubMed
Roy, NS, Benraiss, A, Wang, Set al. Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone. J Neurosci Res 2000;59:321331. 3.0.CO;2-9>CrossRefGoogle ScholarPubMed
Johansson, CB, Svensson, M, Wallstedt, L, Janson, AM, Frisen, J. Neural stem cells in the adult human brain. Exp Cell Res 1999;253:733736. CrossRefGoogle ScholarPubMed
Pagano, SF, Impagnatiello, F, Girelli, Met al. Isolation and characterization of neural stem cells from the adult human olfactory bulb. Stem Cells 2000;18:295300. CrossRefGoogle ScholarPubMed
Nunes, MC, Roy, NS, Keyoung, HMet al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 2003;9:439447. CrossRefGoogle ScholarPubMed
Toma, JG, McKenzie, IA, Bagli, D, Miller, FD. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells 2005;23:727737. CrossRefGoogle ScholarPubMed
Messina, E, De Angelis, L, Frati, Get al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res 2004;95:911921. CrossRefGoogle ScholarPubMed
Dontu, G, Abdallah, WM, Foley, JMet al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003;17:12531270. CrossRefGoogle ScholarPubMed
Ignatova, TN, Kukekov, VG, Laywell, EDet al. Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002;39:193206. CrossRefGoogle ScholarPubMed
Singh, SK, Clarke, ID, Terasaki, Met al. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003;63:58215828. Google ScholarPubMed
Hemmati, HD, Nakano, I, Lazareff, JAet al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A 2003;100:1517815183. CrossRefGoogle ScholarPubMed
Galli, R, Binda, E, Orfanelli, Uet al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 2004;64:70117021. CrossRefGoogle ScholarPubMed
Muller, FJ, Snyder, EY, Loring, JF. Gene therapy: can neural stem cells deliver? Nat Rev Neurosci 2006;7:7584. CrossRefGoogle ScholarPubMed
Martino, G, Pluchino, S. The therapeutic potential of neural stem cells. Nat Rev Neurosci 2006;7:395406. CrossRefGoogle ScholarPubMed
Meissner, KK, Kirkham, DL, Doering, LC. Transplants of neurosphere cell suspensions from aged mice are functional in the mouse model of Parkinson’s. Brain Res 2005;1057:105112. CrossRefGoogle ScholarPubMed
Kelly, S, Bliss, TM, Shah, AKet al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci U S A 2004;101:1183911844. CrossRefGoogle ScholarPubMed
McBride, JL, Behrstock, SP, Chen, EYet al. Human neural stem cell transplants improve motor function in a rat model of Huntington’s disease. J Comp Neurol 2004;475:211219. CrossRefGoogle Scholar
Pluchino, S, Zanotti, L, Rossi, Bet al. Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 2005;436:266271. CrossRefGoogle ScholarPubMed
Jensen, JB, Parmar, M. Strengths and limitations of the neurosphere culture system. Mol Neurobiol 2006;34:153161. CrossRefGoogle ScholarPubMed
Englund, U, Fricker-Gates, RA, Lundberg, Cet al. Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections. Exp Neurol 2002;173:121. CrossRefGoogle ScholarPubMed
Flax, JD, Aurora, S, Yang, Cet al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol 1998;16:10331039. Google ScholarPubMed
Fricker, RA, Carpenter, MK, Winkler, Cet al. Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 1999;19:59906005. Google ScholarPubMed
Suslov, ON, Kukekov, VG, Ignatova, TNet al. Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sci U S A 2002;99:1450614511. CrossRefGoogle ScholarPubMed
Svendsen, CN, Caldwell, MA, Shen, Jet al. Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp Neurol 1997;148:135146. CrossRefGoogle ScholarPubMed
Vroeman, M, Aigner, L, Winkler, Jet al. Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways. Eur J Neurosci 2003;18:743751. CrossRefGoogle Scholar
Winkler, C, Fricker, RA, Gates, MAet al. Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain. Mol Cell Neurosci 1998;11:99116. CrossRefGoogle ScholarPubMed
Valenzuela, M, Sidhu, K, Dean, S, Sachdev, P. Neural stem cell therapy for neuropsychiatric disorders. Acta Neuropsychiatr 2007;19:1126. CrossRefGoogle ScholarPubMed
Bjornson, CR, Rietze, RL, Reynolds, BA, Magli, MC, Vescovi, AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999;283:534537. CrossRefGoogle ScholarPubMed
Clarke, DL, Johansson, CB, Wilbertz, Jet al. Generalized potential of adult neural stem cells. Science 2000;288:16601663. CrossRefGoogle ScholarPubMed
Ostenfeld, T, Tai, YT, Martin, Pet al. Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons. J Neurosci Res 2002;69:955965. CrossRefGoogle ScholarPubMed
Pluchino, S, Quattrini, A, Brambilla, Eet al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003;422:688694. CrossRefGoogle Scholar
Aboody, KS, Brown, A, Rainov, NGet al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S A 2000;97:1284612851. CrossRefGoogle ScholarPubMed
Vescovi, AL, Galli, R, Reynolds, BA. Brain tumour stem cells. Nat Rev Cancer 2006;6:425436. CrossRefGoogle ScholarPubMed
Parmar, M, Skogh, C, Bjorklund, A, Campbell, K. Regional specification of neurosphere cultures derived from subregions of the embryonic telencephalon. Mol Cell Neurosci 2002;21:645656. CrossRefGoogle ScholarPubMed
Klein, C, Fischer, B. Developmental fractionation and differential discrimination of the anti-saccadic direction error. Exp Brain Res 2005;165:132138. CrossRefGoogle ScholarPubMed
Ostenfeld, T, Joly, E, Tai, YTet al. Regional specification of rodent and human neurospheres. Brain Res Dev Brain Res 2002;134:4355. CrossRefGoogle ScholarPubMed
Santa-Olalla, J, Baizabal, JM, Fregoso, M, Del Carmen Cardenas, M, Covarrubias, L. The in vivo positional identity gene expression code is not preserved in neural stem cells grown in culture. Eur J Neurosci 2003;18:10731084. CrossRefGoogle Scholar
Hack, MA, Sugimori, M, Lundberg, C, Nakafuku, M, Gotz, M. Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol Cell Neurosci 2004;25:664678. CrossRefGoogle ScholarPubMed
Gabay, L, Lowell, S, Rubin, LL, Anderson, DJ. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 2003;40:485499. CrossRefGoogle ScholarPubMed
Machon, O, Backman, M, Krauss, S, Kozmik, Z. The cellular fate of cortical progenitors is not maintained in neurosphere cultures. Mol Cell Neurosci 2005;30:388397. CrossRefGoogle Scholar
Spangrude, GJ, Brooks, DM, Tumas, DB. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 1995;85:10061016. Google Scholar
Osawa, M, Hanada, K, Hamada, H, Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242245. CrossRefGoogle ScholarPubMed
Krause, DS, Theise, ND, Collector, MIet al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369377. CrossRefGoogle ScholarPubMed
Reynolds, BA, Rietze, RL. Neural stem cells and neurospheres – re-evaluating the relationship. Nat Methods 2005;2:333336. CrossRefGoogle ScholarPubMed
Seaberg, RM, Van Der Kooy, D. Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 2002;22:17841793. Google ScholarPubMed
Bull, ND, Bartlett, PF. The adult mouse hippocampal progenitor is neurogenic but not a stem cell. J Neurosci 2005;25:1081510821. CrossRefGoogle Scholar
Marshall, GP II, Laywell, ED, Zheng, T, Steindler, DA, Scott, EW. In vitro-derived “neural stem cells” function as neural progenitors without the capacity for self-renewal. Stem Cells 2006;24:731738. CrossRefGoogle ScholarPubMed
Singec, I, Knoth, R, Meyer, RPet al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 2006;3:801806. CrossRefGoogle ScholarPubMed
Tropepe, V, Sibilia, M, Ciruna, BGet al. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 1999;208:166188. CrossRefGoogle ScholarPubMed