Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments


Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.

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Brain-derived neurotrophic factor


Central nervous system




Epidermal growth factor


Embryonic stem cell


Fibroblast growth factor 2


γ-Aminobutyric acid


Growth-associated protein 43


Glial fibrillary acid protein


Lateral ganglionic eminence


Microtubule-associated protein 2


Medial ganglionic eminence



NS cells:

Radial glia-like neural stem cells




Sonic hedgehog


Subventricular zone


  1. 1.

    Wolpert L (1969) Positional information and the spatial pattern of cellular differentiation. J Theor Biol 25:1–47

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Lupo G, Harris WA, Lewis KE (2006) Mechanisms of ventral patterning in the vertebrate nervous system. Nat Rev Neurosci 7:103–114

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Krumlauf R (1993) Hox genes and pattern formation in the branchial region of the vertebrate head. Trends Genet 9:106–112

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Wassef M, Joyner AL (1997) Early mesencephalon/metencephalon patterning and development of the cerebellum. Perspect Dev Neurobiol 5:3–16

    PubMed  CAS  Google Scholar 

  5. 5.

    Rubenstein JL, Beachy PA (1998) Patterning of the embryonic forebrain. Curr Opin Neurobiol 8:18–26

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    McMahon AP (2000) Neural patterning: the role of Nkx genes in the ventral spinal cord. Genes Dev 14:2261–2264

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Campbell K (2003) Dorsal-ventral patterning in the mammalian telencephalon. Curr Opin Neurobiol 13:50–56

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A (2005) Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3:e283

    PubMed  Article  Google Scholar 

  9. 9.

    Pollard SM, Conti L, Sun Y, Goffredo D, Smith A (2006) Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex 16(Suppl 1):i112–i120

    PubMed  Article  Google Scholar 

  10. 10.

    Glaser T, Pollard SM, Smith A, Brustle O (2007) Tripotential differentiation of adherently expandable neural stem (NS) cells. PLoS One 2:e298

    PubMed  Article  Google Scholar 

  11. 11.

    Goffredo D, Conti L, Di Febo F, Biella G, Tosoni A, Vago G, Biunno I, Moiana A, Bolognini D, Toselli M, Cattaneo E (2008) Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells. Cell Death Differ 15:1847–1856

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Onorati M, Camnasio S, Binetti M, Jung CB, Moretti A, Cattaneo E (2010) Neuropotent self-renewing neural stem (NS) cells derived from mouse induced pluripotent stem (iPS) cells. Mol Cell Neurosci 43:287–295

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Kim JB, Zaehres H, Arauzo-Bravo MJ, Scholer HR (2009) Generation of induced pluripotent stem cells from neural stem cells. Nat Protoc 4:1464–1470

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Koch P, Opitz T, Steinbeck JA, Ladewig J, Brustle O (2009) A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci U S A 106:3225–3230

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Hitoshi S, Tropepe V, Ekker M, van der Kooy D (2002) Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain. Development 129:233–244

    PubMed  CAS  Google Scholar 

  16. 16.

    Santa-Olalla J, Baizabal JM, Fregoso M, del Carmen Cardenas M, Covarrubias L (2003) The in vivo positional identity gene expression code is not preserved in neural stem cells grown in culture. Eur J Neurosci 18:1073–1084

    PubMed  Article  Google Scholar 

  17. 17.

    Zappone MV, Galli R, Catena R, Meani N, De Biasi S, Mattei E, Tiveron C, Vescovi AL, Lovell-Badge R, Ottolenghi S, Nicolis SK (2000) Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. Development 127:2367–2382

    PubMed  CAS  Google Scholar 

  18. 18.

    Hack MA, Sugimori M, Lundberg C, Nakafuku M, Gotz M (2004) Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol Cell Neurosci 25:664–678

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Gabay L, Lowell S, Rubin LL, Anderson DJ (2003) Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40:485–499

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Bithell A, Finch SE, Hornby MF, Williams BP (2008) Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes their neuronal subtype specification. Stem Cells 26:1565–1574

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407:313–319

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Spiliotopoulos D, Goffredo D, Conti L, Di Febo F, Biella G, Toselli M, Cattaneo E (2009) An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population. Neurobiol Dis 34:320–331

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046–5061

    PubMed  CAS  Google Scholar 

  24. 24.

    Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3:517–530

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Selkoe D, Kopan R (2003) Notch and Presenilin: regulated intramembrane proteolysis links development and degeneration. Annu Rev Neurosci 26:565–597

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Tao W, Lai E (1992) Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain. Neuron 8:957–966

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Monaghan AP, Grau E, Bock D, Schutz G (1995) The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain. Development 121:839–853

    PubMed  CAS  Google Scholar 

  28. 28.

    Shi Y, Chichung Lie D, Taupin P, Nakashima K, Ray J, Yu RT, Gage FH, Evans RM (2004) Expression and function of orphan nuclear receptor TLX in adult neural stem cells. Nature 427:78–83

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Britz O, Mattar P, Nguyen L, Langevin LM, Zimmer C, Alam S, Guillemot F, Schuurmans C (2006) A role for proneural genes in the maturation of cortical progenitor cells. Cereb Cortex 16(Suppl 1):i138–i151

    PubMed  Article  Google Scholar 

  30. 30.

    Laeng P, Pitts RL, Lemire AL, Drabik CE, Weiner A, Tang H, Thyagarajan R, Mallon BS, Altar CA (2004) The mood stabilizer valproic acid stimulates GABA neurogenesis from rat forebrain stem cells. J Neurochem 91:238–251

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL (2008) Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A 105:16707–16712

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Stenman JM, Wang B, Campbell K (2003) Tlx controls proliferation and patterning of lateral telencephalic progenitor domains. J Neurosci 23:10568–10576

    PubMed  CAS  Google Scholar 

  33. 33.

    Wichterle H, Lieberam I, Porter JA, Jessell TM (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110:385–397

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Stone D, Rosenthal A (2000) Achieving neuronal patterning by repression. Nat Neurosci 3:967–969

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Ericson J, Briscoe J, Rashbass P, van Heyningen V, Jessell TM (1997) Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube. Cold Spring Harb Symp Quant Biol 62:451–466

    PubMed  CAS  Google Scholar 

  36. 36.

    Sander M, Sussel L, Conners J, Scheel D, Kalamaras J, Dela Cruz F, Schwitzgebel V, Hayes-Jordan A, German M (2000) Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 127:5533–5540

    PubMed  CAS  Google Scholar 

  37. 37.

    Jacob J, Briscoe J (2003) Gli proteins and the control of spinal-cord patterning. EMBO Rep 4:761–765

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Kessaris N, Pringle N, Richardson WD (2001) Ventral neurogenesis and the neuron-glial switch. Neuron 31:677–680

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Ariano MA, Cepeda C, Calvert CR, Flores-Hernandez J, Hernandez-Echeagaray E, Klapstein GJ, Chandler SH, Aronin N, DiFiglia M, Levine MS (2005) Striatal potassium channel dysfunction in Huntington’s disease transgenic mice. J Neurophysiol 93:2565–2574

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Gaspard N, Gaillard A, Vanderhaeghen P (2009) Making cortex in a dish: in vitro corticopoiesis from embryonic stem cells. Cell Cycle 8:2491–2496

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Chang HY (2009) Anatomic demarcation of cells: genes to patterns. Science 326:1206–1207

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Conti L, Cattaneo E (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176–187

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    Sun Y, Pollard S, Conti L, Toselli M, Biella G, Parkin G, Willatt L, Falk A, Cattaneo E, Smith A (2008) Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Mol Cell Neurosci 38:245–258

    PubMed  Article  CAS  Google Scholar 

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This research was supported by initial funding from Fondazione Cariplo (Italy) in the context of the awarded N.O.B.E.L. (Operational Network for Biomedicine par Excellence in Lombardy) project entitled “A genetic toolkit for the analyses of neural stem cells—acronym: Mouse NS-toolkit”. This work is also supported by EuroSystem (FP7, European Union Health-F4-2008-200720), Neuroscreen (FP6, European Union LSHB-CT-2007-037766), and NeuroStemcell (FP7, European Union HEALTH-2008-B-222943). The laboratory also acknowledges the contribution of Unicredit Banca S.p.A. (Italy) and of Tavola Valdese (Italy).

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Correspondence to Elena Cattaneo.

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Figure S1. RT–PCR analysis of selected neural progenitor/stem cell markers (JPEG 750 kb)

Figure S2. Immunofluorescence characterization of E14.5 neocortex-derived NSCs. Self-renewing NS14CX cells homogeneously showed double immunoreactivity for Nestin/Vimentin and SOX2/PAX6 (JPEG 1804 kb)

Figure S3. Immunofluorescence characterization of 3 different clones of NS cells derived from E12.5 neocortex (NS12CX) and 3 clones of NS cells derived from E12.5 spinal cord (NS12SC). Self-renewing NS cell clones homogeneously express the typical NS cell markers Nestin, BLBP, RC2, Vimentin, and OLIG2 (JPEG 2075 kb)

Figure S4. Neuronal differentiation of NS12SC. This image shows a GAP-43-positive neuron derived from NS12SC cells differentiated for 23 d (JPEG 646 kb)

Figure S5. RT–PCR analysis of a subset of neurotransmitter receptors, described in striatal and spinal cord neurons in vivo. Neither proliferating (P) nor differentiating (D) NS cells expressed functional glutamatergic, glycinergic, or cholinergic receptors, even if the muscarinic receptor (Chrm4) was detectable (JPEG 579 kb)

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Onorati, M., Binetti, M., Conti, L. et al. Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments. Cell. Mol. Life Sci. 68, 1769–1783 (2011).

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  • Neural stem cells
  • Neural development
  • Positional identity
  • Neuronal differentiation
  • Transcription factors