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Molecular Neurobiology

, Volume 54, Issue 8, pp 6097–6106 | Cite as

Generation of a Functional Human Neural Network by NDM29 Overexpression in Neuroblastoma Cancer Cells

  • Susanna Alloisio
  • Patrizia Garbati
  • Federica Viti
  • Silvia Dante
  • Raffaella Barbieri
  • Giovanni Arnaldi
  • Alessia Petrelli
  • Arianna Gigoni
  • Paolo Giannoni
  • Rodolfo Quarto
  • Mario Nobile
  • Massimo Vassalli
  • Aldo Pagano
Article

Abstract

Recent advances in life sciences suggest that human and rodent cell responses to stimuli might differ significantly. In this context, the results achieved in neurotoxicology and biomedical research practices using neural networks obtained from mouse or rat primary culture of neurons would benefit of the parallel evaluation of the same parameters using fully differentiated neurons with a human genetic background, thus emphasizing the current need of neuronal cells with human origin. In this work, we developed a human functionally active neural network derived by human neuroblastoma cancer cells genetically engineered to overexpress NDM29, a non-coding RNA whose increased synthesis causes the differentiation toward a neuronal phenotype. These cells are here analyzed accurately showing functional and morphological traits of neurons such as the expression of neuron-specific proteins and the possibility to generate the expected neuronal current traces and action potentials. Their morphometrical analysis is carried out by quantitative phase microscopy showing soma and axon sizes compatible with those of functional neurons. The ability of these cells to connect autonomously forming physical junctions recapitulates that of hippocampal neurons, as resulting by connect-ability test. Lastly, these cells self-organize in neural networks able to produce spontaneous firing, in which spikes can be clustered in bursts. Altogether, these results show that the neural network obtained by NDM29-dependent differentiation of neuroblastoma cells is a suitable tool for biomedical research practices.

Keywords

Neuroblastoma Human neuronal network Multi-electrode array (MEA) NDM29 SKNBE2-S1.1 Cancer 

Notes

Acknowledgments

A.P. was supported by the Associazione Italiana per la Lotta al Neuroblastoma/Fondazione Neuroblastoma (Genoa, Italy).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Ciarlo E, Massone S, Penna I et al (2013) An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer’s disease brain samples. Dis Model Mech 6(2):424–33. doi: 10.1242/dmm.009761 CrossRefPubMedGoogle Scholar
  2. 2.
    Mestas J, Hughes CC (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172(5):2731–8, ReviewCrossRefPubMedGoogle Scholar
  3. 3.
    Castelnuovo M, Massone S, Tasso R et al (2010) An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J 24(10):4033–10.1096/fj.10-157032CrossRefPubMedGoogle Scholar
  4. 4.
    Gavazzo P, Vella S, Marchetti C, Nizzari M, Cancedda R, Pagano A (2011) Acquisition of neuron-like electrophysiological properties in neuroblastoma cells by controlled expression of NDM29 ncRNA. J Neurochem 119(5):989–1001. doi: 10.1111/j.1471-4159.2011.07492.x CrossRefPubMedGoogle Scholar
  5. 5.
    Mescola A, Vella S, Scotto M, Gavazzo P, Canale C, Diaspro A, Pagano A, Vassalli M (2012) Probing cytoskeleton organisation of neuroblastoma cells with single-cell force spectroscopy. J Mol Recognit 25(5):270–7. doi: 10.1002/jmr.2173 CrossRefPubMedGoogle Scholar
  6. 6.
    Gavazzo P, Vassalli M, Costa D, Pagano A (2013) Novel ncRNAs transcribed by Pol III and elucidation of their functional relevance by biophysical approaches. Front Cell Neurosci 7(7):203. doi: 10.3389/fncel.2013.00203, ReviewPubMedPubMedCentralGoogle Scholar
  7. 7.
    Costa D, Gigoni A, Würth R, Cancedda R, Florio T, Pagano A (2014) Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA. Cancer Cell Int 2:14–59. doi: 10.1186/1475-2867-14-59 Google Scholar
  8. 8.
    Vella S, Penna I, Longo L, Pioggia G, Garbati P, Florio T, Rossi F, Pagano A (2015) Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA. Sci Rep 17(5):18144. doi: 10.1038/srep18144 Google Scholar
  9. 9.
    Stewart SA, Dykxhoorn DM, Palliser D et al (2003) Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4):493–501CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Duggal N, Hammond RR (2002) Nestin expression in ganglioglioma. Exp Neurol 174(1):89–95CrossRefPubMedGoogle Scholar
  11. 11.
    Paganin D, Nugent KA (1998) Non-interferometric phase imaging with partially coherent light. Phys Rev Lett 80:2586–2589CrossRefGoogle Scholar
  12. 12.
    Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11(7):36–41Google Scholar
  13. 13.
    Yan XX, Jen LS, Garey LJ (1996) NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins. Cereb Cortex 6(3):524–9CrossRefPubMedGoogle Scholar
  14. 14.
    Najdzion J, Wasilewska B, Bogus-Nowakowska K, Rowniak M, Zakowski W, Robak A (2012) A morphometric analysis of the geniculate bodies in selected mammalian species. Bull Vet Inst Pulawy 56:205–210CrossRefGoogle Scholar
  15. 15.
    Guibal C, Baker GE (2009) Abnormal axons in the albino optic tract. Invest Ophthalmol Vis Sci 50(12):5516–21. doi: 10.1167/io CrossRefPubMedGoogle Scholar
  16. 16.
    Petrelli A, Marconi E, Salerno M, De Pietri TD, Berdondini L, Dante S (2013) Nano-volume drop patterning for rapid on-chip neuronal connect-ability assays. Lab Chip 13(22):4419–29. doi: 10.1039/c3lc50564b CrossRefPubMedGoogle Scholar
  17. 17.
    Alloisio S, Nobile M, Novellino A (2015) Multiparametric characterisation of neuronal network activity for in vitro agrochemical neurotoxicity assessment. Neurotoxicology 48:152–65. doi: 10.1016/j.neuro.2015.03.013 CrossRefPubMedGoogle Scholar
  18. 18.
    Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2:3081–3089CrossRefPubMedGoogle Scholar
  19. 19.
    Gore A, Li Z, Fung HL et al (2011) Somatic coding mutations in human induced pluripotent stem cells. Nature 471:63–37CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hussein SM, Batada NN, Vuoristo S et al (2011) Copy number variation and selection during reprogramming to pluripotency. Nature 471:58–62CrossRefPubMedGoogle Scholar
  21. 21.
    Salimi A, Nadri S, Ghollasi M et al (2014) Comparison of different protocols for neural differentiation of human induced pluripotent stem cells. Mol Biol Rep 41:1713CrossRefPubMedGoogle Scholar
  22. 22.
    Odawara A, Katoh H, Matsuda N, Suzuki I (2016) Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture. Sci Rep 6:26181CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lister R, Pelizzola M, Kida YS et al (2011) Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471:68–73CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Pagano A, Giannoni P, Zambotti A, Randazzo N, Zerega B, Cancedda R, Dozin B (2002) CALbeta, a novel lipocalin associated with chondrogenesis and inflammation. Eur J Cell Biol 81(5):264–72CrossRefPubMedGoogle Scholar
  25. 25.
    Scharf SJ, Horn GT, Erlich HA (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233(4768):1076–8CrossRefPubMedGoogle Scholar
  26. 26.
    Kaufman DL, Evans GA (1990) Restriction endonuclease cleavage at the termini of PCR products. Biotechniques 9(3):304–306, Erratum in: Biotechniques 9(6):720PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Susanna Alloisio
    • 1
    • 2
  • Patrizia Garbati
    • 3
  • Federica Viti
    • 2
  • Silvia Dante
    • 4
  • Raffaella Barbieri
    • 3
  • Giovanni Arnaldi
    • 3
    • 5
  • Alessia Petrelli
    • 4
  • Arianna Gigoni
    • 3
    • 5
  • Paolo Giannoni
    • 5
  • Rodolfo Quarto
    • 3
    • 5
  • Mario Nobile
    • 2
  • Massimo Vassalli
    • 2
  • Aldo Pagano
    • 3
    • 5
  1. 1.ETT SpaGenoaItaly
  2. 2.National Research CouncilInstitute of BiophysicsGenoaItaly
  3. 3.IRCCS-AOU San Martino-ISTGenovaItaly
  4. 4.Istituto Italiano di TecnologiaGenovaItaly
  5. 5.Department of Experimental Medicine (DIMES)University of GenovaGenovaItaly

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