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Combined Method of Neuronal Cell-Inducible Vector and Valproic Acid for Enhanced Gene Expression under Hypoxic Conditions

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Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury.


To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo.


The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo.


In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

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  1. 1.

    Hagg T, Oudega M. Degenerative and spontaneous regenerative processes after spinal cord injury. J Neurotrauma. 2006;23:264–80.

  2. 2.

    Lu P, Kadoya K, Tuszynski MH. Axonal growth and connectivity from neural stem cell grafts in models of spinal cord injury. Curr Opin Neurobiol. 2014;27:103–9.

  3. 3.

    Park JH, Kim JY. Iatrogenic spinal subarachnoid hematoma after diagnostic lumbar puncture. Korean J Spine. 2017;14:158–61.

  4. 4.

    Bae HJ, Cho TG, Kim CH, Lee HK, Moon JG, Choi JI. Aortic injury during transforaminal lumbar interbody fusion. Korean J Spine. 2017;14:118–20.

  5. 5.

    Lu P, Jones LL, Snyder EY, Tuszynski MH. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol. 2003;181:115–29.

  6. 6.

    Iwai H, Nori S, Nishimura S, Yasuda A, Takano M, Tsuji O, et al. Transplantation of neural stem/progenitor cells at different locations in mice with spinal cord injury. Cell Transplant. 2014;23:1451–64.

  7. 7.

    Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D, et al. Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell. 2012;150:1264–73.

  8. 8.

    Abematsu M, Tsujimura K, Yamano M, Saito M, Kohno K, Kohyama J, et al. Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury. J Clin Invest. 2010;120:3255–66.

  9. 9.

    Liu ML, Oh JS, An SS, Pennant WA, Kim HJ, Gwak SJ, et al. Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia-inducible gene expression system. J Gene Med. 2010;12:990–1001.

  10. 10.

    Lee HJ, Kim KS, Park IH, Kim SU. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS One. 2007;2:e156.

  11. 11.

    Zhu S, Ambasudhan R, Sun W, Kim HJ, Talantova M, Wang X, et al. Small molecules enable OCT4-mediated direct reprogramming into expandable human neural stem cells. Cell Res. 2014;24:126–9.

  12. 12.

    Han DW, Tapia N, Hermann A, Hemmer K, Hoing S, Araúzo-Bravo MJ, et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell. 2012;10:465–72.

  13. 13.

    Hong JY, Lee SH, Lee SC, Kim JW, Kim KP, Kim SM, et al. Therapeutic potential of induced neural stem cells for spinal cord injury. J Biol Chem. 2014;289:32512–25.

  14. 14.

    Oh J, You Y, Yun Y, Lee HL, Yoon DH, Lee M, et al. A gene and neural stem cell therapy platform based on neuronal cell type-inducible gene overexpression. Yonsei Med J. 2015;56:1036–43.

  15. 15.

    Yun Y, Oh J, Kim Y, Kim G, Lee M, Ha Y. Characterization of neural stem cells modified with hypoxia/neuron-specific VEGF expression system for spinal cord injury. Gene Ther. 2018;25:27–38.

  16. 16.

    You Y, Che L, Lee HY, Lee HL, Yun Y, Lee M, et al. Antiapoptotic effect of highly secreted GMCSF from neuronal cell-specific GMCSF overexpressing neural stem cells in spinal cord injury model. Spine (Phila Pa 1976). 2015;40:E1284–91.

  17. 17.

    Kim HM, Hwang DH, Lee JE, Kim SU, Kim BG. Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue sparing after spinal cord injury. PLoS One. 2009;4:e4987.

  18. 18.

    Brockington A, Lewis C, Wharton S, Shaw PJ. Vascular endothelial growth factor and the nervous system. Neuropathol Appl Neurobiol. 2004;30:427–46.

  19. 19.

    Göttlicher M, Minucci S, Zhu P, Krämer OH, Schimpf A, Giavara S, et al. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001;20:6969–78.

  20. 20.

    Glass CK, Rosenfeld MG. The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 2000;14:121–41.

  21. 21.

    Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? EMBO J. 2000;19:1176–9.

  22. 22.

    Grunstein M. Histone acetylation in chromatin structure and transcription. Nature. 1997;389:349–52.

  23. 23.

    Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001;276:36734–41.

  24. 24.

    Suraweera A, O’Byrne KJ, Richard DJ. Combination therapy with histone deacetylase inhibitors (HDACi) for the treatment of cancer: achieving the full therapeutic potential of HDACi. Front Oncol. 2018;8:92.

  25. 25.

    Oh J, Lee KI, Kim HT, You Y, Yoon DH, Song KY, et al. Human-induced pluripotent stem cells generated from intervertebral disc cells improve neurologic functions in spinal cord injury. Stem Cell Res Ther. 2015;6:125.

  26. 26.

    Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci U S A. 2004;101:16659–64.

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This work was partly supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University; Basic Science Research Program through the National Research Foundation of Korea (NRF) (No. 2015R1D1A1A02059821); and a National Research Foundation of Korea Grant, funded by the Korean Government (NRF-2015R1A6A3A01018883). This research was also supported by grants from the National Research Foundation of Korea (2013M3A9B4076483 and 2016K1A3A1A61006001) and KRIBB research initiative program funded by the Ministry of Science and ICT.

Author information

JSO and YH organized and supervised the study; YY and DB performed the experiments and wrote the manuscript; DL and EC performed electrophysiological studies; JK contributed to material preparation.

Correspondence to Jinsoo Oh or Yoon Ha.

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The authors declare no financial conflicts of interest.

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The Animal Care and Use Committee of the Medical Research Institute of Yonsei University College of Medicine approved all protocols (IACUC approval 291 No. 2017-0114) in the current study.

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Yun, Y., Baek, D., Lee, D. et al. Combined Method of Neuronal Cell-Inducible Vector and Valproic Acid for Enhanced Gene Expression under Hypoxic Conditions. Tissue Eng Regen Med (2019). https://doi.org/10.1007/s13770-019-00223-w

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  • Induced neural stem cells (iNSCs)
  • Neuron-specific enolase promoter (pNSE)
  • Hypoxia
  • Valproic acid (VPA)
  • Spinal cord injury (SCI)