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Hypoxia-specific GM-CSF-overexpressing neural stem cells improve graft survival and functional recovery in spinal cord injury

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the differentiation and function of hematopoietic cells. GM-CSF has been implicated in nervous system function. The goal of the present study was to understand the effects of hypoxia-induced GM-CSF on neural stem cells (NSCs) in a model of spinal cord injury (SCI). GM-CSF-overexpressing NSCs were engineered utilizing a hypoxia-inducible gene expression plasmid, including an Epo enhancer ahead of an SV promoter (EpoSV-GM-CSF). Cells were then subjected to hypoxia (pO2, 1%) or a hypoxia-mimicking reagent (CoCl2) in vitro. The progression of time of GM-CSF expression was tracked in EpoSV-GM-CSF-transfected NSCs. Overexpression of GM-CSF in undifferentiated and differentiated NSCs created resistance to H2O2-induced apoptosis in hypoxia. NSCs transfected with EpoSV-GM-CSF or SV-GM-CSF were transplanted into rats after SCI to assess the effect of GM-CSF on NSC survival and restoration of function. Moreover, a significantly higher amount of surviving NSCs and neuronal differentiation was observed in the EpoSV-GM-CSF-treated group. Significant improvement in locomotor function was also found in this group. Thus, GM-CSF overexpression by the Epo enhancer in hypoxia was beneficial to transplanted NSC survival and to behavioral improvement, pointing toward a possible role for GM-CSF in the treatment of SCI.

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References

  1. Kan EM, Ling EA, Lu J . Stem cell therapy for spinal cord injury. Curr Med Chem 2010; 17: 4492–4510.

    Article  CAS  PubMed  Google Scholar 

  2. Goldman S . Stem and progenitor cell-based therapy of the human central nervous system. Nat Biotechnol 2005; 23: 862–871.

    Article  CAS  PubMed  Google Scholar 

  3. Carlstedt T . Nerve root replantation. Neurosurg Clin N Am 2009; 20: 39–50; vi.

    Article  PubMed  Google Scholar 

  4. Nandoe Tewarie RS, Hurtado A, Bartels RH, Grotenhuis A, Oudega M . Stem cell-based therapies for spinal cord injury. J Spinal Cord Med 2009; 32: 105–114.

    Article  PubMed  Google Scholar 

  5. Lindvall O, Kokaia Z . Stem cells for the treatment of neurological disorders. Nature 2006; 441: 1094–1096.

    Article  CAS  PubMed  Google Scholar 

  6. Bantubungi K, Blum D, Cuvelier L, Wislet-Gendebien S, Rogister B, Brouillet E et al. Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington′s disease. Mol Cell Neurosci 2008; 37: 454–470.

    Article  CAS  PubMed  Google Scholar 

  7. Oh JS, Ha Y, An SS, Khan M, Pennant WA, Kim HJ et al. Hypoxia-preconditioned adipose tissue-derived mesenchymal stem cell increase the survival and gene expression of engineered neural stem cells in a spinal cord injury model. Neurosci Lett 2010; 472: 215–219.

    Article  CAS  PubMed  Google Scholar 

  8. Oh JS, Kim KN, An SS, Pennant WA, Kim HJ, Gwak SJ et al. Co-transplantation of mouse neural stem cells (mNSCs) with adipose tissue-derived mesenchymal stem sells improves mNSC survival in a rat spinal cord injury model. Cell Transplant 2010; 472: 215–219.

    CAS  Google Scholar 

  9. Lee M, Lee ES, Kim YS, Choi BH, Park SR, Park HS et al. Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system. Spine (Phila Pa 1976) 2005; 30: 2729–2734.

    Article  Google Scholar 

  10. Jin H, Liu ML, Kim HA, Lee M, An S, Oh J et al. Role of the oxygen-dependent degradation domain in a hypoxia-inducible gene expression system in vascular endothelial growth factor gene therapy. Spine (Phila Pa 1976) 2009; 34: E952–E958.

    Article  Google Scholar 

  11. Iwai M, Stetler RA, Xing J, Hu X, Gao Y, Zhang W et al. Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin after neonatal hypoxic/ischemic brain injury. Stroke 2010; 41: 1032–1037.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Schneider A, Kruger C, Steigleder T, Weber D, Pitzer C, Laage R et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J Clin Invest 2005; 115: 2083–2098.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Kim NK, Choi BH, Huang X, Snyder BJ, Bukhari S, Kong TH et al. Granulocyte-macrophage colony-stimulating factor promotes survival of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced murine Parkinson′s disease model. Eur J Neurosci 2009; 29: 891–900.

    Article  PubMed  Google Scholar 

  14. Bouhy D, Malgrange B, Multon S, Poirrier AL, Scholtes F, Schoenen J et al. Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB J 2006; 20: 1239–1241.

    Article  CAS  PubMed  Google Scholar 

  15. Hayashi K, Ohta S, Kawakami Y, Toda M . Activation of dendritic-like cells and neural stem/progenitor cells in injured spinal cord by GM-CSF. Neurosci Res 2009; 64: 96–103.

    Article  CAS  PubMed  Google Scholar 

  16. 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.

    Article  CAS  PubMed  Google Scholar 

  17. An SS, Jin HL, Kim KN, Kim DS, Cho J, Liu ML et al. Neuroprotective effect of combined hypoxia-induced VEGF and bone marrow-derived mesenchymal stem cell treatment. Childs Nerv Syst 2010; 26: 323–331.

    Article  PubMed  Google Scholar 

  18. Schlett K, Madarasz E . Retinoic acid induced neural differentiation in a neuroectodermal cell line immortalized by p53 deficiency. J Neurosci Res 1997; 47: 405–415.

    Article  CAS  PubMed  Google Scholar 

  19. Ogawa D, Okada Y, Nakamura M, Kanemura Y, Okano HJ, Matsuzaki Y et al. Evaluation of human fetal neural stem/progenitor cells as a source for cell replacement therapy for neurological disorders: properties and tumorigenicity after long-term in vitro maintenance. J Neurosci Res 2009; 87: 307–317.

    Article  CAS  PubMed  Google Scholar 

  20. Cao QL, Zhang YP, Howard RM, Walters WM, Tsoulfas P, Whittemore SR . Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001; 167: 48–58.

    Article  CAS  PubMed  Google Scholar 

  21. Mautes AE, Liu J, Brandewiede J, Manville J, Snyder E, Schachner M . Regional energy metabolism following short-term neural stem cell transplantation into the injured spinal cord. J Mol Neurosci 2004; 24: 227–236.

    Article  CAS  PubMed  Google Scholar 

  22. Zhang X, Zeng Y, Zhang W, Wang J, Wu J, Li J . Co-transplantation of neural stem cells and NT-3-overexpressing Schwann cells in transected spinal cord. J Neurotrauma 2007; 24: 1863–1877.

    Article  PubMed  Google Scholar 

  23. Zhang L, Gu S, Zhao C, Wen T . Combined treatment of neurotrophin-3 gene and neural stem cells is propitious to functional recovery after spinal cord injury. Cell Transplant 2007; 16: 475–481.

    Article  CAS  PubMed  Google Scholar 

  24. McLay RN, Kimura M, Banks WA, Kastin AJ . Granulocyte-macrophage colony-stimulating factor crosses the blood--brain and blood--spinal cord barriers. Brain 1997; 120 (Pt 11): 2083–2091.

    Article  PubMed  Google Scholar 

  25. Schermer C, Humpel C . Granulocyte macrophage-colony stimulating factor activates microglia in rat cortex organotypic brain slices. Neurosci Lett 2002; 328: 180–184.

    Article  CAS  PubMed  Google Scholar 

  26. Mantovani G, Massa E, Astara G, Murgia V, Gramignano G, Lusso MR et al. Phase II clinical trial of local use of GM-CSF for prevention and treatment of chemotherapy- and concomitant chemoradiotherapy-induced severe oral mucositis in advanced head and neck cancer patients: an evaluation of effectiveness, safety and costs. Oncol Rep 2003; 10: 197–206.

    CAS  PubMed  Google Scholar 

  27. Schmeler KM, Vadhan-Raj S, Ramirez PT, Apte SM, Cohen L, Bassett RL et al. A phase II study of GM-CSF and rIFN-gamma1b plus carboplatin for the treatment of recurrent, platinum-sensitive ovarian, fallopian tube and primary peritoneal cancer. Gynecol Oncol 2009; 113: 210–215.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Xiaowei H, Ninghui Z, Wei X, Yiping T, Linfeng X . The experimental study of hypoxia-inducible factor-1alpha and its target genes in spinal cord injury. Spinal Cord 2006; 44: 35–43.

    Article  CAS  PubMed  Google Scholar 

  29. Ju Y, He M, Mao B . Sequential changes of hypoxia-inducible factor 1 alpha in experimental spinal cord injury and its significance. Chin J Traumatol 2002; 5: 103–106.

    PubMed  Google Scholar 

  30. Choi BH, Ha Y, Ahn CH, Huang X, Kim JM, Park SR et al. A hypoxia-inducible gene expression system using erythropoietin 3′ untranslated region for the gene therapy of rat spinal cord injury. Neurosci Lett 2007; 412: 118–122.

    Article  CAS  PubMed  Google Scholar 

  31. Lee M, Choi D, Choi MJ, Jeong JH, Kim WJ, Oh S et al. Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3′-untranslated region for the VEGF gene therapy. J Control Release 2006; 115: 113–119.

    Article  CAS  PubMed  Google Scholar 

  32. Wang MJ, Lin S . A region within the 5′-untranslated region of hypoxia-inducible factor-1alpha mRNA mediates its turnover in lung adenocarcinoma cells. J Biol Chem 2009; 284: 36500–36510.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Groot AJ, Verheesen P, Westerlaken EJ, Gort EH, van der Groep P, Bovenschen N et al. Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha. Lab Invest 2006; 86: 345–356.

    Article  CAS  PubMed  Google Scholar 

  34. Percy MJ, Mooney SM, McMullin MF, Flores A, Lappin TR, Lee FS . A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation. Mol Cancer 2003; 2: 31.

    Article  PubMed Central  PubMed  Google Scholar 

  35. Lee M, Bikram M, Oh S, Bull DA, Kim SW . Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease. Pharm Res 2004; 21: 736–741.

    Article  CAS  PubMed  Google Scholar 

  36. Lipnik K, Greco O, Scott S, Knapp E, Mayrhofer E, Rosenfellner D et al. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors. Virology 2006; 349: 121–133.

    Article  CAS  PubMed  Google Scholar 

  37. Grasselli F, Basini G, Bussolati S, Bianco F . Cobalt chloride, a hypoxia-mimicking agent, modulates redox status and functional parameters of cultured swine granulosa cells. Reprod Fertil Dev 2005; 17: 715–720.

    Article  CAS  PubMed  Google Scholar 

  38. Hara A, Niwa M, Aoki H, Kumada M, Kunisada T, Oyama T et al. A new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent, cobalt chloride. Brain Res 2006; 1109: 192–200.

    Article  CAS  PubMed  Google Scholar 

  39. Lee H, Bien CM, Hughes AL, Espenshade PJ, Kwon-Chung KJ, Chang YC . Cobalt chloride, a hypoxia-mimicking agent, targets sterol synthesis in the pathogenic fungus Cryptococcus neoformans. Mol Microbiol 2007; 65: 1018–1033.

    Article  CAS  PubMed  Google Scholar 

  40. Halliwell B . Oxidative stress and neurodegeneration: where are we now? J Neurochem 2006; 97: 1634–1658.

    Article  CAS  PubMed  Google Scholar 

  41. Hyslop PA, Zhang Z, Pearson DV, Phebus LA . Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro. Brain Res 1995; 671: 181–186.

    Article  CAS  PubMed  Google Scholar 

  42. Schallenberg M, Charalambous P, Thanos S . GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death. Exp Eye Res 2009; 89: 665–677.

    Article  CAS  PubMed  Google Scholar 

  43. Giulian D, Li J, Li X, George J, Rutecki PA . The impact of microglia-derived cytokines upon gliosis in the CNS. Dev Neurosci 1994; 16: 128–136.

    Article  CAS  PubMed  Google Scholar 

  44. Schabitz WR, Kruger C, Pitzer C, Weber D, Laage R, Gassler N et al. A neuroprotective function for the hematopoietic protein granulocyte-macrophage colony stimulating factor (GM-CSF). J Cereb Blood Flow Metab 2008; 28: 29–43.

    Article  PubMed  Google Scholar 

  45. Ha Y, Kim YS, Cho JM, Yoon SH, Park SR, Yoon DH et al. Role of granulocyte-macrophage colony-stimulating factor in preventing apoptosis and improving functional outcome in experimental spinal cord contusion injury. J Neurosurg Spine 2005; 2: 55–61.

    Article  PubMed  Google Scholar 

  46. Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported by grants from the Stem Cell Research Center of the 21st Century Frontier Research Program (SC-4180), funded by the Ministry of Education, Science and Technology (2010-000-2213). Funding was also provided by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2010K001350).

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Kim, H., Oh, J., An, S. et al. Hypoxia-specific GM-CSF-overexpressing neural stem cells improve graft survival and functional recovery in spinal cord injury. Gene Ther 19, 513–521 (2012). https://doi.org/10.1038/gt.2011.137

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