Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat

Stem Cell Research & Therapy

Eva Sykova²  Jack Price³  Graham Cocks³  Miroslava Kapcalova¹  Pavel Prochazka¹  Karolina Turnovcova¹  Vit Herynek⁴  Pavla Jendelova²  Nataliya Romanyuk¹  Takashi Amemori¹ 

[1] Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic;Department of Neuroscience, Second Faculty of Medicine, Charles University, Prague, Czech Republic;Institute of Psychiatry, King’s College London, London, UK;MR-Unit, Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic

关键词: neuroregeneration;    trophic support;    motor neuron differentiation;    spinal cord injury;    Human fetal neural stem cells;   

Others  :  847445
DOI  :  10.1186/scrt219

 received in 2012-12-13, accepted in 2013-06-04,  发布年份 2013

【 摘 要 】

Introduction

A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI).

Methods

A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes.

Results

Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)⁺ axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2⁺ and choline acetyltransferase (ChAT)⁺ neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T₂-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2.

Conclusions

The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.

【 授权许可】

   
2013 Amemori et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Donnelly EM, Lamanna J, Boulis NM: Stem cell therapy for the spinal cord. Stem Cell Res Ther 2012, 3:24. BioMed Central Full Text
• [2]Willerth SM: Neural tissue engineering using embryonic and induced pluripotent stem cells. Stem Cell Res Ther 2011, 2:17. BioMed Central Full Text
• [3]Lindvall O, Kokaia Z: Stem cells in human neurodegenerative disorders: time for clinical translation? J Clin Invest 2010, 120:29-40.
• [4]Ronaghi M, Erceg S, Moreno-Manzano V, Stojkovic M: Challenges of stem cell therapy for spinal cord injury: human embryonic stem cells, endogenous neural stem cells, or induced pluripotent stem cells? Stem Cells 2010, 28:93-99.
• [5]Thomas KE, Moon LD: Will stem cell therapies be safe and effective for treating spinal cord injuries? Br Med Bull 2011, 98:127-142.
• [6]Einstein O, Ben-Hur T: The changing face of neural stem cell therapy in neurologic diseases. Arch Neurol 2008, 65:452-456.
• [7]Einstein O, Fainstein N, Vaknin I, Mizrachi-Kol R, Reihartz E, Grigoriadis N, Lavon I, Baniyash M, Lassmann H, Ben-Hur T: Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression. Ann Neurol 2007, 61:209-218.
• [8]Rossi SL, Keirstead HS: Stem cells and spinal cord regeneration. Curr Opin Biotechnol 2009, 20:552-562.
• [9]Arboleda D, Forostyak S, Jendelova P, Marekova D, Amemori T, Pivonkova H, Masinova K, Sykova E: Transplantation of predifferentiated adipose-derived stromal cells for the treatment of spinal cord injury. Cell Mol Neurobiol 2011, 31:1113-1122.
• [10]Hofstetter CP, Schwarz EJ, Hess D, Widenfalk J, El Manira A, Prockop DJ, Olson L: Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A 2002, 99:2199-2204.
• [11]Urdzikova L, Jendelova P, Glogarova K, Burian M, Hajek M, Sykova E: Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats. J Neurotrauma 2006, 23:1379-1391.
• [12]Amemori T, Jendelova P, Ruzickova K, Arboleda D, Sykova E: Co-transplantation of olfactory ensheathing glia and mesenchymal stromal cells does not have synergistic effects after spinal cord injury in the rat. Cytotherapy 2010, 12:212-225.
• [13]Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, Summers R, Gage FH, Anderson AJ: Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A 2005, 102:14069-14074.
• [14]Hooshmand MJ, Sontag CJ, Uchida N, Tamaki S, Anderson AJ, Cummings BJ: Analysis of host-mediated repair mechanisms after human CNS-stem cell transplantation for spinal cord injury: correlation of engraftment with recovery. PLoS One 2009, 4:e5871.
• [15]Yan J, Xu L, Welsh AM, Hatfield G, Hazel T, Johe K, Koliatsos VE: Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS Med 2007, 4:e39.
• [16]Yasuda A, Tsuji O, Shibata S, Nori S, Takano M, Kobayashi Y, Takahashi Y, Fujiyoshi K, Hara CM, Miyawaki A, Okano HJ, Toyama Y, Nakamura M, Okano H: Significance of remyelination by neural stem/progenitor cells transplanted into the injured spinal cord. Stem Cells 2011, 29:1983-1994.
• [17]Lee SH, Chung YN, Kim YH, Kim YJ, Park JP, Kwon DK, Kwon OS, Heo JH, Ryu S, Kang HJ, Paek SH, Wang KC, Kim SU, Yoon BW: Effects of human neural stem cell transplantation in canine spinal cord hemisection. Neurol Res 2009, 31:996-1002.
• [18]Pollock K, Stroemer P, Patel S, Stevanato L, Hope A, Miljan E, Dong Z, Hodges H, Price J, Sinden JD: A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol 2006, 199:143-155.
• [19]Boer GJ: Ethical guidelines for the use of human embryonic or fetal tissue for experimental and clinical neurotransplantation and research: Network of European CNS transplantation and restoration (NECTAR). J Neurol 1994, 242:1-13.
• [20]Britain G: Human Tissue Act 2004. London: HMSO; 2004.
• [21]Zhang F, Frost AR, Blundell MP, Bales O, Antoniou MN, Thrasher AJ: A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors. Mol Ther 2010, 18:1640-1649.
• [22]Babic M, Horak D, Trchova M, Jendelova P, Glogarova K, Lesny P, Herynek V, Hajek M, Sykova E: Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling. Bioconjug Chem 2008, 19:740-750.
• [23]Nakamura M, Okano H: Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells. Cell Res 2013, 23:70-80.
• [24]Pedersen EB, Zimmer J, Finsen B: Triple immunosuppression protects murine intracerebral, hippocampal xenografts in adult rat hosts: effects on cellular infiltration, major histocompatibility complex antigen induction and blood–brain barrier leakage. Neuroscience 1997, 78:685-701.
• [25]Basso DM, Beattie MS, Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995, 12:1-21.
• [26]Goldstein LB: Effects of bilateral and unilateral locus coeruleus lesions on beam-walking recovery after subsequent unilateral sensorimotor cortex suction-ablation in the rat. Restor Neurol Neurosci 1997, 11:55-63.
• [27]Benowitz LI, Routtenberg A: GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci 1997, 20:84-91.
• [28]Andersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245-5250.
• [29]Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002, 30:e36.
• [30]Erceg S, Ronaghi M, Oria M, Rosello MG, Arago MA, Lopez MG, Radojevic I, Moreno-Manzano V, Rodriguez-Jimenez FJ, Bhattacharya SS, Cordoba J, Stojkovic M: Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection. Stem Cells 2010, 28:1541-1549.
• [31]Parr AM, Kulbatski I, Tator CH: Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury. J Neurotrauma 2007, 24:835-845.
• [32]Sykova E, Jendelova P: In vivo tracking of stem cells in brain and spinal cord injury. Prog Brain Res 2007, 161:367-383.
• [33]Sykova E, Jendelova P: Migration, fate and in vivo imaging of adult stem cells in the CNS. Cell Death Differ 2007, 14:1336-1342.
• [34]Kubinova S, Sykova E: Nanotechnology for treatment of stroke and spinal cord injury. Nanomedicine (Lond) 2010, 5:99-108.
• [35]Guzman R, Uchida N, Bliss TM, He D, Christopherson KK, Stellwagen D, Capela A, Greve J, Malenka RC, Moseley ME, Palmer TD, Steinberg GK: Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proc Natl Acad Sci U S A 2007, 104:10211-10216.
• [36]Zhang ZG, Jiang Q, Zhang R, Zhang L, Wang L, Arniego P, Ho KL, Chopp M: Magnetic resonance imaging and neurosphere therapy of stroke in rat. Ann Neurol 2003, 53:259-263.
• [37]van Buul GM, Kotek G, Wielopolski PA, Farrell E, Bos PK, Weinans H, Grohnert AU, Jahr H, Verhaar JA, Krestin GP, van Osch GJ, Bernsen MR: Clinically translatable cell tracking and quantification by MRI in cartilage repair using superparamagnetic iron oxides. PLoS One 2011, 6:e17001.
• [38]Jakeman LB, Wei P, Guan Z, Stokes BT: Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol 1998, 154:170-184.
• [39]Jones LL, Oudega M, Bunge MB, Tuszynski MH: Neurotrophic factors, cellular bridges and gene therapy for spinal cord injury. J Physiol 2001, 533:83-89.
• [40]Tuszynski MH: Gene therapy for nervous system disease. Ann N Y Acad Sci 1997, 835:1-11.
• [41]Tuszynski MH, Gabriel K, Gage FH, Suhr S, Meyer S, Rosetti A: Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after adult spinal cord injury. Exp Neurol 1996, 137:157-173.
• [42]Polentes J, Jendelova P, Cailleret M, Braun H, Romanyuk N, Tropel P, Brenot M, Itier V, Seminatore C, Baldauf K, Turnovcova K, Jirak D, Teletin M, Côme J, Tournois J, Reymann K, Sykova E, Viville S, Onteniente B: Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain. Cell Transplant 2012, 21:2587-2602.
• [43]Althaus HH, Kloppner S, Klopfleisch S, Schmitz M: Oligodendroglial cells and neurotrophins: a polyphonic cantata in major and minor. J Mol Neurosci 2008, 35:65-79.