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  • Spinal Grafting of Human Spinal Stem Cells in a Porcine L3 Contusion Model: Effect of High Dose Immunosuppression Treatment on Cell Graft Survival and Maturation in the Acutely Injured Spinal Cord.

    Final Number:
    1643

    Authors:
    David Weingarten MD; Stefan Juhas; Jan Motlik; Jana Juhasova; Sylvia Marsala; Joseph D. Ciacci MD; Martin Marsala MD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2013 Annual Meeting

    Introduction: Spinal regenerative therapies, including cell replacement therapies (CRT), are rapidly gaining traction as viable treatments for acute and chronic spinal cord injury (SCI). Before such a treatments can effectively be translated into clinical practice, large animal data is needed to characterize effective immunosuppression protocols and long-term survival of grafted cells in the potentially inhospitable milieu of the acutely injured spinal cord. In the present study, we characterize the survival and maturation of clinical grade human spinal stem cells (hNPCs) grafted in and around the injury epicenter using a porcine L3 contusion model.

    Methods: Isoflurane-anesthetized adult Gottingen-Minnesota minipigs (n=10) underwent 2-level laminectomies (L2-L5) followed by L3 spinal contusion using a 5-mm-diameter circular bar (peak force of 2.5kg at a velocity of 3cm/sec). At 24 hours post-injury, animals received 12 bilateral injections of hNPCs targeted in and around the injury epicenter. After cell grafting, animals were continuously immunosuppressed with tacrolimus (targeted blood level 50-60ng/ml) and mycophenolate mofetil (30mg/kg/day). During recovery, motor and sensory function were periodically monitored for 4 weeks. After survival, the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or non-specific (DCX, MAP2, CHAT, GFAP, APC) antibodies.

    Results: In all cell-grafted animals, hNUMA-positive cells were readily identified. Numerous terminally differentiated grafted neurons with extensive axo-dendritic sprouting were seen; these exhibited hNSE and HO14 immunoreactivity. Similarly, a high density of hSYN-positive terminals derived from grafted neurons and residing in the vicinity of host neurons were also seen. A moderate degree of inflammatory change, as evidenced by the appearance of reactive astrocytes and microglia, was also identified.

    Conclusions: These data demonstrate that, using this immunosuppression protocol, cells grafted into the acutely injured spinal cord can survive a minimum of 4 weeks despite the inflammatory, post-traumatic environment.

    Patient Care: This study demonstrates that cell replacement therapy (CRT) can be performed within 24hrs post-injury, despite the potentially hostile, inflammatory milieu of the acutely injured cord. This demonstrates that the timing of treatment may be more flexible than previously believed, and hence that acute SCI may be amenable to cell replacement therapy. This brings us closer to realizing stem cell therapy as a viable therapeutic options for patients with acute SCI.

    Learning Objectives: By the conclusion of this session, participants should be able to 1) Identify some of the potential barriers to cell replacement therapies (CRT) in patients with acute spinal cord injury (SCI), 2) Appreciate the means by which CRT may benefit SCI patients, 3) Describe some of the goals of future large animal and human trials necessary to bring CRT to fruition as an active therapeutic option.

    References: Akesson, E., Sandelin, M., Kanaykina, N., Aldskogius, H., Kozlova, E. N., & El Masry, W. S. (2008). Long-Term Survival , Robust Neuronal Differentiation , and Extensive Migration of Human Forebrain Stem / Progenitor Cells Transplanted to the Adult Rat Dorsal Root Ganglion Cavity. Cell transplantation, 17(10-11), 1115–1123. Basso, D. M., Beattie, M. S., Bresnahan, J. C., Anderson, D. K., Faden, A. I., Gruner, J. A., Holford, T. R., et al. (1996). MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. Journal of neurotrauma, 13(7), 343–59. Bontadini, A. (2012). HLA techniques: typing and antibody detection in the laboratory of immunogenetics. Methods (San Diego, Calif.), 56(4), 471–6. Boulenguez, P., & Vinay, L. (2009). Strategies to restore motor functions after spinal cord injury. Current opinion in neurobiology, 19(6), 587–600. Cizkova, D., Kakinohana, O., Kucharova, K., Marsala, S., Johe, K., Hazel, T., Hefferan, M. P., et al. (2007). Functional Recovery in Rats with Ischemic Paraplegia after Spinal Grafting of Human Spinal Stem Cells. Neuroscience, 147(2), 546–560. Dumont, R. J., Okonkwo, D. O., Verma, S., Hurlbert, R. J., Boulos, P. T., Ellegala, D. B., & Dumont, a S. (2001). Acute spinal cord injury, part I: pathophysiologic mechanisms. Clinical neuropharmacology, 24(5), 254–64. Haase, A., Olmer, R., Schwanke, K., Wunderlich, S., Merkert, S., Hess, C., Zweigerdt, R., et al. (2009). Generation of induced pluripotent stem cells from human cord blood. Cell stem cell, 5(4), 434–41. Hawryluk, G. W. J., Rowland, J., Kwon, B. K., & Fehlings, M. G. (2008). Protection and repair of the injured spinal cord: a review of completed, ongoing, and planned clinical trials for acute spinal cord injury. Neurosurgical focus, 25(5), Hofstetter, C. P., Holmström, N. A. V, Lilja, J. A., Schweinhardt, P., Hao, J., Spenger, C., Wiesenfeld-Hallin, Z., et al. (2005). Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nature neuroscience, 8(3), 346–53. Kakinohana, O., Juhasova, J., Juhas, S., Motlik, J., Platoshyn, O., Galik, J., Hefferan, M., et al. (2012). Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naïve immunosuppressed minipigs: a qualitative and quantitative study. Cell transplantation. doi:10.3727/096368912X653200 Keirstead, H. S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K., & Steward, O. (2005). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. The Journal of neuroscience?: the official journal of the Society for Neuroscience, 25(19), 4694–705. doi:10.1523/JNEUROSCI.0311-05.2005 Lin, T. C. Y., & Lee, O. K. S. (2008). Stem cells: a primer. The Chinese journal of physiology, 51(4), 197–207. Lu, P., Wang, Y., Graham, L., McHale, K., Gao, M., Wu, D., Brock, J., et al. (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell, 150(6), 1264–73. Navarro, R., Juhas, S., Keshavarzi, S., Juhasova, J., Motlik, J., Johe, K., Marsala, S., et al. (2012a). Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study. Journal of neurotrauma, 29(3), 499–513. Navarro, R., Juhas, S., Keshavarzi, S., Juhasova, J., Motlik, J., Johe, K., Marsala, S., et al. (2012b). Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study. Journal of neurotrauma, 29(3), 499–513. Okamura, R. M., Lebkowski, J., Au, M., Priest, C. A., Denham, J., & Majumdar, A. S. (2007). Immunological properties of human embryonic stem cell-derived oligodendrocyte progenitor cells. Journal of neuroimmunology, 192(1-2), 134–44. Olmer, R., Haase, A., Merkert, S., Cui, W., Palecek, J., Ran, C., Kirschning, A., et al. (2010). Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem cell research, 5(1), 51–64. Riley, J., Federici, T., Polak, M., Kelly, C., Glass, J., Raore, B., Taub, J., et al. (2012). Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: a phase I safety trial, technical note, and lumbar safety outcomes. Neurosurgery, 71(2), 405–16; discussion 416. Usvald, D., Vodicka, P., Hlucilova, J., Prochazka, R., Motlik, J., Kuchorova, K., Johe, K., et al. (2010). Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell transplantation, 19(9), 1103–22. Yuan, S. H., Martin, J., Elia, J., Flippin, J., Paramban, R. I., Hefferan, M. P., Vidal, J. G., et al. (2011). Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PloS one, 6(3), e17540. Zhang, W., Dahlberg, J. E., & Tam, W. (2007). MicroRNAs in tumorigenesis: a primer. The American journal of pathology, 171(3), 728–38. Zhang, Y. W., Denham, J., & Thies, R. S. (2006a). Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors. Stem cells and development, 15(6), 943–52. Zhang, Y. W., Denham, J., & Thies, R. S. (2006b). Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors. Stem cells and …, 952, 943–952.

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