Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration

Abstract

The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In murine models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of SCI. Corticospinal regeneration requires grafts to be driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, a process that is potentially related to the attenuation of the glial scar. Rat corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord 'replacement' with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of SCI research and offering new possibilities for clinical translation.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Corticospinal axons extensively regenerate into NPC grafts.
Figure 2: Axonal regeneration beyond the graft in focal CST lesions.
Figure 3: Electrophysiological connectivity between regenerating corticospinal axons and grafted neurons.
Figure 4: Corticospinal regeneration requires an injury signal and contact with neural grafts.
Figure 5: Corticospinal regeneration requires caudalized, homotypic grafts and enables functional improvement.
Figure 6: Homotypic human NPC grafts support corticospinal regeneration.

References

  1. Liu, K., Tedeschi, A., Park, K.K. & He, Z. Neuronal intrinsic mechanisms of axon regeneration. Annu. Rev. Neurosci. 34, 131–152 (2011).

    PubMed  Google Scholar 

  2. Tuszynski, M.H. & Steward, O. Concepts and methods for the study of axonal regeneration in the CNS. Neuron 74, 777–791 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bareyre, F.M. et al. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat. Neurosci. 7, 269–277 (2004).

    CAS  PubMed  Google Scholar 

  4. Rosenzweig, E.S. et al. Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury. Nat. Neurosci. 13, 1505–1510 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Weidner, N., Ner, A., Salimi, N. & Tuszynski, M.H. Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury. Proc. Natl. Acad. Sci. USA 98, 3513–3518 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Schnell, L. & Schwab, M.E. Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 343, 269–272 (1990).

    CAS  PubMed  Google Scholar 

  7. GrandPré, T., Li, S. & Strittmatter, S.M. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417, 547–551 (2002).

    PubMed  Google Scholar 

  8. Liu, K. et al. PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat. Neurosci. 13, 1075–1081 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zukor, K. et al. Short hairpin RNA against PTEN enhances regenerative growth of corticospinal tract axons after spinal cord injury. J. Neurosci. 33, 15350–15361 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ohtake, Y. et al. The effect of systemic PTEN antagonist peptides on axon growth and functional recovery after spinal cord injury. Biomaterials 35, 4610–4626 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Starkey, M.L. & Schwab, M.E. Anti-Nogo-A and training: can one plus one equal three? Exp. Neurol. 235, 53–61 (2012).

    CAS  PubMed  Google Scholar 

  12. Hollis, E.R. II, Jamshidi, P., Löw, K., Blesch, A. & Tuszynski, M.H. Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation. Proc. Natl. Acad. Sci. USA 106, 7215–7220 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Xu, X.M., Guénard, V., Kleitman, N., Aebischer, P. & Bunge, M.B. A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord. Exp. Neurol. 134, 261–272 (1995).

    CAS  PubMed  Google Scholar 

  14. Vavrek, R., Pearse, D.D. & Fouad, K. Neuronal populations capable of regeneration following a combined treatment in rats with spinal cord transection. J. Neurotrauma 24, 1667–1673 (2007).

    PubMed  Google Scholar 

  15. Lu, P. et al. Motor axonal regeneration after partial and complete spinal cord transection. J. Neurosci. 32, 8208–8218 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee, Y.S. et al. Nerve regeneration restores supraspinal control of bladder function after complete spinal cord injury. J. Neurosci. 33, 10591–10606 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Cummings, B.J. et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord–injured mice. Proc. Natl. Acad. Sci. USA 102, 14069–14074 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bonner, J.F. et al. Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord. J. Neurosci. 31, 4675–4686 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lu, P. et al. Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150, 1264–1273 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Lu, P. et al. Long-distance axonal growth from human induced pluripotent stem cells after spinal cord injury. Neuron 83, 789–796 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Peljto, M., Dasen, J.S., Mazzoni, E.O., Jessell, T.M. & Wichterle, H. Functional diversity of ESC-derived motor neuron subtypes revealed through intraspinal transplantation. Cell Stem Cell 7, 355–366 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ma, L. et al. Human embryonic stem cell–derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice. Cell Stem Cell 10, 455–464 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Mayer-Proschel, M., Kalyani, A.J., Mujtaba, T. & Rao, M.S. Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19, 773–785 (1997).

    CAS  PubMed  Google Scholar 

  24. Du Beau, A. et al. Neurotransmitter phenotypes of descending systems in the rat lumbar spinal cord. Neuroscience 227, 67–79 (2012).

    CAS  PubMed  Google Scholar 

  25. Perry, R.B. et al. Subcellular knockout of importin β1 perturbs axonal retrograde signaling. Neuron 75, 294–305 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Cho, Y., Sloutsky, R., Naegle, K.M. & Cavalli, V. Injury-induced HDAC5 nuclear export is essential for axon regeneration. Cell 155, 894–908 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Fawcett, J.W., Schwab, M.E., Montani, L., Brazda, N. & Müller, H.W. Defeating inhibition of regeneration by scar and myelin components. Handb. Clin. Neurol. 109, 503–522 (2012).

    PubMed  Google Scholar 

  28. Liu, Y. & Rao, M.S. Glial progenitors in the CNS and possible lineage relationships among them. Biol. Cell 96, 279–290 (2004).

    CAS  PubMed  Google Scholar 

  29. Cao, Q.L., Howard, R.M., Dennison, J.B. & Whittemore, S.R. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord. Exp. Neurol. 177, 349–359 (2002).

    CAS  PubMed  Google Scholar 

  30. García-Alías, G., Barkhuysen, S., Buckle, M. & Fawcett, J.W. Chondroitinase ABC treatment opens a window of opportunity for task-specific rehabilitation. Nat. Neurosci. 12, 1145–1151 (2009).

    PubMed  Google Scholar 

  31. Conner, J.M., Chiba, A.A. & Tuszynski, M.H. The basal forebrain cholinergic system is essential for cortical plasticity and functional recovery following brain injury. Neuron 46, 173–179 (2005).

    CAS  PubMed  Google Scholar 

  32. Montoya, C.P., Campbell-Hope, L.J., Pemberton, K.D. & Dunnett, S.B. The “staircase test”: a measure of independent forelimb reaching and grasping abilities in rats. J. Neurosci. Methods 36, 219–228 (1991).

    CAS  PubMed  Google Scholar 

  33. Wahl, A.S. et al. Neuronal repair. Asynchronous therapy restores motor control by rewiring of the rat corticospinal tract after stroke. Science 344, 1250–1255 (2014).

    CAS  PubMed  Google Scholar 

  34. Alaverdashvili, M. & Whishaw, I.Q. Motor cortex stroke impairs individual digit movement in skilled reaching by the rat. Eur. J. Neurosci. 28, 311–322 (2008).

    PubMed  Google Scholar 

  35. Whishaw, I.Q., Gorny, B. & Sarna, J. Paw and limb use in skilled and spontaneous reaching after pyramidal tract, red nucleus and combined lesions in the rat: behavioral and anatomical dissociations. Behav. Brain Res. 93, 167–183 (1998).

    CAS  PubMed  Google Scholar 

  36. Girgis, J. et al. Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery. Brain 130, 2993–3003 (2007).

    CAS  PubMed  Google Scholar 

  37. Krajacic, A., Weishaupt, N., Girgis, J., Tetzlaff, W. & Fouad, K. Training-induced plasticity in rats with cervical spinal cord injury: effects and side effects. Behav. Brain Res. 214, 323–331 (2010).

    PubMed  Google Scholar 

  38. Steward, O., Sharp, K.G. & Matsudaira Yee, K. Long-distance migration and colonization of transplanted neural stem cells. Cell 156, 385–387 (2014).

    CAS  PubMed  Google Scholar 

  39. Tuszynski, M.H. et al. Neural stem cell dissemination after grafting to CNS injury sites. Cell 156, 388–389 (2014).

    CAS  PubMed  Google Scholar 

  40. Tuszynski, M.H. et al. Neural stem cells in models of spinal cord injury. Exp. Neurol. 261, 494–500 (2014).

    CAS  PubMed  Google Scholar 

  41. Steward, O., Sharp, K.G., Yee, K.M., Hatch, M.N. & Bonner, J.F. Characterization of ectopic colonies that form in widespread areas of the nervous system with neural stem cell transplants into the site of a severe spinal cord injury. J. Neurosci. 34, 14013–14021 (2014).

    PubMed  PubMed Central  Google Scholar 

  42. Garcia-Ovejero, D. et al. The ependymal region of the adult human spinal cord differs from other species and shows ependymoma-like features. Brain 138, 1583–1597 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. Conti, L. & Cattaneo, E. Neural stem cell systems: physiological players or in vitro entities? Nat. Rev. Neurosci. 11, 176–187 (2010).

    CAS  PubMed  Google Scholar 

  44. Chen, H. et al. Human-derived neural progenitors functionally replace astrocytes in adult mice. J. Clin. Invest. 125, 1033–1042 (2015).

    PubMed  PubMed Central  Google Scholar 

  45. Blackmore, M.G. et al. Krüppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract. Proc. Natl. Acad. Sci. USA 109, 7517–7522 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Norenberg, M.D., Smith, J. & Marcillo, A. The pathology of human spinal cord injury: defining the problems. J. Neurotrauma 21, 429–440 (2004).

    PubMed  Google Scholar 

  47. Coumans, J.V. et al. Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J. Neurosci. 21, 9334–9344 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Molofsky, A.V. et al. Astrocyte-encoded positional cues maintain sensorimotor circuit integrity. Nature 509, 189–194 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Wu, Y., Liu, Y., Chesnut, J.D. & Rao, M.S. Isolation of neural stem and precursor cells from rodent tissue. Methods Mol. Biol. 438, 39–53 (2008).

    CAS  PubMed  Google Scholar 

  50. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  PubMed  Google Scholar 

  51. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  PubMed  Google Scholar 

  52. Zhang, S.C., Wernig, M., Duncan, I.D., Brüstle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

    CAS  PubMed  Google Scholar 

  53. Hu, B.Y. & Zhang, S.C. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat. Protoc. 4, 1295–1304 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Weidner, N., Blesch, A., Grill, R.J. & Tuszynski, M.H. Nerve growth factor–hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1. J. Comp. Neurol. 413, 495–506 (1999).

    CAS  PubMed  Google Scholar 

  55. Kadoya, K. et al. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury. Neuron 64, 165–172 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Jones, L.L. & Tuszynski, M.H. Spinal cord injury elicits expression of keratan sulfate proteoglycans by macrophages, reactive microglia, and oligodendrocyte progenitors. J. Neurosci. 22, 4611–4624 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Wang, D., Ichiyama, R.M., Zhao, R., Andrews, M.R. & Fawcett, J.W. Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury. J. Neurosci. 31, 9332–9344 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Graham, Y. Yang, E. Boehle, J.K. Lee, M. Kim, E. Liu, R. Pope and T. Moynihan for their technical assistance; Planned Parenthood; the Rat Resource and Research Center, University of Missouri, Columbia, Missouri, for providing GFP rats; James Thompson at the University of Wisconsin–Madison, for providing hiPSC line IMR90; K. Wewetzer, University of Freiburg, Germany, for providing 27C7 antibody; R. Darnell, the Rockefeller University, New York, for providing Hu antibody; Y. Jones for use of the electron-microscopy core facility at Cellular and Molecular Medicine, University of California, San Diego; and the Nikon Imaging Center at Hokkaido University for use of the confocal laser microscope. This work was supported by the US Veterans Administration (Gordon Mansfield Spinal Cord Injury Consortium; to M.H.T. and P.L.) the US National Institutes of Health (NS042291 to M.H.T. and GM008349 to J.K.); the Craig H. Neilsen Foundation (to K.K., H.K. and J.N.D.); the Bernard and Anne Spitzer Charitable Trust (to M.H.T.); the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to M.H.T. and J.C.); Kobayashi Hospital Furate Research Fund (to K.K.); the Japan Society for the Promotion of Science (to H.K.); and the Busta Family and Bleser Family funds (to S.C.Z.).

Author information

Authors and Affiliations

Authors

Contributions

K.K. conceived and carried out experiments, interpreted the results and wrote the manuscript. P.L. contributed to the conception of the project and performed complete-transection experiments. K.N. and H.S. carried out experiments. C.L.-K. and J.N.D. contributed to behavior analysis. G.P. contributed to mouse experiments. H.K. contributed to the characterization of human NPCs. L.Y., J.K. and S.-C.Z. contributed to the generation of human NPCs from iPSCs. J.B., Y.T. and J.C. performed the electrophysiological analysis. M.H.T. contributed to the conception of the project and the interpretation of results, and wrote the manuscript.

Corresponding author

Correspondence to Mark H Tuszynski.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 20425 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kadoya, K., Lu, P., Nguyen, K. et al. Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration. Nat Med 22, 479–487 (2016). https://doi.org/10.1038/nm.4066

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4066

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing