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Human Neuronal Stem Cells Differentiate and Promote Locomotor Recovery in Spinal Cord-Injured Mice

Human Neuronal Stem Cells Differentiate and Promote Locomotor Recovery in Spinal Cord-Injured Mice. Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen Anderson Journal Club Presentation: Alvin P. Penalosa, MD

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Human Neuronal Stem Cells Differentiate and Promote Locomotor Recovery in Spinal Cord-Injured Mice

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  1. Human Neuronal Stem Cells Differentiate and Promote Locomotor Recovery in Spinal Cord-Injured Mice • Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen Anderson • Journal Club Presentation: • Alvin P. Penalosa, MD • Neurosurgery Senior House Officer • Newcastle General Hospital

  2. Objectives • Definitive identification of transplanted cells • Long-term survival and Engraftment data • Evidence of Differentiation • Direct evidence of functional integration of cells in injured spinal cord

  3. Endpoints • Selected stem cell neurospheres survive, engraft, differentiate, and are associated with locomotor improvements • Selective ablation by Diptheria toxin results in loss of locomotor recovery • Transplanted stem cells remyelinate axons and differentiate into neurons based on electron microscopy

  4. Methods: Contusion Injuries • Mice received a laminectomy at the T9 spine • One Cohort: 50kd contusion spinal cord injury + randomization to either hCNS-SCns (n=16) or vehicle (n=19) • 2nd Cohort: 60kd contusion spinal cord injury + randomization to stem cell or vehicle

  5. Stem Cells: Harvesting

  6. Methods: stem cell injections • Extract from fetal brain concentrated to 75,000 cells/microlitre in injection buffer • 9 days post-injury, 4 injections (250nl of cells vs vehicle) bilaterally 0.75mm from midline at both anterior aspect of T10 and posterior aspect of T8

  7. Methods: Blinded Assessment • Functional recovery via BBB locomotor rating scale weekly for 1 month by observers blinded to treatment • 16 weeks: videotaped on horizontal ladder beam task and scored blind for step errors • Euthanasia as per ethics guidelines

  8. Differentiation and Engraftment • Immunostaining for human nuclear antigen (SC101) or human cytoplasmic antigen (SC121) • Electron Microscopy

  9. RESULTS

  10. Engraftment • 24h, 48h, 4 weeks, 17 weeks • extensive survival and engraftment in both gray and white matter • no migration in lesion epicenter with a rim surounding part of contused cord • ?myelination or regeneration of spared axons form bridge circuits=recovery

  11. Locomotor Recovery • 16 weeks: BBB scoring suggested recovery of coordinated forelimb-hindlimb locomotor function in transplanted mice (BBB>12) vs vehicle P<0.05; Chi square=3.94) • horizontal ladder beam task: grafted mice (n=9) exhibited fewer mistakes averaging 4.2 (SE 1.2) vs 13.5 (SE 4.1) in the vehicle group (n=12)

  12. Discussion

  13. Cell-based Therapeutics • Diptheria Toxin: reversal of locomotor improvement suggests survival of stem cells vital in maintenance of improved performance • differentiation into myelinating oligodendrocytes and neurons with EM criteria=?mechanism for sustained locomotor recovery

  14. Conclusions • survival and differentiation in a traumatically-injured surrounding without contributing to scarring • plays a role in locomotor recovery

  15. Issues • further studies to prove the exact mechanism by which locomotor recovery is achieved • application in human models and the issue of graft/tissue/cell rejection

  16. The Future • Geron Corporation: the first Biotech company given USFDA approval to use this technique on 10 selected spinal cord-injured human patients

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