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 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

ObjectivesObjectives 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 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

EndpointsEndpoints 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 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

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) 2 nd Cohort: 60kd contusion spinal cord injury + randomization to stem cell or vehicle 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) 2 nd Cohort: 60kd contusion spinal cord injury + randomization to stem cell or vehicle

Stem Cells: Harvesting

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 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

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 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

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

RESULTS

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 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

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) 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)

Discussion

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 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

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

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 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

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