Human Neuronal Stem Cells Human Neuronal Stem Cells Differentiate and Promote Differentiate and Promote
Locomotor Recovery in Spinal Locomotor Recovery in Spinal Cord-Injured MiceCord-Injured Mice
Human Neuronal Stem Cells Human Neuronal Stem Cells Differentiate and Promote Differentiate and Promote
Locomotor Recovery in Spinal Locomotor Recovery in Spinal Cord-Injured MiceCord-Injured Mice
Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen
AndersonAnderson
Journal Club Presentation:Journal Club Presentation:
Alvin P. Penalosa, MDAlvin P. Penalosa, MD
Neurosurgery Senior House OfficerNeurosurgery Senior House OfficerNewcastle General HospitalNewcastle General Hospital
Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Brian J Cummings, Nobuko Uchida, Stanley Tamaki, Desiree Salazar, Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen Mitra Hooshmand, Robert Summers, Fred Gage, and Aileen
AndersonAnderson
Journal Club Presentation:Journal Club Presentation:
Alvin P. Penalosa, MDAlvin P. Penalosa, MD
Neurosurgery Senior House OfficerNeurosurgery Senior House OfficerNewcastle General HospitalNewcastle General Hospital
ObjectivesObjectivesObjectivesObjectives
Definitive identification of transplanted cellsDefinitive identification of transplanted cells
Long-term survival and Engraftment dataLong-term survival and Engraftment data
Evidence of DifferentiationEvidence of Differentiation
Direct evidence of functional integration of Direct evidence of functional integration of cells in injured spinal cordcells in injured spinal cord
Definitive identification of transplanted cellsDefinitive identification of transplanted cells
Long-term survival and Engraftment dataLong-term survival and Engraftment data
Evidence of DifferentiationEvidence of Differentiation
Direct evidence of functional integration of Direct evidence of functional integration of cells in injured spinal cordcells in injured spinal cord
EndpointsEndpointsEndpointsEndpoints
Selected stem cell neurospheres survive, Selected stem cell neurospheres survive, engraft, differentiate, and are associated engraft, differentiate, and are associated with locomotor improvementswith locomotor improvements
Selective ablation by Diptheria toxin results Selective ablation by Diptheria toxin results in loss of locomotor recoveryin loss of locomotor recovery
Transplanted stem cells remyelinate axons Transplanted stem cells remyelinate axons and differentiate into neurons based on and differentiate into neurons based on electron microscopyelectron microscopy
Selected stem cell neurospheres survive, Selected stem cell neurospheres survive, engraft, differentiate, and are associated engraft, differentiate, and are associated with locomotor improvementswith locomotor improvements
Selective ablation by Diptheria toxin results Selective ablation by Diptheria toxin results in loss of locomotor recoveryin loss of locomotor recovery
Transplanted stem cells remyelinate axons Transplanted stem cells remyelinate axons and differentiate into neurons based on and differentiate into neurons based on electron microscopyelectron microscopy
Methods: Contusion Methods: Contusion InjuriesInjuries
Methods: Contusion Methods: Contusion InjuriesInjuries
Mice received a laminectomy at the T9 spineMice received a laminectomy at the T9 spine
One Cohort: 50kd contusion spinal cord One Cohort: 50kd contusion spinal cord injury + randomization to either hCNS-SCns injury + randomization to either hCNS-SCns (n=16) or vehicle (n=19)(n=16) or vehicle (n=19)
22ndnd Cohort: 60kd contusion spinal cord injury Cohort: 60kd contusion spinal cord injury + randomization to stem cell or vehicle+ randomization to stem cell or vehicle
Mice received a laminectomy at the T9 spineMice received a laminectomy at the T9 spine
One Cohort: 50kd contusion spinal cord One Cohort: 50kd contusion spinal cord injury + randomization to either hCNS-SCns injury + randomization to either hCNS-SCns (n=16) or vehicle (n=19)(n=16) or vehicle (n=19)
22ndnd Cohort: 60kd contusion spinal cord injury Cohort: 60kd contusion spinal cord injury + randomization to stem cell or vehicle+ randomization to stem cell or vehicle
Stem Cells: HarvestingStem Cells: Harvesting
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Methods: stem cell Methods: stem cell injectionsinjections
Methods: stem cell Methods: stem cell injectionsinjections
Extract from fetal brain concentrated to Extract from fetal brain concentrated to 75,000 cells/microlitre in injection buffer75,000 cells/microlitre in injection buffer
9 days post-injury, 4 injections (250nl of cells 9 days post-injury, 4 injections (250nl of cells vs vehicle) bilaterally 0.75mm from midline vs vehicle) bilaterally 0.75mm from midline at both anterior aspect of T10 and posterior at both anterior aspect of T10 and posterior aspect of T8aspect of T8
Extract from fetal brain concentrated to Extract from fetal brain concentrated to 75,000 cells/microlitre in injection buffer75,000 cells/microlitre in injection buffer
9 days post-injury, 4 injections (250nl of cells 9 days post-injury, 4 injections (250nl of cells vs vehicle) bilaterally 0.75mm from midline vs vehicle) bilaterally 0.75mm from midline at both anterior aspect of T10 and posterior at both anterior aspect of T10 and posterior aspect of T8aspect of T8
Methods: Blinded Methods: Blinded AssessmentAssessment
Methods: Blinded Methods: Blinded AssessmentAssessment
Functional recovery via BBB locomotor rating Functional recovery via BBB locomotor rating scale weekly for 1 month by observers scale weekly for 1 month by observers blinded to treatmentblinded to treatment
16 weeks: videotaped on horizontal ladder 16 weeks: videotaped on horizontal ladder beam task and scored blind for step errorsbeam task and scored blind for step errors
Euthanasia as per ethics guidelinesEuthanasia as per ethics guidelines
Functional recovery via BBB locomotor rating Functional recovery via BBB locomotor rating scale weekly for 1 month by observers scale weekly for 1 month by observers blinded to treatmentblinded to treatment
16 weeks: videotaped on horizontal ladder 16 weeks: videotaped on horizontal ladder beam task and scored blind for step errorsbeam task and scored blind for step errors
Euthanasia as per ethics guidelinesEuthanasia as per ethics guidelines
Differentiation and Differentiation and EngraftmentEngraftment
Differentiation and Differentiation and EngraftmentEngraftment
Immunostaining for human nuclear antigen Immunostaining for human nuclear antigen (SC101) or human cytoplasmic antigen (SC101) or human cytoplasmic antigen (SC121)(SC121)
Electron MicroscopyElectron Microscopy
Immunostaining for human nuclear antigen Immunostaining for human nuclear antigen (SC101) or human cytoplasmic antigen (SC101) or human cytoplasmic antigen (SC121)(SC121)
Electron MicroscopyElectron Microscopy
RESULTSRESULTS
EngraftmentEngraftment
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 RecoveryLocomotor 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)
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DiscussionDiscussion
Cell-based TherapeuticsCell-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
ConclusionsConclusions
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
IssuesIssues
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 FutureThe Future
Geron Corporation: the first Biotech company given USFDA approval to use this technique on 10 selected spinal cord-injured human patients
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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