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

QuickTime™ and aH.264 decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

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)

QuickTime™ and a decompressor

are needed to see this picture.

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