cell transplantation to the rescue: repairing the injured...
TRANSCRIPT
![Page 1: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/1.jpg)
®
Cell transplantation to the rescue: Repairing the injured spinal cord
Mary Bartlett Bunge, PhD
Christine E. Lynn Distinguished Professor of Neuroscience
Professor of Cell Biology, Neurological Surgery, and Neurology
The Miami Project to Cure Paralysis University of Miami Miller School of Medicine
![Page 2: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/2.jpg)
Founders (1985) Current Staff -Barth Green, M.D. - 21 basic science faculty -Donald Misner - 16 clinical science faculty -Beth Roscoe - 12 management -Nick Buoniconti - 18 post-docs -Marc Buoniconti - 17 graduate students
- 104 research staffScientific Directors - 41 support staff
-ÅkeSeiger, M.D., Ph.D. - other students (high -Richard Bunge, M.D. school, undergrad, -W. Dalton Dietrich, Ph.D. medical, residents)
![Page 3: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/3.jpg)
![Page 4: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/4.jpg)
![Page 5: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/5.jpg)
![Page 6: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/6.jpg)
Spinal Cord Injury (SCI) • 12,000 new cases / year in the U.S.• Approx 1.275 million Americans with
paralysis due to SCI; 5.3 million with paralysis due to some type of CNS injury/disorder
• Nearly 50%, ages 16-30; 80%,males• Higher occurrence in military, Dept of
Defense funding • MVAs (36.5%), falls (28.5), violence (14)
![Page 7: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/7.jpg)
Quality of Life Issues with SCI
Muscle paralysis, loss of feeling Reduced pulmonary function Early cardiovascular disease, stroke, DVTsLack of bowel and bladder control; infection Temperature, blood pressure vary widelyPressure ulcers, osteoporosis Sexual function/fertility impaired Pain long–term, 80 %Obesity, diabetes, cognitive decline
![Page 8: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/8.jpg)
Cultured cells for CNS repair
“If we have normal cell types available in culture and if a nervous system disease stems from an absence or deficiency of one of these cell types, then it is an obvious step to transfer cultured cells into the diseased animal to attempt to correct the deficiency. Whereas at present this would not seem practical for many types of neurons, it may be practical for supporting cells (e.g. Schwann or glial).”
Richard P Bunge, The changing uses of nerve tissue culture 1950-1975 in The Nervous System, Donald B Tower, editor in chief, Vol 1 the Basic Neurosciences, 1975.
![Page 9: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/9.jpg)
![Page 10: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/10.jpg)
Nerve cell (neuron)
![Page 11: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/11.jpg)
Peripheral nerve
![Page 12: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/12.jpg)
![Page 13: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/13.jpg)
Peripheral nerve Schwann cell myelinated axons
![Page 14: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/14.jpg)
Myelin formation by Schwann cells
![Page 15: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/15.jpg)
![Page 16: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/16.jpg)
Albert J. Aguayo, M.D.
![Page 17: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/17.jpg)
![Page 18: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/18.jpg)
![Page 19: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/19.jpg)
Photo of St. Louis Arch
![Page 20: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/20.jpg)
Patrick M.Wood, PhD
![Page 21: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/21.jpg)
Schwann cell biology in vitro
• SCs generate a number of ECM components• SCs require contact with ECM to
differentiate• SCs require contact with axons to form
basal lamina• SCs require basal lamina to form myelin• SCs require ascorbic acid to form myelin
(Type IV collagen)
[Defined medium (N2) supported SC profileration but not differentiation]
![Page 22: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/22.jpg)
Schwann cell biology in vitro - 2
• Mitogenic signal on axons for SC proliferation (control of SC #)
• Perineurium formed in cultures of neurons, SCs and fibroblasts
• Perineurium formed from fibroblasts, not SCs (Lac-Z retrovirus)
• Circumnavigation by inner lip of SC cytoplasm a part of myelin formation mechanism
![Page 23: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/23.jpg)
![Page 24: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/24.jpg)
![Page 25: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/25.jpg)
![Page 26: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/26.jpg)
Why Schwann cells?• Promote axon regeneration/PN• Aguayo team; CNS axons regenerated into PN• Are readily accessible in PN• Produce growth factors, ECM • Myelinate axons in CNS, restore activity• Can obtain large numbers in culture• Can transplant person’s own cells• Can genetically engineer cells• Promising pre-clinical data in multiple species
(rats, pigs, primates) ; FDA approval for human SCs
![Page 27: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/27.jpg)
Xu XM, Guénard V, Kleitman N, Bunge MB (1995) Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. J Comp Neurol 351:145-160.
Xu XM, Guénard V, Kleitman N, Aebischer P, Bunge MB (1995) 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.
Xu XM, Chen A, Guénard V, Kleitman N, Bunge MB (1997) Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord. J Neurocytol 26:1-16.
Xiao-Ming Xu, M.D., Ph.D.
![Page 28: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/28.jpg)
PAN/PVC tube
SC
Scanning EM
*
![Page 29: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/29.jpg)
How do we measure outcome?-Behavior: Walking? Pain assessment?-Histology: Lesion volume, overall tissue changes -Transplanted SC survival and number?-Count SC-myelinated axons? Total # axons?-Immunostaining
Scar formation?Types of axons in implant, and beyond?Inflammatory response?
-Neuroanatomical tracing What spinal and supraspinal neurons responded? What types of axons regenerated into implant?Did these axons leave implant?
![Page 30: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/30.jpg)
SC/Matrigel bridge at 30 dpt
![Page 31: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/31.jpg)
![Page 32: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/32.jpg)
Thoracic complete transection model: Schwann cells
• Axons grow onto SC bridge from both stumps
• 2000 myelinated axons on bridge• 8x more non-myelinated axons/bridge
![Page 33: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/33.jpg)
Transplants at 12 wk after contusion injury
LV-GFP
Dr. Damien Pearse
![Page 34: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/34.jpg)
Thoracic contusion model: Schwann cells
• Reduce cyst formation• Protect spinal cord tissue from
secondary damage • Support axonal growth into SC graft• ~5000 SC-myelinated axons in graft• Some improvement in walking after
paralysis
![Page 35: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/35.jpg)
Injured spinal cord tissue environment -1
• Excessive EAA (e.g., glutamate) release• EAA receptor activation• Excessive Ca++ entry into cells• Ca++ stimulated activation of proteolytic and
other degradative enzymes• Cytoskeletal protein breakdown• Oxygen free radical release leading to
membrane damage• Cytokine, chemokine release
![Page 36: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/36.jpg)
Injured spinal cord tissue environment-2
• Many tissue damaging sequelae initiated, leading to secondary damage
• Substances inhibitory to repair appear• Inflammation begins• Barrier to axon growth (scar) forms• Sustenance for surviving nerve cells cut off• Cyst created due to tissue loss, preventing
axon regrowth
![Page 37: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/37.jpg)
To repair the cord - 1
• Halt spread of secondary tissue damage
• Save as many nerve cells as possible• Curb inflammation• Reduce scar formation• Neutralize inhibitory factors
![Page 38: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/38.jpg)
To repair the cord - 2
• Awaken nerve cells to regrow axons• Provide sustenance to nerve cells• Promote axon growth across injury• Guide growth to appropriate area• Enable formation of connections
![Page 39: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/39.jpg)
Combination strategies: SCs +
• Steroid (used clinically)• Variety of growth factors• Another cell type (olfactory ensheathing
cells)• An enzyme (to reduce scar formation)• Elevation of a cell signaling molecule, cAMP• Introduction of genes to improve Schwann
cell efficacy
ALL THESE IMPROVED REPAIR/FUNCTION
![Page 40: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/40.jpg)
Combination strategies: SCs +
• More SC-myelinated axons in graft • Increase in regenerated axons from
neurons above spinal cord (Distance factor overcome)
• Exit of regenerated axons from graft into the cord
• More improvement in walking
![Page 41: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/41.jpg)
SCs with added growth factor gene survive better and improve axon growth
Control SCs
SCs + growth factor
![Page 42: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/42.jpg)
GF levels signif. increased in cord at 2 dpt, low levels up to 6 wpt
GF led to 5X increase in graft vol, SC #GF led to a signif. increase in myel.
axons (SC 5,100; LV-GFP SCs 4,600; LV-GFP/GF SCS 26,000) and total axons (~75,000)
DβH+ fiber length signif. increased with GFNo improvement in walking
Viral Vector/growth factor (GF)-
engineered SC study (2007)
![Page 43: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/43.jpg)
Combined engineering of Schwann cell implantsto secrete neurotrophin and chondroitinase promotes
axonal regeneration and locomotion after SCI
H Kanno, Y Pressman, A Moody, R Berg, E Muir, J Rogers, H Ozawa, E Itoi, D Pearse, M Bunge
![Page 44: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/44.jpg)
Haruo Kanno, M.D., Ph.D.
![Page 45: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/45.jpg)
Experiment Adult female Fischer rats (160-180g)
• Moderate contusion injury at thoracic 8 level
Schwann cell transplantation:• 1 week after injury, 1+1 million cells
Experimental groups:1. Vehicle (no cells) (control)2. green-SCs + red-SCs (control)3. green/GF-SCs + red-SCs4. green-SCs + red/ENZ-SCs5. green/GF-SCs + red/ENZ-SCs
![Page 46: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/46.jpg)
![Page 47: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/47.jpg)
2B6 staining
GFPmCherry/ChABC2B6
1000μm
Chondroitinase activity in vivo
![Page 48: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/48.jpg)
SC-transplanted spinal cord
Combination strategyControl
![Page 49: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/49.jpg)
0
10000
20000
30000
40000
50000
60000
70000
80000
1) DMEM/F122) GFP + mCherry3) GFP/D15A + mCherry4) GFP + mCherry/Ch'ase5) GFP/D15A + mCherry/Ch'ase
# SC
mye
linat
ed a
xons
/ se
ctio
n
SC myelination in implant
**
DMEM/F12GFP + mCherryGFP/GF+ mCherryGFP + mCherry/ChABCGFP/GF + mCherry/ChABC
#
![Page 50: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/50.jpg)
Combination vs single treatments
• Further increase in implant compared to GF or ENZ
• Further increase in SC myel axons compared to ENZ
• Higher number of responding neurons above cord than with GF or ENZ
• Higher number of their axons in implant and in cord beyond than with GF or ENZ
• Improved walking scores• Lessened pain in hindpaws
![Page 51: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/51.jpg)
Experimental Neurology 1997;148:502-22
The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cordGuest JD, Rao A, Olson L, Bunge MB, Bunge RP
The Miami Project to Cure Paralysis, The Organ Procurement Team, Department of Neurological Surgery, University of Miami School of Medicine, Miami, Florida__________________________________________________________________________
Nude athymic rats Human SCs/Matrigel, with or without PAN/PVC
channels T8 complete transection, T9-10 cord removed Methylprednisolone
![Page 52: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/52.jpg)
Myelinated axons, ~2,000
Propriospinal, sensory, motoneuronal, brainstem
(DβH, 5HT) axons in SC bridgePropriospinal and sensory axons re-entered the
spinal cord (up to 2.6 mm)Walking scores significantly improved __________________________________________________
Robust axon growth into SC bridge, some growth beyond and modest improvement in locomotion
![Page 53: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/53.jpg)
Human Schwann cells
Can be isolated and readily increased in # in culture Up to 10 m SCs, single adult sural nerve biopsy
100 m SCs in 3-4 wk Variation in rate of increase from donor to donor Preparation: Careful dissection of bundles Intro culture medium with forskolin (↑cAMP) and heregulin
(mitogenic), 1 wk Overnight incubation in collagenase and dispase; then
mechanical dissociation of fascicles by trituration Onto laminin, with forskolin and heregulin (all reduce
fibroblasts, leading to 95% SCs or higher)
![Page 54: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/54.jpg)
APPROVAL FROM THE FDA FOR OUR FIRST SC TRIAL
Phase 1 (for safety) Clinical trial: to inject person’s own Schwann cells (from peripheral nerve*) into site of SCI
SC number increased in culture for 3-5 wkSCs injected into lesion epicenter_______________*From single adult sural nerve biopsy, 100 m
SCs/ 3-4 wk
![Page 55: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/55.jpg)
Feasibility for trial? (Many rodent studies over nearly 20 years)Detailed characterization of manufactured hSCsPreclinical safety studies (rodents, pigs,
primates)Development of clinical cell injection methodAdequate outcome assessment methods for a
valid appraisal of patient
Guest et al. 2013
![Page 56: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/56.jpg)
Miami Project Schwann Cell Team
![Page 57: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/57.jpg)
MilestonesDec ‘07 Organized SC clinical trial team, weekly meetings
‘08- ‘09 Developed GMP cell manufacturing procedures for human SCs
‘09-’10 -Tested toxicology and tumorigenicity in rats -SCI studies in pigs to determine safest cell
injection procedure
‘10-’11 -More toxicology and tumorigenicity studies on rats-Studies in pigs to determine safest dose and
volume for clinical protocol
July ‘11 Submitted IND application to the FDA
July ‘12 Approval!
Dec ‘12 Performed 1st transplantation
![Page 58: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/58.jpg)
IND submission
![Page 59: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/59.jpg)
The autologous hSC trial • Open label, unblinded, non-randomized and
non- placebo dose escalation study, looking at safety of transplantation of autologous hSCs with long term follow-up in subjects with subacute SCI
• Cell preparation protocols are FDA approved and cell processing is conducted in a facility with extensive experience producing clinical grade cells (GMP)
• Piece of sural nerve is harvested within 5 days post- injury
![Page 60: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/60.jpg)
What did the FDA require?
Studies to detect: Tumors? Biodistribution?Appropriate dose (MTD)? Toxicity?Cell survival for 6 mo?
Characterization of cell product (GMP)Every substance FDA approvedEach step in protocol recorded + witnessedMode of SC injection
![Page 61: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/61.jpg)
Challenges• $$$, rats, ramping up personnel (training),
paper work• Documentation of outcome of EVERY rat,
including cause of death• SOPs for every step in every procedure• Sectioning of entire brain and spinal cord
to detect migration of transplanted SCs• Certified Pathologist to examine
sections
![Page 62: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/62.jpg)
![Page 63: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/63.jpg)
Next steps?New clinical trial for combination approachTranslatable:
-Improve implant survival-Slow release of added components in
implant and beyond-Growth factor gradients beyond implant-Provide environment for axon
growth/implant and beyond-Provide oxygen for early implant
Collaborate with bioengineers
![Page 64: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/64.jpg)
“Dream” biomaterialNon-toxic, biocompatible, biodegradableNon-inflammatory, non–scar response Injectable as fluid (with cells) for closed
injuryGels upon filling injury site, linear
arrangement (tunnels) Promotes axon growth Protects viability of cells Slowly releases growth factors, other
![Page 65: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/65.jpg)
Funding: MBB• NIH Fellowship• NIH research grant 1971-2017 (3 Javits
Awards)Renewed every 5 or 7 yr
• Private FoundationsDonors to The Miami Project, 25 yrThe Buoniconti Foundation The C and D Reeve Foundation, 15 yr
Christine E. Lynn Disting Professor, 11 yr• State of Florida
![Page 66: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/66.jpg)
“If you want to go quickly, go alone. If you want to go far, go together.” – African Proverb
![Page 67: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/67.jpg)
Thank You !
The Miami Project to Cure Paralysis
![Page 68: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/68.jpg)
![Page 69: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/69.jpg)
Neurotrophins• Family Members:
-Nerve Growth Factor (NGF)-Brain Derived Neurotrophic Factor (BDNF)-Neurotrophin 3 (NT–3)-Neurotrophin 4 and/or 5 (NT- 4/5)
• Functions:-Control of proliferation, survival, differentiation-Central and peripheral nervous systems-Developing and adult nervous systems
![Page 70: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/70.jpg)
Inclusion Criteria• Traumatic thoracic SCI between levels T3-T11• ASIA grade A, 18-50 years of age • Consents to spinal nerve biopsy, implantation,
one year follow-up and 5-year protocol
Cohort Target dose Injections Total volume # subjects 1 5 x 106 1 50 µl 22 10 x 106 1 100 µl 33 15 x 106 1 150 µl 3
Dose Escalation
![Page 71: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/71.jpg)
Exclusion criteria
• Penetrating injury• Spinal cord transection• Lesions involving conus• Multi-system trauma• Closed head injury• Severe neuropathic pain at admission• BMI < 35
![Page 72: Cell transplantation to the rescue: Repairing the injured ...129.171.203.12/cms/file/bungepre.pdf · • MVAs (36.5%), falls (28.5), violence (14) Quality of Life Issues with SCI](https://reader035.vdocuments.site/reader035/viewer/2022071105/5fdef2894f2861129f2c656d/html5/thumbnails/72.jpg)
Outcomes• 1° outcome – assess safety
– Serious adverse events– Changes in sensory and motor scores – Changes in pain or spasticity– Electrophysiology for MEP and SSEP– MRI
• 2° outcome – evaluate functional scales– Functional Independence Measure (FIM)– Spinal Cord Independence Measure (SCIM)– Autonomic (BP, HR, tilt table response, etc.)
• Follow-up of above for one year• Follow-up (MRI annually for 4 years after first year)