Stem Cell Therapy of Spinal Cord Injury


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This is a lecture that Dr. Young gave in Seoul, Korea on November 23, 2008.

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Stem Cell Therapy of Spinal Cord Injury

  1. 1. Stem Cell Treatment of Spinal Cord Injury Wise Young, Ph.D., M.D. Keck Center for Collaborative Neuroscience Rutgers University, Piscataway, New Jersey
  2. 2. Sources of Available Cells • Bone marrow (or peripheral blood) stem cells – Advantages. Autologous bone marrow cells are immune- compatible. Proven efficacy for hematological conditions. – Disadvantages. Expensive to isolate and grow for transplants. • Umbilical cord blood cells – Advantages. HLA-matched cells are rapidly available. Proven efficacy for hematological conditions. Relatively inexpensive. – Disadvantages. Difficult to grow and differentiate. • Embryonic and fetal stem cells – Advantages. Easy to grow and differentiate. – Disadvantages. Limited availability. Not available in sufficient numbers for HLA-matching. Socially controversial.
  3. 3. Preclinical UCB Studies • Umbilical cord blood (UCB) cells may be beneficial when transplanted shortly after spinal cord injury: – Intravenous infusion of human UCB improves function in rat SCI model (Saporta, et al., 2004) – Intraspinal transplants of human CD34+ UCB cells improve recovery in hemisected rats (Li, et al., 2004; Zhao, et al, 2004) – Intraspinal transplants of human CD34+ UCB cells plus BDNF improves recovery in injured rat spinal cords (Kuh, et al. 2005) – CD34+ UCB cells transplanted to contused spinal cords survived 3 weeks and significantly improve neurological function in rats (Nishio, et al., 2006) – UCB cells transplanted into spinal cord 7 days after contusion differentiated into cells with neural phenotypes, myelinated axons, and improved locomotor recovery (Dasan, et al., 2007). • UCB cells are beneficial in other CNS injury models including stroke, traumatic brain injury, and a mouse model of ALS.
  4. 4. Neurological UCB Therapy • Recent studies indicate the UCB cells are beneficial in patients with Duchenne’s muscular dystrophy (Kong, et al., 2004; Zhang, et al., 2005) and infantile Krabbe’s disease (Escolar, et al., 2005). • Several clinical groups are transplanting UCB cells into patients with chronic spinal cord injury, e.g. – Kang, et al. (2006) in Inchon, Seoul transplanted HLA-matched umbilical cord blood cells into a woman with chronic spinal cord injury. – Beike BioTech is infusing non-HLA-matched CD34+ cells intravenously or intrathecally into hundreds of patients with spinal cord injury in Shenzhen, Shenyang, Kungming, and Hongzhou. – In Mexico, Bahamas, and India, many doctors are tranfusing unmatched umbilical cord blood into patients with spinal cord injury.
  5. 5. Lithium Treatment of SCI • Yick LW, So KF, Cheung PT and Wu WT (2004). Lithium chloride reinforces the regeneration-promoting effect of chondroitinase ABC on rubrospinal neurons after spinal cord injury. J Neurotrauma. 21: 932-43. After spinal cord injury, enzymatic digestion of chondroitin sulfate proteoglycans promotes axonal regeneration of central nervous system neurons across the lesion scar. We examined whether chondroitinase ABC (ChABC) promotes the axonal regeneration of rubrospinal tract (RST) neurons following injury to the spinal cord. The effect of a GSK-3beta inhibitor, lithium chloride (LiCl), on the regeneration of axotomized RST neurons was also assessed. Adult rats received a unilateral hemisection at the seventh cervical spinal cord segment (C7). Four weeks after different treatments, regeneration of RST axons across the lesion scar was examined by injection of Fluoro-Gold at spinal segment T2, and locomotor recovery was studied by a test of forelimb usage. Injured RST axons did not regenerate spontaneously after spinal cord injury, and intraperitoneal injection of LiCl alone did not promote the regeneration of RST axons. Administration of ChABC at the lesion site enhanced the regeneration of RST axons by 20%. Combined treatment of LiCl together with ChABC significantly increased the regeneration of RST axons to 42%. Animals receiving combined treatment used both forelimbs together more often than animals that received sham or single treatment. Immunoblotting and immunohistochemical analysis revealed that LiCl induced the expression of inactive GSK-3beta as well as the upregulation of Bcl-2 in injured RST neurons. These results indicate that in vivo, LiCl inhibits GSK-3beta and reinforces the regeneration-promoting function of ChABC through a Bcl-2-dependent mechanism. Combined use of LiCl together with ChABC could be a novel treatment for spinal cord injury. 5
  6. 6. Lithium Effects on Messengers
  7. 7. Lithium Stimulates N01.1 Growth Lithium treatment of N01.1 (7 days) 180000 160000 140000 120000 cell number 100000 N01.1 80000 60000 40000 20000 0 Control Lithium (3mM) N01.1 cells were cultured in 3 mM lithium chloride for 7 days. Lithium-treated cultures had 359% more cells than control cultures grown without lithium.
  8. 8. Lithium Effects on N01.1 in SCI Lithium Saline Lithium (100 mg/kg) markedly improves survival of N01.1 cells transplanted into spinal cords. The cells were injected above and below the injury site, shortly after injury. The rats were perfused with paraformaldehyde at 2 weeks after injury and the spinal cords were viewed with an epi-fluorescent dissecting microscope. In saline-treated rats, there was very little fluorescence.
  9. 9. Lithium-treated N01.1 Transplants Oral lithium chloride (LiCl) treatment markedly increased survival of N01.1 cells transplanted in injured rat spinal cords. Rats were injured with a 25.0 mm weight drop contusion at T9-10 and transplanted with N01.1 cells shortly after injury. The rat received daily intraperitoneal injections of 100 mg/kg of LiCl. At 2 weeks after injury, green fluorescent N01.1 cells filled the entire injury site but did not invade into surrounding cord. The rats did not receive cyclosporin. The horizontal bar represents 1 mm.
  10. 10. Lithium Boosts Growth Factors Growth Factor Lithium Per GAPDH Saline (mRNA levels) Real-time PCR showed that LIF, GDNF, NT3, NGFa, and NGFb mRNA levels were 3-5 times higher in spinal cords of N01.1-transplanted and lithium-treated rats (n=6, blue) than in N01.1-transplanted and saline-treated rat (n=6, red). The mRNA concentrations were normalized to GAPDH levels.
  11. 11. Recent Lithium Studies • Lithium stimulates proliferation of neural and other stem cells but it stimulates neurotrophin production only in umbilical cord blood cells. • Other glycogen synthetase kinase beta-3 (GSK- b3) inhibitors have similar effects as lithium. • Calcineurin inhibitors (cyclosporin and FK506) block the effects of lithium, suggesting that the GSK-b3 acts through NFAT (the nuclear factor of activated t-cells).
  12. 12. Lithium Effects on ALS • Fornai, et al (2008). Lithium delays progression of amyotrophic lateral sclerosis. Proc. Nat. Acad. Sci. 105: 2052-2057. – 44 patients with ALS randomized to either riluzole or riluzole + lithium – Daily oral doses of lithium (serum 0.8 mM) – At the end of 15 months, 29% of control group died – Lithium group had no significant decrease of motor scores.
  13. 13. ChinaSCINet Trials • Phase 0 Observational Study. This trial collected about 600 patients with spinal cord injury and collected standardized data on them for up to a year. • Phase 1 Open-label Lithium. This trial assessed a 6-week course daily oral lithium treatment in 20 subjects with chronic SCI. • Phase 2 Lithium vs Placebo. This double-blind trial will randomize 60 subjects with chronic SCI to 6-week oral lithium vs. placebo (start January 2008). • Phase 2 Escalating dose of cord blood mononuclear cell (CBMC). This trial will evaluate safety and efficacy of 1.6-6.4 million HLA- matched CBMC cells transplanted to spinal cords of 40 subjects with chronic SCI with methylprednisolone and lithium. • Phase 3 HLA-matched CBMC transplants ± Lithium. This trial will randomize 400 subjects that have received CBMC transplants to lithium or placebo.
  14. 14. MP & CyA Effects 2 weeks 14 weeks
  15. 15. N01.1 Transplantation N01.1 cells at 1 week after transplantation into uninjured spinal cord.
  16. 16. Diameter vs. Volume
  17. 17. Injectate Volume Rat Human
  18. 18. Dorsal Root Entry Zones
  19. 19. Central Injection Sites Bevel Down Bevel Up 45˚ angle, 1.5 mm depth, 0.5µl over 10 minutes • Bevel direction is important. Down-bevel result in dye localizing to ventral gray while up-bevel has result in dye in dorsal gray matter. • There is greater spread of dye with lumbar cord (right).
  20. 20. Cord Blood Mononuclear (CBM) Cells HLA matched cord blood >4/6 match 20 subjects chronic SCI
  21. 21. ChinaSCINet Advantages • Rapid clinical trials. Capacity to randomize as many as 3000 chronic and 3000 acute SCI patients per year. • High standards. China SFDA and U.S. FDA registration of clinical trials, fulfilling international GCP criteria. • Experience. Chinese spinal surgeons have more cell transplantation experience than any others. • Low costs. Estimated $22,000 per subject for cell transplant, surgery, hospitalization, and rehabilitation. • Rigorous. The trials are the first randomized controlled trials to asses safety and efficacy of individual and combination cell transplants and drug therapies.
  22. 22. Intradural Decompression Title: Neurosurgical Treatment and Rehabiitation of Spinal Cord Injury: Analysis of 30 cases Authors: Hui Zhu, Yaping Feng, Wise Young**, Siwei You***, Xuefeng Sheng*, Yansheng Liu*, Gong Ju*** * PLA Kunming General Hospital, Kunming, Yunnan ** Rutgers University, Piscataway, NJ *** Fourth Military Univ., Xi’an, China Abstract: 30 subjects with “complete” injury (ASIA A) had surgery at 2-65 days after injury, including stabilization, laminectomy, and intradural decompression. After 3 months of intensive rehabilitation, 43% walked with crutch or cane and 17% walked without assistance; 47% converted to B, C, or D.
  23. 23. Surgical Approach Orthopedic surgeons first stabilized the injury site. Incise the dura and inspect cord: Mild Contusion. The injury site is intact and firm. Severe Contusion. Soft necrotic cord below surface. Lacerated Contusion. Cord surface is disrupted. Remove arachnoid adhesions to restore pulsative cerebrospinal fluid flow If cord has soft necrotic zone, a lateral myelotomy was done and necrotic tissues were removed.
  24. 24. Kunming Locomotor Scale I. Unable to stand II. Stand with wheeled walker and assistance III. Stand with wheeled walker, no assistance IV. Walk with wheeled walker, lock knees V. Walk with wheeled walker, no assistance VI. Walk with 4-point walker, no assistance VII. Walk with crutches, no assistance VIII. Walk with cane, no assistance IX. Walk unstably without aid or assistance X. Walk stably without aid or assistance
  25. 25. List of subjects sorted by surgery time, showing KLS and ASIA classification and scores.
  26. 26. Subjects with Subjects with MC injuries MC injuries had the best had the best motor score walking score improvement recovery but all groups showed some walking recovery Change in walking, motor, pin, and touch scores by ASIA scores Subjects that Subjects with with MC had the MC injuries best pin sensory had the best recovery touch score improvement Change in walking, motor, pin, and touch scores segregated by injury type
  27. 27. No Complications None had a major surgical complications, such as death, wound infections, meningitis, pneumonia. All started rehabilitation 17 days after surgery. During rehabilitation, there were no major adverse events, such as decubiti, urinary tract infections that required antibiotics, deep venous thromboses. Such a low complication rate is unusual. This is better than normal in this population.
  28. 28. Motor Recovery Intradural decompression of spinal cord improves motor and walking recovery of ASIA A injuries: ASIA A conversion to C or D should be <10% but is 33% Locomotor recovery to KLS VII (no braces or assistance) was 29% ASIA A to D conversion occurred in 20% of the subjects that received intradural decompression. Recovery of unassisted walking with no braces or devices occurred in 17% of the cases. Motor and sensory recovery correlated with the KLS scores.
  29. 29. Locomotor Recovery Kunming Locomotor Scale (KLS) is a reliable indicator of locomotor recovery. At 17 days after surgery but before starting rehabilitation, 63% KLS IV. Walking with wheeled support & assistance 23% KLS V. Walking with wheeled support, no assistance 10% KLS IX or X. Walking without assistance or device. All recovered locomotion after rehabilitation: 40% KLS IV. Walking with wheeled support & assistance 43% KLS VI or VII. Walking with crutches or cane. 17% KLS X. Walking without assistance or aid.
  30. 30. ASIA Classification Changes All subjects were ASIA A on admission. 17 days after surgery: 60% A, 13% B, 17% C, 10% D After 1 month: 60% A, 13% B, 17% C, 10% D After 2 months: 57% A, 10% B, 17% C, 17% D After 3 months: 53% A, 13% B, 13% C, 20% D ASIA conversion From ASIA A to B, C, or D: 47% From ASIA A to C or D: 33% From ASIA A to C: 13% From ASIA A to D: 20% Normally, only 5% of patients should convert from ASIA A to C and none should convert to D.
  31. 31. Proposal of a Clinical Trial A phase 3 randomized multicenter trial randomize to intradural decompression vs. laminectomy locomotor rehabilitation program similar to Kunming 6-month outcomes: ASIA, WISCI, KLS, SCIM, VAS, MAS. Entry criteria Inclusion: <65 years, <3 months SCI, ASIA A, C4-T10. Exclusion: transected cord, other medical conditions, Hypotheses Intradural decompression is safe The treatment can restore locootor function to as many as half of patients with subacute spinal cord injury.
  32. 32. Cell Transplantation Trials The discovery that intradural decompression is beneficial for spinal cord injury has provided an extraordinary opportunity to assess cell transplants. Most of our centers operate on more than 500 patients with spinal cord injury per year. We can randomize over 500*20 = 10,000 patients/year. This will allow large scale testing of various cell transplants and other combination therapies to maximize recovery in subacute spinal cord injury.
  33. 33. Summary of Kunming Study Subdural intramedullary decompression is not only safe but may be beneficial for some patients after severe spinal cord injury at 2-65 days after injury. Intensive locomotor training in these patients is feasible and markedly improves motor and walking scores in three months after surgery. Transplantation of cells can be easily included in this procedure in a double-blind randomized clinical trial.
  34. 34. Subjects with Subjects with 3-month 3-month ASIA ASIA D had D had early earlier motor walking recovery recovery Subjects with 3-month ASIA Subjects with C had later 3-month ASIA motor C had late recovery walking recovery Subjects with Subjects with 3-month ASIA 3-month ASIA B recovered A had little touch over sensory time recovery Change in walking, motor, pin, and touch scores segregated by 3-month ASIA classification
  35. 35. Isolating Neonatal Rat Blood Cells Growth Curves - RNBC 25.0 DMEM 10% FCS bFGF Plasma EGF layer 12.5 Clonal isolation N01.1 Buffy coat layer Transplant Erythrocytes, neutrophils 0 0 1 2 34567 8 9 Weeks after Plating 50 ml conical vial N01 - WT G02 - GFP after centrifugation 5 mm Isolating rat neonatal blood mononuclear Growth rates of rat neonatal cells using Ficoll gradient to isolate blood cells (RNBC) from mononuclear cells (buffy coat layer), Sprague Dawley rats (N01 culture, and transplantation. (non-GFP rats) and GFP rats.
  36. 36. ChinaSCINet Activities • 9/04 – First investigator meeting • 12/05 – First ISCITT in Hong Kong • 8/06 – First Impactor Workshop (HKU) • 12/06 – Second ISCITT in Guangzhou • 4/07 - Cell Transplant Workshop • 2/08 - Clinical Trial Workshop • 5/08 - Second Impactor Workshop (Xi’an) • 10/08 - Third ISCITT in Beijing
  37. 37. LiCl treated N01.1 cells N01.1 cells were grown in media with 3 mM lithium chloride for 7 days. The cultures retained nestin expression (green), a stem cell marker.
  38. 38. Neonatal Rat Blood N01.1 cells N01 cells were isolated from rat neonatal blood cells of Sprague-Dawley (SD) rats and cultured in DMEM, 10%FBS, EGF and bFGF. At 6 weeks, 60% of N01 cells were nestin-positive. A clonal line was isolated and named N01.1. All N01.1 cells expressed nestin (A, green). N01.1 cells can be cultured for long period of time without changes in morphology or nestin expression. When serum was withdrawn from the growth media and the cells were passaged, N01.1 cells formed spherical structures (B & C), similar to neurospheres formed by neural stem cells (presented by Dongming Sun at the First Scientific Annual Meeting, Stem Cell Research in NJ)
  39. 39. N01.1 Large scale expansion N01.1 cells grow on microcarrier beads and can be expanded by over a million times. From Vista Biologicals
  40. 40. N01.1 Transfection with GFP N01.1 cells transfected with the GFP gene and visualized with phase contrast and epifluorescence.
  41. 41. Summary of Lithium Results • Animal studies suggest that umbilical cord blood cell transplants improve functional recovery in mouse and rat spinal cord injury models. • Nestin-expressing neonatal rat blood cells (N01) were isolated, cloned (N01.1), expanded, and transfected with the green fluorescent protein (GFP) gene. • Lithium inhibits GSK-3-beta and markedly increases N01.1 proliferation in culture without losing nestin expression in the cells. • Lithium increase N01.1 survival in injured spinal cords and boosts expression of growth factors including NT-3, GDNF, NGFA, and LIF.
  42. 42. Trial Hypotheses • Phase 1 Lithium. A six-week course of oral lithium can be given safely in patients with chronic SCI. • Phase 2 Lithium vs. placebo. Lithium improves neurological function in subjects with chronic SCI. • Phase 2 Cord blood mononuclear cell (CBMC) ± MP. A pre-operative 30 mg/kg methylprednisolone (MP) bolus improves safety and survival of HLA-matched CBMC transplants in subjects with chronic SCI. • Phase 3. CBMC transplants ± lithium. Lithium improves neurological function in subjects with chronic SCI transplanted with CBMC.
  43. 43. Clinical Trial Rationale • Preclinical animal studies indicate that: – neonatal blood mononuclear cells (N01.1) are safe to transplant to spinal cord and – lithium stimulates transplanted N01.1 to produce growth factors that can enhance regeneration. • The ChinaSCINet trials will assess whether – Lithium and HLA-matched cord blood mononuclear cell (CBMC) transplants are safe in chronic SCI, – CBMC alone improves neurological function, and – lithium improves neurological function after CBMC transplants.
  44. 44. Spinal Cord Injury
  45. 45. Conclusions Many therapies show promise in regenerating and remyelinating the spinal cord. Several therapies are ready to go to clinical trial. Combination therapies are likely to be needed. Much work needs to be done to prepare other therapies for clinical trial. Industry sponsorship of spinal cord injury clinical trials is beginning. Rigorous clinical trials are necessary for progress.