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

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. 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. 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. 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. 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. Lithium Effects on Messengers

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. 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. 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. 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. 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).

13. 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.

15. 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.

20. MP & CyA Effects
2 weeks
14 weeks

21. N01.1 Transplantation
N01.1 cells at 1 week after transplantation into uninjured spinal cord.

22. Diameter vs. Volume

23. Injectate Volume
Rat Human

24. Dorsal Root Entry Zones

25. 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).

26. Cord Blood
Mononuclear
(CBM) Cells
HLA matched
cord blood
>4/6 match
20 subjects
chronic SCI

28. 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.

29. 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.

30. 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.

31. 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

33. List of subjects sorted by surgery time, showing KLS and ASIA classification and scores.

34. 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

35. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.

41. 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.

43. 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

44. 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.

45. 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

46. 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.

47. 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)

48. N01.1 Large scale expansion
N01.1 cells grow on microcarrier beads and
can be expanded by over a million times.
From Vista Biologicals

49. N01.1 Transfection with GFP
N01.1 cells transfected with the GFP gene and
visualized with phase contrast and epifluorescence.

50. 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.

51. 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.

52. 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.

53. Spinal Cord Injury

54. 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.