1. Art and Diseño

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Bone Marrow Stromal cells for repair of
the spinal cord
R.D.S. Nandoe Tewarie
Bone M a rro w Strom al ce lls for re p air o f the sp in a l cord
ISBN 9 78 -9 0 -9 0 2 5 19 2 -9
Cover design: T.A. N andoe Tew arie
Printed by:
Q u ick p rin t N ijm eg en
This thesis is funded by the N e th erlan d s O rg a n iza tio n for Scien tific Research (N W O ) grant
num ber 0 1 7.0 0 1 .2 6 5 . Further fin a n cia l su p p o rt is provided by The M iam i Project to Cure
Paralysis at the U niversity o f M iam i, the In te rn a tio n a l C e n te r for S p in al Cord In ju ry at
Johns
H o p k in s U n iversity and the
D e p a rtm e n t o f N eurosurgery from the
Radboud
U niversity M ed ical C e n te r N ijm eg en.
© 2 010, R.D.S. N andoe Tew arie, N ijm eg e n , The N eth erlan d s
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Bone Marrow Stromal cells for repair of
the spinal cord
Een w e te n sch a p p e lijk e proeve op het gebied van de
M ed ische W e te n sch a p p e n
Proefschrift
ter verkrijg in g van de graad van doctor
aan de Radboud U n iversite it N ijm eg en
op g ezag van de Rector M a g n ificu s prof. m r. S.C.J.J. K ortm ann,
volgens b esluit van het college van decanen
in het o p e n b a a r te verdedigen op w o e n sd ag 7 ap ril 2 0 1 0
om 15.30 uur precies
door
Rishi Devindredath Sharma Nandoe Tewarie
geboren op 2 m ei 19 78
te Z e v e n a a r
Promotor:
Prof. dr. J.A. G ro te nh u is
Co-promotores:
Dr. M . O udega (U n iversity o f Pittsburg)
Dr. R .H .M .A . Bartels
Manuscriptcommissie:
Prof. dr. A .C .H . G eurts
Prof. dr. G .W .A .M . Padberg
Prof. dr. W .C. Peul (Leids U n iv e rsita ir M edisch C e n tru m / M edisch C e ntru m H a a g lan d e n )
Prof. dr. R .P.H . Veth
Prof. dr. P. W e sse lin g
Paranimfen:
M r. drs. D.R. Buis
M w. M r. G.A. N and oe Tew arie
D it werk draag ik op aan w ijlen m ijn m o ed er E.K. Nandoe-Bissessur,
28-08-1950 t
23-04-2004
Introduction and Outline Thesis
9
S ubm itted N e u ro re h a b ilita tio n (2010)
J Spin C o rd M ed. 2009; 32 (2): 105-114
Review of Literature on Bone Marrow Stromal Cells
31
C ell Transplant. 2006; 15 (7): 563-577
Gene expression pattern and suitability
47
S ubm itted Restorative N eurology (2010)
Survival and neuroprotection
65
J N e u ro tra u m a 2009; 26 (12): 2313-2322
Inflammation and cell survival
79
N eu ro rep ort 2010; 21 (3): 221-226
Functional recovery after transplantation
89
S ubm itted C ell Transplantation (2010)
Summary and general discussion
105
Nederlandse samenvatting
113
Synthesis
119
A cknow led gem ents
Curriculum vitae
A bb reviatio ns
List o f p u blication s
Reference list
126
N e u ro c h iru rg is c h C e n tru m N ijm e g e n
1
General introduction to spinal cord injury and stem cells.
Outline of the thesis.
A clin ical p e rsp ective o f sp in a l cord injury.
Subm itted N e u ro re h a b ilita tio n (2010)
Stem cell based th erap ie s for sp in a l cord injury.
J Spin C o rd M e d 2009; 32 (2): 105-114
R.D.S. N andoe Tew arie
A. H urtado
R .H .M .A . Bartels
J.A. G ro tenhu is
M . O udega
Chapter 1
A. Introduction to spinal cord injury
Each year, m any people worldw ide suffer from sp inal cord injury (SCI). These injuries cause
death o f neural cells, severance and dem yelination o f descending and ascend ing axons, and,
consequently, loss o f m otor and sensory function. Endogenous repair efforts fail to repair the
spinal cord and, as a result, the functional im p airm ents are p erm anent. M ost people who
experience SCI are destined to spend the rem ainder o f their life in a w heelchair3, 35, ” 8, 145, 340 345.
Potential treatm ents for SCI are being tested in the clin ic but so far none o f these have
em erged as one that reverses the devastating functional consequences o f SCI. H ere we review
SCI with an em p hasis on the current status o f clinical care and clinical trials. In an
accom panying review we discuss the ap p lication o f stem cells for sp inal cord repair.
E P ID E M IO LO G Y A N D ETIO LOGY O F SCI
In co n sisten t data reporting m akes it difficult to accurately estim ate the worldw ide incidence o f
S C I3, ” 4, 254, 301 315 388. The an nual incidence in the United States is about 4 0 cases per m illion
population or about 12,000 cases per year2’6, 4’7, 452. O ver 77 % o f SCI occurred am ong m ales. A
num ber o f studies profiling the epidem iology o f SCI indicated that the population o f SCI
people has grown over 255,000 (in 2 0 0 7 ) with estim ates between 2 2 7.0 8 0 and 300,938
patients. In the United States and m ost W estern European countries, the average age at
injury has increased over the last 3 decades from 28.7 to 39.5 years. M ost injuries occur
between the ages o f 16 and 30. The percentage o f people older than 6 0 that suffered from SCI
has increased from 4 .7% in 1980 to 11.5% am ong injuries since 2 0 0 0 .
In the United States, the m ain causes o f SCI are m otor vehicle crashes (42% ), falls (27.1% ),
violence (15.3%), unknown (8.1% ), sports (7.4% ). These num bers are sim ilar in other countries
although the percentage o f violence may be sm aller216, 417, 452. Over 70 % o f injuries are
contusive in ju rie s54, 195.
CONSEQUENCES O F SCI
Pathophysiological and anatomical consequences
A force to the vertebral colum n causes dam age to the ligam ents and vertebrae (Fig. 1A, B). The
torn ligam ents cause instability o f the vertebral colum n. Dislocated bone fragm ents of
dam aged vertebrae m ay com press the sp in al cord (Fig. 1A, B). This causes im m ediate neural
cell death, axon dam age and d em yelin ation155 (Fig. 2A). The cellular dam age results in instant
loss o f m otor and sensory function. After the first destructive events, a sequence o f m olecular
and cellular pathophysiological events
(Fig. 2A)
including an aggressive inflam m atory
respon se w ithin the dam aged tissue leads to additional tissue loss at the injury epicen ter and
at distant sites34, 65, 155, 192 (secondary injury). On the other hand, there are also various cellular
10
Introduction and Outline Thesis
events during the early and later stages o f SCI that could be interpreted as attem pts to correct
for the inflicted dam age (Fig. 2B).
Functional consequences
The functional consequences o f SCI are highly variable and depend on the degree o f tissue
dam age, which in turn d ep end s on the im pact severity. In patients with SCI with a relatively
sm all am ount o f tissue dam age, som e endogenous recovery offu nction can be observed, which
is m ost likely resulting from plasticity o f the sp inal nervous tissue102, '°7. In people with SCI
with large tissue dam age the neurological deficits are generally m ajor and perm anent. There
are very few reports o f people with a large injury that regain m otor function to a degree that
indep en dence can be achieved. In these few cases the injury was generally inflicted to the
lower (lum bar) level o f the sp in al cord'97.
F ig . 1 .
Imaging o f human spinal cord compressive injury.
(A) S ag ittal view o f th e cervical spinal cord on m a g n e tic
re so n a n c e im ag in g d e m o n stra tin g the d islocated C 5 an d C 6 v erteb ra c o m p re ssin g the sp inal cord (a rro w ). T h e
fo rm ation o f a h a e m a to m a ventral to the spinal co lu m n C 1 -C 4 in d icates p o ssible lig a m e n t d a m a g e (a s te ris k ). (B )
C lo s e r view o f the d a m a g e d C 5 and C 6 cervical verteb rae on co m p u te d to m o g rap h y sc a n , cle arly sh o w in g bony d a m a g e
o f the v e rte b ra e , w ith d islo catin g fractu re s in the sp inal ca n a l ( a r ro w ). (C ) C o n v e n tio n al X -ray lateral v iew o f th e cervical
spinal colu m n a fte r dorsal stab ilizatio n from C 2 to T h 2 . B e c a u s e o f the d a m a g e to ten d o n s an d v e rteb rae stab ilizatio n
su rg ery n e e d s to be im p le m en ted in a large n u m b e r o f c a s e s o f S C I . (D ) C o n v e n tio n al X -ra y a n tero -p o ste rio r view o f
the s a m e patie n t with stab ilize d cervical v e rteb rae a s in pan el C .
11
Chapter 1
A
F ig . 2 .
A C U T E - S E C O N D A R Y - C H R O N IC
Degenerative and regenerative events after spinal cord injury.
A s c h e m a tic rep re se n tatio n o f the d e g e n e ra tiv e (A)
an d re g e n e rativ e (B ) e v e n ts th at ta k e p la ce afte r spinal cord co n tu sion injury. R ostral is to the left. In B , the
re g e n e rativ e e v e n ts th at ta k e p la ce relatively e arly afte r injury an d over a lim ited tim e period are u pregu lation o f
reg e n e ra tio n -a sso c ia te d g e n e s
( R A G s ) , axon sprouting, a n g io g e n e sis, trop hic factor u p reg u lation , S ch w a n n cell
invasion. O t h e r re g e n e rativ e e v e n ts su ch a s d e b ris rem oval, ste m cell birth/proliferation, m yelin atio n , an d plasticity m ay
a lso occur at later tim e points an d over a longer tim e period.
Social consequences
The critical-care m edicine practice for people with SCI has considerably im proved during the
last decade and is nowadays more widely available. Accordingly, m ore than 95% o f SCI
patients survive their initial hosp italizatio n. SCI decreases the lifespan by about 7% each
year401. A functionally com plete and high level (cervical) injury im pact the lifespan m ore
dram atically than a functionally incom plete or low level (thoracic-lum bar) injury40’. Together,
the relatively young age when SCI occurs, the im proved m edical care, and the lack o f effective
therapies are respon sib le for the con tinually increasing num ber o f paralyzed people with SCI.
This puts a high financial burden on the patient, h is/her fam ily, and society3, ’3’ 348. SCI is the
second m ost expensive condition to treat in the United States after respiratory distress
syndrom e in infants and is ranked third in m edical conditions requiring the longest stay in
hospitals442. The costs o f lifetim e care for a SCI p atient varies between ’ and 3 m illion
dollars263. The Centre for D isease Control in the United States estim ated that about ’ 0 billion
dollars are sp en t yearly on SCI treatm ent excluding the m anag em en t o f pressure ulcers, a
com m on side-effect o f SCI, which adds another billion dollars per year263.
12
Introduction and Outline Thesis
C LIN IC A L ASSESSMENT O F FUNCTION AFTER SCI
The A m erican S p in a l Injury Association (ASIA) im p airm en t scale is often used to assess the
level and the com pleteness o f S C I’54, 2° 5, 251 259. This scale grades the preserved derm atom e for
sensory function and the strength o f 2 ° “key” m uscles in the upper and lower lim bs25’. It
provides clinician s with a standard for grading sensory and motor function im p airm en t after
SCI. Table ’ provides the 5 A SIA scores and their im p licatio ns. Testin g the intrin sic foot
muscle, could com plem ent the A SIA score as, in the m ajority o f SCI patients, it provides an
earlier and superior indicator of supraspinal influence over m otoneurons projecting to lower
extremity m uscles57.
Another frequently used scale is the A SIA Lower Extremity M otor Score (LEM S), an ASIA
subscore, which provides a prediction of the ability to walk. The LEM S scale is com m only used
together with and supplem ents the A SIA scale. A person without neurological deficits scores
5 ° on the LEM S scale. A score of 3 ° or more is predicative for com m unity am bulation ’ year
after injury and a score of 2 ° or less predicts lim ited am bulation’97, 251 252 259, 428.
Classification o f SCI can also be achieved by m easuring functional ability using the
Functional
Independence
M easure
(F IM )’° 5; a 7-p oint scale that m easures
’ 8 item s
concerning mobility, locomotion, self-care, bowel a n d /o r bladder function, com m unication,
and social cognition. A score of ’ indicates total dependence on a caregiver and a score of 7
indicates com plete ind ep endence’° 6, 264 O ther scales to assess functional ability are the
Q u adriplegic Index of Function (Q IF), Modified Barthel Index (M BI), and W alking Index for
SCI (W ISCI), C apab ilities o f Upper Extremity Instrum ent (CUE), Sp inal Cord Independence
M easure
(SCIM )
and
the
C anad ian
O ccupational
Performance
M easure
(CO PM ).
13
Chapter 1
ASIA
grade
Classification
A
Com plete
B
Incom plete
C
Incom plete
D
Incom plete
E
Norm al
Level of impairment
No motor or sensory function preserved in the S4 and S5 segm ents
Sensory but not motor function preserved below neurological level
and including S4 and S5 segm ents
Motor function preserved below neurological level and more than
h alf o f key m uscles below that level have a muscle grade o f < 3
Motor function preserved below neurological level and at least h alf of
key m uscles below that level have a m uscle grade o f > 3
Motor and sensory functions are normal
Table 1 . Standard neurological classification o f spinal cord injury. The presence of motor and sensory function per
dermatome (neurological level) can be tested with the ASIA (American Spinal Injury Association) scale. For motor
function, 10 key muscles in all four limbs are scored 0-5 (0 = total paralysis; 5 = normal) for a maximum score of 10 0 .
For sensory function, light touch and pin prick are being used at key sensory points on the right and left side o f 28
dermatomes to assess the absence (score will be 0), impaired presence (1) or normal presence (2) o f sensory function.
The maximum sensory score is 224 (112 for each o f the tests; 56 for each side). The scoring sheet can be found at
http://www.asia-spinalinjury.org/publications/2006_Classif_worksheet.pdf
TREATMENT O F SCI
An acute and a chronic phase can be distinguished after SCI. Since SCI is often a consequence
of severe accidents, clinical care during the acute phase is generally focused on stabilization of
the patient. D uring the chronic phase the m ain attention will need to be on preventing and, if
unsuccessful, treating SCI consequences such as pain, infections, and pressure ulcers am ong
others.
Clinical care acutely after SCI
To date there is insufficient evidence that would support standards o f care during the acute
phase o f SCI. It is advised to m aintain patients in an intensive care unit for close m onitoring
of respiratory and hem odynam ic com plications. For adequate spinal perfusion, which is at risk
due to
injury-induced
edema,
a
m ean
arterial
pressure
of 85-90
mmHg
should
be
m ain tained42. D epen d in g on the type o f injury, surgical interventions should be considered to
relieve the spinal cord from com pressing bone fragm ents49, 126. The physician m ay decide to
14
Introduction and Outline Thesis
perform surgery to decom press or stabilize dislocated vertebrae and the vertebral column (Fig.
1C, D). Decom pression surgeries49, 126 m ay accelerate functional im provem ents and result in
shorter hospitalization and rehabilitation periods264, 316. However, it does not necessarily result
in an improved final outcome71.
The lack of standards o f care during the acute phase of SCI is in part due to the large
variability am ong injuries and m akes its early m anagem ent com plicated. If bone fragm ents
continue to com press the spinal cord, early surgery m ay be vital to prevent exacerbation of
spinal cord tissue destruction. However, in cases without a clear sign of such urgency there is
no consensus on whether and what type of early surg ical/clinical interventions m ust be
im p le m ented 126. The lack of standards of care is dem onstrated by a case presented in figure 1.
Due to a fall this patient had m ultiple fractures of the cervical spinal cord, including
dislocation fractures of the C5 and C6 vertebrae resulting in com pression of the spinal cord. In
the acute phase, the patient was admitted at the intensive care unit and monitored. At first, a
decom pression lam inectom y and dorsal spondylodesis from C2-TI12 was performed. However,
the C5 dislocation fracture was not repositioned sufficiently requiring a second surgery where a
corporectomy C5 and C6 including a ventral spondylodesis was performed. The type o f surgical
intervention should be considered on a case-to-case basis, which makes it com plicated to study
the efficacy of intervention in the acute phase after SCI in random ized and controlled clinical
trials.
Besides surgical interventions, pharm acological treatm ents to lim it the secondary injury
after SCI
are
often
considered. The
best-known
treatm ent
is
a
high
dose o f the
glucocorticosteroid, m ethylprednisolone sodium succinate (M PSS) within 8 hours after the
injury44'46. Experim entally it was dem onstrated that a high dose o f M PSS reduces the
inflam m atory response and lim it tissue loss after dam age to the spinal cord311. The effects of
M PSS in patients with SCI were investigated in 3 consecutive N ational Acute Sp inal Cord
Injury Studies44'46 (N A SCIS). The results dem onstrated that M PSS treatm ent in the acute
phase of SCI resulted in neurological im provem ents up to 6 months after injury. M PSS was
the standard of care in the United States and other countries. After a thorough review of the
results from the N A S C IS studies and a more com prehensive assessm ent o f the benefits and
risks involved in high dose M PSS treatm ent, the therapeutic benefits became disputed64, 225, 287,
288, 344, 425. Especially in patients with com plete SCI high dose steroid treatm ent can lead to
adverse effects such as myopathy and wound infection that m ay negatively influence
functional outcome and in som e cases m ay be life-threatening225, 344. Currently, m any SCI
clinics worldwide have discontinued the ‘standard' acute adm inistration o f M PSS after SCI.
The debate on the use of M PSS should be accom panied by efforts to develop alternative
treatm ents that counteract the early destructive events occurring during the acute phase of
SCI.
15
Chapter 1
Clinical care at later stages after SCI: Preventing complications
Different com plications may occur during the later stages of SCI (Fig. 3) that each dem ands
specific actions a n d /o r interventions. For instance, SCI can lead to p ain 439, 44°, decreased
fertility327, and autonom ic dysreflexia with loss of bladder and bowel control436. It has to be
taken into consideration that m any SCI patients get accustomed to the specific injury-related
pain they experience and as a result reveal their distress to their physician often at a late
stage250 374. For some SCI-related conditions, such as decreased fertility, it is the patient's
personal desire that should guide the physician's actions.
Other common problems that arise after SCI are septicem ia, respiratory insufficiency, and
pneum onia
due to muscle atrophy (Fig.
3). These com plications
may cause clinical
deterioration and could eventually result in death. They often occur without typical sym ptom s.
For example, pyelonefritis can occur without flank pain or a fem ur fracture can occur without
pain. This may lead to delay or errors in diagnosis and treatm ent’04, ’56. It is im perative that
SCI patients receive annual screenings and long-term follow-ups to prevent these secondary
com plications. It is advised to treat patients on a regular basis with pneum ococcal and
influenza vaccine to prevent opportunistic infections20’ 2°2. M onitoring the skin and urinary
tract and im p le m enting aggressive treatm ents against pressure ulcers and urinary tract
infections is needed to reduce the risk of septicem ia20’ 2°2. A ppropriate nutrition and exercise
should also be incorporated in the (new) lifestyle. Rehabilitation
program s should be
im plem ented to reduce the risk o f cardiovascular disease40’.
G enerally, the possible medical com plications of SCI
patients are known, mostly
recognizable, and their treatm ent often straightforward. It is different for the psychological
problems that arise after S C I37, 397. It m ay be possible to recognize some o f these but treatm ent
and responses to the treatm ent are depending greatly on the individual. O ne can expect an
initial period o f denial a n d /o r inability to fully com prehend the functional consequences
caused by the injury. Next a period of acceptance will have to run its course37, 397. The patient
needs to learn to live with the disabilities and this may be accom panied by bouts of
depression. The m ental state o f the patient can have its effect on m edical treatm ents439. The
psychological consequences o f SCI should not be underestim ated and appropriate guidance of
patient and fam ily should have an im portant place in the late care m anagem en t of S C I37, 44’.
16
Introduction and Outline Thesis
First years
Chronic
Fig. 3. Complications after spinal cord injury. The most common complications that occur during the first years after SCI
are listed on the left and those that occur mostly at later (chronic) stages are listed on the right. Spasticity and pressure
sores occur during the first years but are also common at chronic stages of SCI (UTI = urinary tract infection).
C LIN ICA LLY TESTED APPROACHES TO ELICIT FU N CTIO N AL RECOVERY
Con tin u in g m edical care after SCI is necessary to m aintain the patient's health and quality of
life. However, this generally does not result in dram atic im provem ents in function that would
allow the patient to live an ind ependent life. Repair-prom oting pharm aceutical a n d /o r
surgical interventions will be necessary to significantly change the functional outcome after
SCI (Fig. 4). Here we will review some o f the current treatm ents that are aim ed at lim iting
functional loss and /o r im proving outcome after SCI. In addition we discuss possible future
treatm ents for spinal cord repair. Table 2 provides a list o f clinical treatm ents for SCI.
17
Chapter 1
Neuroprotection
Lim it cell/tissue loss
Plasticity
Form ation new circuits
1
Transplantation
Substitute/replace cells
Functional
— ► recovery
\
Rehabilitation
Recruitm ent new circuits
t
Axon regeneration
G row th-m yelination-synaptogenesis
Fig. 4 . Different approaches that could result directly and/or indirectly in functional recovery after S C I
Neuroprotective approaches
D uring the last 30 years, m any experim ental studies have targeted neuroprotection (i.e.,
tissue sparing) early after SCI to improve outcome. Experim ental evidence has shown that the
functional loss after SCI can be lim ited by im p le m enting neuroprotective approaches. The
best known neuroprotective approach is acute adm inistration o f M PSS. This has been tested
clinically and is still being used around the world44-46. M PSS treatm ent after SCI was first
thought to improve functional outcome, but at present its true therapeutic potential is
intensely debated225, 287, 288, 344, 425. The m ain goal o f M PSS treatm ent after SCI is to decrease the
aggressive inflam m atory response norm ally present w ithin the damaged tissue. This would
lim it the contribution o f macrophages and activated microglia to the secondary loss o f nervous
tissue.
Another exam ple o f a m olecule that could elicit neuroprotective effects after SCI is the
tetracycline derivative, m inocycline’8-20. M inocycline m ay exert its protective effects through
m echanism s that decrease injury-induced
glutam ate-m ediated
excitotoxicity’8, 421 a n d /o r
im m unom odulatory m echanism s such as blocking m icroglial activation’9, ’’0. Moreover,
m inocycline m ay reduce oligodendrocyte and neuronal apoptosis as well as dieback of
damaged axons’30 399. These experim entally studies have established m inocycline as a
prom ising candidate for early treatm ent after SCI. Currently, a phase I/II clinical study is
underway in
the
United
States
to assess the efficacy of intravenously adm inistered
m inocycline in the acute phase after S C I2’.
18
Introduction and Outline Thesis
A- % ? r
B
Loss o f function
A'
3#
B’
C ’~ § V
Im proved function
Fig. 5. Axon regeneration after spinal cord injury. Schematic representation o f axon regeneration that could contribute to
functional recovery after SCI. There are 3 types of damages that are inflicted to axons after SCI. Axons may be still in
contact with their target neurons but demyelinated (A) due to immediate or delayed death of oligodendrocytes. These
axons can become ‘functional’ and contribute to motor recovery when they are remyelinated (A’) by either endogenous
oligodendrocytes derived from local stem cells or oligodendrocyte precursor cells, or by transplanted stem/precursor
cells or Schwann cells. Axons may be severed and thereby devoid o f contact with their target neurons and demyelinated
(B ). In that case, the axons need to regenerate across/beyond the injury, establish synaptic contacts with target
neurons, and be myelinated by endogenous or transplanted cells (B’). Unmyelinated axons may be severed and
without contact with target neurons (C). These need to regenerate and establish synaptic connections with the original
or new target neurons (C’).
Axon growth-promoting approaches
Functional im provem ents after SCI could be elicited by axon growth-prom oting approaches as
this could result in either restoration of damaged axonal circuits or elicit p lastic events’36, ’6’ 363
(Fig. 5). Examples of axon growth- or plasticity-prom oting treatm ents are the adm inistration of
Cethrin® or N O G O antibodies. Cethrin® is a Rho antagonist that reduces the levels of
intracellular GTPase-associated sign aling proteins Rho and Rac to physiological levels’’6, 443.
Elevated Rho has axon growth-inhibitory effects through the above m entioned pathway294.
N O G O antibodies neutralize axon growth-inhibitory effects o f oligodendrocyte m yelin-bound
Nogo2’ 72. Thus, both Cethrin® and N O G O antibodies m ay result in enhanced axon growth
a n d /o r axon plasticity after SCI. Both treatm ents are currently tested clin ically for their efficacy
to repair the injured spinal cord2’.
After SCI axons m ay still be intact but not functional due to injury-induced conduction
block. A dm inistration o f the potassium channel blocker, 4-am inopyridine (4-AP) may restore
such a conduction block and this could restore axon function and thus contribute to improved
19
Chapter 1
function. The efficacy of 4-AP in SCI patients has been tested clinically but so far the outcome
has been m odest95, 1,2 ,57.
Cell transplantation-based approaches
Neuroprotection as well as axonal regeneration could be achieved by tra n sp lan tin g growthprom oting cellular or a-cellular substrates. Exam ples of cellular substrates that are clinically
tested are olfactory ensheathing cells, peripheral nerves, and activated m acrophages. Grafting
olfactory ensheathing cells into the spinal cord is being exam ined in Ch in a, Australia, and
Portugal94, ,27, 231. Autologous peripheral nerves are being grafted into the injured spinal cord in
Taiw an80 173 and activated autologous m acrophages in Israel378 and Belgium 209. Thus far, there
is no clear evidence that these transp lan tatio n strategies elicit m ajor functional changes.
Approach
Elicit neuroprotection
Main objectives
Limit cell/tissue loss
Examples
*M P , ^minocycline, riluzole
Elicit axon regeneration
Promote growth, myelination,
*Cetrin^, *N O G O
Provide growth substrate
(transplantation)
Support axon regeneration
Promote cell survival / axon growth
SCs, *O EG , *PN G
^Activated macrophages
Facilitate plasticity
Promote formation new circuits
*Cetrin^, *anti-NOGO-A
Restore conduction block
Limit spasticity
Limit osteoporosis
Improve bowel/bladder
Increase axon excitability
Decrease reflex activity
Prevent fractures
Limit uncontrolled release
Alleviate (neuropathic) pain
Manage infertility
Elicit neuroprotection
Add/silence repair genes
Decrease hyperexcitability
Facilitate erection, ejaculation
Limit cell death
Promote regenerative events
Enhance effects other interventions
Replace the lost neural cells
Increase proliferation endogenous cells
* 4-AP
*Baclofen, Fampridine
Risedronate, vibration
Gut stimulants, Ditropan
Colostomy tube
Amitryptaline, botulinum toxin
Sildenafil, vibration, levitra
Hypothermia
Gene therapy: Viral vectors
siRNA
Stem cells/progenitors
*AIT-o82 (Neotrofin)
Prevent muscle atrophy
Promote regenerative events?
Facilitate (controlled) movements
Facilitate muscle action
Promote regenerative events?
Targeted physical therapy:
*Locomat, *treadmill training
Robotic prosthetics
Electrical stimulation:
^Alternating currents, IST-12
Cell replacement
Elicit muscle strength/use
Enable movements
Enable movements
Table 2. Therapies fo r the injured spinal cord. Most of the listed approaches are or have been extensively studied in
experimental models of spinal cord injury. For each a few examples are given that are investigated in the laboratory or
clinic (indicated by asterisks). The approaches listed under the line are comparatively novel.
20
Introduction and Outline Thesis
Frontiers in treatment o f SCI
A relatively new concept that does not focus on anatom ical a n d /o r functional repair but rather
on supporting the patient to achieve some degree of independence is the use o f robotics to
enable execution of specific motor tasks. Currently, there are concerted efforts to em ploy
cerebral
(cortical) control for steering robotic devices in com bination with
micro-chip
technologies that would enable fine-tuning of the robotic m ovem ents depending on the
tasks’", 227 33’.
Other com paratively novel approaches im p le m ent physical a n d /o r electrical activity to
elicit spinal cord repair. Although these approaches are generally designed to improve muscle
strength/use, it has been hypothesized that these particular approaches could also elicit
regenerative cellular events that could contribute to improved outcome’32 ’82. Moreover,
locomotor activity27, 67, 362 446 and electrical stim ulatio n5’ 342 369 m ay promote spinal cord repair
via stim ulation of plastic m echanism within existing axon circuits involved in motor function.
A relatively new concept within the more conventional field of cell-based approaches to
repair the spinal cord is the transp lantatio n of stem cells to either replace lost cells or elicit
regenerative cellular events after SCI. Stem cells have been studied for their potential to
restore degenerative diseases in the central nervous system, such as Parkinson's disease,
A lzheim er's disease, am yotrophic lateral sclerosis, and m ultiple sclerosis2’4, 276, 3’7 or in
traum atic injuries such as transient brain traum a235, 357 or S C I’2’ 122 2° 4.
Stem cells hold prom ise for spinal cord repair, but their true potential has not yet clearly
been shown. At this time, stem cell-based therapies are at an early stage, and the associated
risks are still unclear. To enable future use of stem cells for therapeutic purposes, discussions
on all related issues and especially the moral aspects need to be held today. As with any
medical intervention, the questions to be asked are whether this approach is the m ost likely
one to achieve success and whether the risks justify the benefits. The challenges are to further
develop these concepts within current ethical and social boundaries to increase our knowledge
and, through experim ental research, evaluate if they provide any clinical benefit for patients.
B. Stem cell-based therapies
Stem cells proliferate, migrate, and differentiate to form organism s during em bryogenesis.
D uring adulthood, stem cells are present within tissues/organs including the central nervous
system6, ’5’ 26’ 347, 4’6 where they m ay differentiate into neurons’23. After the identification and
characterization o f stem cells, a great deal of interest has been given to their potential for
treatm ent o f spinal cord injury (SCI), traum atic brain injury, and degenerative brain d iseases52
’33, 141 339, 393, 394. Considering their characteristic abilities to self-renew and differentiate into any
cell type in the body the therapeutic promise o f stem cells is justified. Before effective
therapies can be developed there are several issues that need to be addressed and resolved.
21
Chapter 1
These issues range from increasing our basic knowledge about the stem cell's biology to
prevailing over moral concerns fueled by religious a n d /o r political ideas.
STEM CELL D EFIN ITIO N S
A stem cell is defined by its ability o f self-renewal and its totipotency. Self-renewal is
characterized by the ability to go through an asym m etric division in which one of the resulting
cells rem ains a ‘stem cell', without signs of aging, and the other (daughter) cell becomes
restricted to one o f the germ layers. A stem cell m ay become quiescent and at later stages
reenter the cycle o f cell division309, 343.
A true stem cell is a totipotent cell; it can become any cell type present in an organism .
By m any the zygote is considered to be the only true totipotent (stem) cell because it is able
to differentiate into either a placenta cell or an em bryonic cell. Others define the cells o f the
inner cell m ass within the blastocyst as em bryonic stem cells (ESC). These cells are
pluripotent as they cannot become a placenta cell (Fig. 6). Besides ESCs, undifferentiated
cells can be found am ong differentiated cells o f a specific tissue after birth. These cells are
known as adult stem cells, although a better term would be ‘som atic stem cell' since they are
also present in children and um bilical cords. There is am ple evidence that adult stem cells are
not restricted to a particular germ layer and can transdifferentiate82 2,1 2,7,282, 46°. An im portant
advantage o f adult stem cells over ESC is that they can be harvested without destruction of an
embryo. As a result, adult stem cells have gained am ple interest for their application in a
variety o f disorders.
DIFFERENTIATION
The pluripotent stem cell differentiates into a m ultipotent cell o f the three germ layers. These
three layers are the ectodermal layer (from which skin and neural tissue originate), the
mesodermal layer (connective tissue, muscle, bone and blood cells), and the endodermal layer
(gastrointestinal
tract and
internal
glandular organs)
(Fig.
7). The
m ultipotent cell
differentiates into a unipotent cell of a particular cell lineage within its own germ layer. The
unipotent cell is capable of becom ing a cell type within that particular cell lineage. At the
successive phases of differentiation (or determ ination), the resulting progeny are known as
progenitor cells; ‘stem cell-like' cells capable of self-renewal. W ithin the central nervous
system, unipotent neural progenitors become the neurons and glial cells present in brain and
spinal cord (Fig. 6).
In the classic embryology, the totipotent stem cell becomes unipotent through successive
phases of fate restriction. The steps in this process were thought to be irreversible. However,
recently it was shown in vitro that the fate of m ultipotent cells can be chanced to another
22
Introduction and Outline Thesis
germ layer82' 2"' 2’7'282' 46°. This process is known as transdifferentiation. The unlim ited potential of
transdifferentiation prompted m any investigators to obtain cells that norm ally derive from
stem cells that are more difficult to harvest from stem cells that are easier to harvest. For
instance, it is less com plicated to harvest stem cells from skin262' 447 or bone marrow382' 4’° than
from the b rain ’90' 336.
Transdifferentiation has often been shown using non-specific markers and ignoring
possible artifacts due to culturing m ethods122,335. Therefore, the existence of transdifferentiation
is still debated335,412. It should be kept in m ind that forced differentiation into a cell from a
lineage within an unnatural germ layer could result in abnorm al phenotypes that after
grafting could induce carcinogenesis392.
P r o g e n it o r
N e u ra l
P r o g e n it o r
N e u ro n
Fig. 6. From embryonic stem cell to
differentiated neural cell. Embryonic stem cells
from the inner cell mass o f the blastocyst are
pluripotent and go through phases of
differentiation that changes them into unipotent
cells. Here this process is depicted for the
generation o f neural cells; oligodendrocytes,
neurons, and astrocytes.
23
Chapter 1
^ B la s t o c y s t
Zygote
G astrula
Muscle
Bone
Blood
Connective tissue
G-I tract
Glandular organs
Fig. 7. All tissues in an organism originate from the
3 germ layers. These layers are the ectoderm layer,
endoderm layer, and the mesoderm layer. Neural
cells that form the central and peripheral nervous
system derive from the ectoderm.
POTENTIAL O F STEM CELLS FOR SPINAL CO RD REPAIR
After SCI, endogenous regenerative events occur ind icating that the spinal cord attem pts to
repair itself. Schwann
cells, the m yelin atin g
and
regeneration-prom oting cell
in
the
peripheral nervous system, m igrate from spinal roots into the dam aged tissue and m yelinate
spinal cord axons’38, 4o8. The expression of regeneration-associated genes is increased in
damaged neurons’44. There is a surge in proliferation o f local adult stem cells and progenitor
cells’98, 253, 278
However,
axonal
growth
is thwarted
by growth-inhibitors
present
on
oligodendrocyte myelin debris and on cells that form scar tissue’42 389, 420. Also, the new-born
stem cells and progenitor cells do not integrate functionally into the injured spinal cord
tissue. Thus, the endogenous regenerative events that occur after injury fail to repair the
spinal cord.
Improved functional outcome after SCI m ay be elicited by neuroprotective approaches
that lim it secondary tissue loss and thus the loss of function. Alternatively, functional recovery
could be elicited by axon growth-promoting approaches that result in restoration of damaged
a n d /o r formation o f new axon circuits that could become involved in function. There is little
doubt that stem cells and neural progenitor cells could become invaluable com ponents of
repair strategies for the spinal cord. They can become neural cells that m ay support
anatom ical/functional recovery. A lternatively, they m ay secrete growth factors that could
support neuroprotection a n d /o r axon regenerat ion. The potential o f stem cells or progenitor
24
Introduction and Outline Thesis
cells to support spinal
cord repair has been
investigated
extensively90' ’59, 4’4. Their
shortcomings for repair are also understood75' 47’. Over the last decade, stem cells have often
been studied without im p le m enting explicit criteria that would define the used cells as such.
Consequently, the therapeutic potential o f true stem /progenitor cells is still unknown. Other
matters related to the use of stem /progenitor cells for SCI also need to be resolved before
effective therapies can be developed. How can the cells be best obtained? Do they need to be
differentiated in vitro before transp lan tatio n? How can survival of grafted stem /progenitor
cells be improved and uncontrolled division and differentiation be prevented’99? How can
functional integration of the transplanted cells be im proved?
Cell replacement in the injured spinal cord
Considering the ability of stem cells to become any cell type, their potential use for cell
replacem ent strategies is com m onsense. W ith the appropriate com bination of (growth)
factors (induction cocktail), ESC can be used to obtain neurons and glial cells3’' 232. ES-derived
neurons can survive and integrate following injection into the injured rat spinal cord’0’. It was
shown that transplanted mouse ESC m yelinate axons in the m yelin-deficient shiverer rat
spinal cord53. Also, mouse ESC grafted into the injured (normal) rat spinal cord result in
improved functional recovery260. Im portantly, ESC were found to survive well w ithin the injured
spinal cord suggesting that long-term treatm ents could be achieved using this approach’84.
H um an ESC can be directed towards m ultipotent neural precursors63, motor neurons224, 228
and oligodendrocyte progenitor cells200. The latter were found to differentiate into mature
oligodendrocytes in vitro and in vivo297. Moreover, these cells are able to m yelinate axons after
transp lantatio n into the spinal cord of m yelin-deficient shiverer m ice and adult rats200.
Neural progenitor cells (i.e., m ultipotent cells from which the cells o f the central nervous
system arise) often aggregate into neurospheres. Cao and colleagues58 showed that neural
progenitor cells transplanted into the injured rat spinal cord favored differentiation into
astrocytes. These results indicated the need for differentiation protocols prior to grafting59.
Fetal neural precursor cells genetically modified to express noggin, an antagonist o f bone
m orphogenetic
protein,
differentiate
preferably
into
neurons
and
oligodendrocytes383.
Transp lan tatio n of these cells into the injured mouse spinal cord resulted in improved
functional outcome383. However, this result could not be shown by others using the sam e
approach’22.
H um an neural progenitor cells can be harvested from blastocyst-stage embryos and
m anipulated to generate functional neurons and glia299. W hen human neural progenitor cells
were grafted into the injured rat spinal cord some of them were found to differentiate into
oligodendrocytes92 93. Moreover, this finding was accom panied by improved functional
outcome92 93 (Fig. 8).
25
Chapter 1
M esenchym al stem cells from bone m arrow may also have therapeutic prom ise for S C I285,
322. Although still debated66, these particular adult stem cells have been shown to differentiate
into bone, fat, tendon and cartilage cells337. It has been published that these cells can also
transdifferentiate in vitro into liver333, skeletal’28, ,29, 432 and cardiac m uscle249, 3,0 cells, and into
central nervous system cells’3, 47, 2,2 270 27’ 333, 37°. This makes m esenchym al bone marrow stromal
stem cells interesting for strategies for repair of the injured spinal cord. M any m edical fields
are exploring m esenchym al stem cells for instance for repair of the heart after myocardial
infarction’’9, 365, osteogenesis imperfecta in orthopedics68, ’72, organogenesis in internal
m edicine255, 3° 7, intervertebral disc disease in neurosurgery’64, 366-368, and stroke/
neurodegenerative diseases in neurology222, 404.
Neuroprotection
A neuroprotective strategy im plem ented soon after SCI would be the first line o f defense
against injury-induced tissue loss, and could contribute to an improved neurological outcome.
It has been demonstrated that neural progenitor cells can protect against excitotoxicity233, 24’.
Also, neural progenitor cells secrete a variety o f molecules that could protect neural cells from
death m echanism s other than excitotoxicity233, 24’. Thus, transplantation o f these cells into the
injured spinal cord could in fact exert neuroprotective effects. Bone marrow stromal cells have
also been shown to elicit neuroprotective effects as grafting into the injured adult rat spinal
cord resulted in tissue sp arin g ’70, 3° 4. This may have resulted from the secretion of a num ber of
growth factors’43, 2’9, 247, 457.
Axon regeneration
Prom oting axon growth in the injured spinal cord could contribute to restoring function. The
ability of neural progenitor cells to secrete a variety of neurotrophic factors indicates that they
could promote growth of damaged axons233, 24’. Adult neural progenitor cells were found to
provide a perm issive guiding substrate for corticospinal axon regeneration after spinal cord
injury334. The stem cell-like olfactory ensheathing cells assist axon regeneration in the injured
spinal cord in a different m anner. These cells are capable o f preventing axons from
recognizing growth-inhibitory m olecules thereby allow ing them to elongate into otherwise
inhibitory terrain346, 349.
26
Introduction and Outline Thesis
Totipotent
em bryonic stem cell
Fig. 8. Potential effects o f stem cells on spinal cord repair. Although transplanted stem cells could elicit axon
regeneration and/or neuroprotection through secretion o f growth factors, the most logical contribution to repair could
come from their ability to replace lost neural cells. This could result in remyelination of de-myelinated axons if they
become oligodendrocytes, restoration of (new) circuits if they become neurons, and providing scaffolding and nutrition
o f the injured area if they become astrocytes. Generally, the latter is not preferred because astrocytes express a number
o f axon growth inhibitory molecules that could prevent axon regeneration and thus limit the overall restoration.
C LIN IC A L APPLICATION O F STEM CELLS FOR SCI
The translation of approaches developed in the laboratory involving stem cells into the clin ic
is in progress. The use o f stem cells harvested from tissue from an adult has facilitated the use
of stem cells in the clin ic as it has practically dism issed the moral objections surrounding the
use o f stem cells derived from an embryo. Nevertheless, for reasons described below the use of
ESC is often preferred over that o f adult stem cells. The use of human ESC for spinal cord
repair in the United States has been proposed by Geron, a California-based biotechnology
com pany. The application o f adult human stem cells for treatm ent o f SCI is in progress in
m any countries around the world257. For instance, autologous bone marrow-derived stem cells
have been transplanted in the injured spinal cord o f 25 patients in Guayaquil, Ecuador, a trial
that is supported by a California-based biotechnology com pany, Prim eCell Therapeutics LLC.
Encouraging results have been reported such as improved w alking and sensory perception. It
has been suggested that surm ounting the ethical hurdles (see below) could benefit the clinical
application of ESC2’.
27
Chapter 1
EM BRYO NIC VERSUS AD U LT STEM CELLS
ESC can develop into more than 2 0 0 different cell types present in the hum an body379 and
under the appropriate circum stances into an entire organism 283. H um an ESC have been
isolated from blastocyst-stage embryos4’9. They have also been created using som atic cell
nuclear transfer’4, ’5 or parthenogenetic activation o f eggs83, 43’. Isolated ESC do not undergo
senescence and retain high telom erase activity and normal cell cycle signaling, which explains
their rapid proliferation in culture28’ 320. These p lastic characteristics make the ESC suitable for
central nervous system repair strategies. However, transp lantatio n o f ESC can result in
teratom as due to uncontrollable cell proliferation300 353, 386. Also, ESC in culture m ay undergo
genom ic and epigenetic changes that could lead to transform ation although this can be
prevented using proper culture techniques467. Transp lanted ESC are prone to be rejected after
injection into adult tissue and long-term treatm ent with im m unosuppressive drugs may be
required to prevent this loss300. These findings have to some extent tem pered the enthusiasm
for application o f ESC in repair strategies for the central nervous system, despite the fact that
ESC possess by far the greatest potential and could be applied in a broad selection of
reparative cell therapies.
An alternative for ESC are stem cells obtained from tissue after birth. For instance, neural
progenitor cells have been harvested from adult brain234, 426 and spinal cord258. However, adult
stem cells are less plastic than ESC and divide less frequently in culture’08. Also, their
differentiation potential m ay decrease in tim e450. This makes them a possible but somewhat
lim ited alternative for ESC. On the other hand, they offer the advantage that they can be
transplanted without genetic m odifications or pre-treatm ents. Im m une rejection would not be
an issue with adult stem cells when the cells are isolated from the p atient’49 (autografting).
Also, adult stem cells show a high degree o f genom ic stability during culture’35, 429 and usually
do not result in tum or form ation’35. Finally, there is much less moral concern surrounding the
use of adult stem cells because they can be harvested from the patient. These latter features
support the use of adult stem cells over ESC for strategies aim ed at rep airing the central
nervous system. This is certainly true if strategies can be developed that circum vent the
potential drawbacks of using adult stem cells such as the lower p lastic ability and lower rate of
proliferation in vitro compared to ESC.
ETHICAL A N D SOCIAL CO N CERN S
O n e o f the issues that surround the use of ESC is the tim e point at which we consider an
embryo a person’48, 332 36°. According to the Roman Catholic Church and other religious
institutions an embryo “m ust be treated from conception as a living person”36’. This im plies
that a blastocyst cannot be used to harvest. Others consider an embryo to be a person only
after the 2 0 th week of gestation’48, 332 im p lying that ESC can be harvested from blastocysts. Also,
28
Introduction and Outline Thesis
in that case, ESC could be harvested form embryos that were generated but not selected for in
vitro fertilization. These would otherwise be discarded.
D iscussions on what constitutes ‘life' and when does ‘life' start are often intense as they
are driven by moral concerns fueled by religious and political ideas. These issues need to be
addressed with respect to all opponents. Rules regarding the harvest and use o f stem cells can
only be set after full agreem ent by all groups within a society.
Ethical issues that surround the use o f adult stem cells mostly involve their possible
m isuse437. For instance, oocytes can be derived from stem cells o f m ale origin which allows the
production o f a child from one or two male biological p aren ts’74, 293, 438. The potential biological
problems and psychological effects on the child are unknown. It would also be possible that
the offspring develops defects due to acquisition of pairs o f (recessive) genes’74, 293, 438.
Therapeutic cloning and genetic m anipulation are other issues that surround the use of
stem cells. C lo n in g o f cells, genetically matched for the host, could in theory be beneficial for
organ transplantatio n as it may solve issues such as organ shortage and rejection. G enetic
m anipulation could convert ESC into gametes, which would allow germ line gene therapy
(G LGT)293.
IN D U C ED PLURI POTENT STEM CELLS
It is now possible to obtain pluripotent cells by reprogram m ing differentiated cells, such as
fibroblasts, via the introduction o f 4 transcription factors, O C T3/4, SO X 2, KLF4, and M YC
(induced pluripotent stem (iPS) cells)295, 3’9. This new technology which was first described by
Takahashi and Yam an aka407 for mouse fibroblasts and has now been applied for other mouse
cells306 and for human som atic cells465. O f the 4 transcription factors, M YC and KLF4 can be
substituted by others33, 466. The underlying m echanism s for this typically straightforward and
robust reprogram m ing procedure are still unknown and intensely debated. At present it is still
unclear in how closely iPS-cells resem ble conventional ESC and whether application of iPScells would result in sim ila r functional results as can be obtained with ESC. Com parative gene­
expression profiles o f human ESC and human iPS-cells is now ongoing239, 465. Several hurdles
need to be overcome before iPS-cell technology can produce cells for clinical use295 such as the
use of retroviral vectors to introduce the transcription factors and the need for selection
markers to identify the reprogram m ed cells, as well as the use of the oncogene M Y C and the
integration o f retroviral vectors into the genome. These needs are required for proper
reprogram m ing but they modify the cell genetically and modified cells face regulatory
obstacles for therapeutic app lication s. Nevertheless, it is evident that the iPS-cell technology
is prom ising and has opened exciting avenues for the clinical application o f pluripotent cells
without the ethical obstacles that come along with the use of ESC.
29
Chapter 1
C. Outline ofthe thesis
In Chapter 1 a general introduction to the etiology, clinical grading, and treatm ent options for
spinal cord injury are reviewed in Part A. Aspects o f stem cell term inology, advantages and
disadvantages of stem cell app lication are reviewed in Part B. I n Chapter 2 the literature on
the use of the Bone M arrow Stromal Cell (BM SC), a specific type o f adult stem cells, for repair
of the injured spinal cord is reviewed. Transdifferentiation, neural induction based on
morphology and electrophysiological properties are som e o f the aspects that are discussed. In
Chapter 3 we compared the gene profiles o f B M SC early and late in culture and discuss their
potential for spinal cord repair in light o f our results. In Chapter 4 we present an in vivo study,
in which we have investigated B M SC survival as well as their neuroprotectyive effects in the
contused adult rat spinal cord. Chapter 5 describes a study on the effects of different
im m unosuppressive agents on B M SC survival w ithin the contused adult rat spinal cord. In
Chapter 6 we studied whether a com bination therapy of BM SC transp lantatio n and
im m unosuppression would improve the overall outcome in term o f locomotor and sensory
function after a contusive injury in the rat spinal cord. Finally, in Chapter 7 and 8 the findings
are sum m arized and discussed in light o f future scientific anc clinical perspectives
30
THE MIAMI PROJECT
TO CURE PARALYSIS
2
Review of literature on the use of bone marrow stromal cells.
Bone marrow stromal cells for repair o f the sp inal cord: towards clinical application.
Cell Transplantation 2006; 15 (7): 563-577
R.D.S. Nandoe Tewarie
A. Hurtado
A .D .O . Levi
J.A. Grotenhuis
M. Oudega
Chapter 2
IN TRO D U CTIO N
Stem cells are defined by their capacity for self-renewal and differentiation into different cell
types343, 355, 379. In the early em bryonic phase, stem cells are totipotent but after a few divisions
the cells are determ ined to become specific for one of the three germ layers; the ectodermal
layer, which will give rise to skin and neural tissue, the mesoderm al layer, which will give rise
to connective tissue, muscle, bone and blood cells, and the endodermal layer, which will give
rise to gastrointestinal tract and internal glandular organ cells. In the classic embryology, this
‘determ ination' o f the stem cells is thought to be an irreversible process. Recently, it has
become clear that the determ ined stem cell is in fact phenotypically p lastic and is able to give
rise to cells from different germ layers, a process known as transdifferentation30, ,9,, 270 27’ 277.
Because of their versatility, stem cells have gained am ple attention over the last years for
their potential in rep lace m en t/re p air approaches. However, the term ‘stem cells' has been
used loosely without clear and appropriate criteria that define the used cell types. For
exam ple, CNS-derived neurospheres have been used extensively as a source for neural stem
cells (N SCs), whereas it is now clear that they are in fact heterogeneous cell populations
consisting m ostly of neural progenitors and precursors, i.e., cells that are already directed
towards the neural lineage. Recently, Parker and colleagues321 elegantly dem onstrated an
overlap of , 8 % o f stem ness genes between CNS-derived neurospheres and the O 7 .2 N S C
clone, which fulfills the in vitro and in vivo operational definition of a stem cell32’ 395, 413.
Interestingly, this percentage of overlap increased two-fold when the O 7 .2 N S C clone was
cultured as a neurosphere reflecting a shift from a “stem -like” to a “differentiated” gene
expression pattern32,.
The stroma o f bone marrow houses m ultipotent cells that can differentiate into lineages
of blood cells, stromal and skeletal tissue88, 96, 152 ,78, ,79, 245. It has been reported that these stem
cells can also transdifferentiate into liver cells333, skeletal129, 432 and cardiac249, 3,0 muscle cells,
and C N S cells13, 47, ,29, ,66 ,8°, 2,2 238, 27°, but this is still debated66, 435. Bone m arrow is relatively easy
to obtain, which circum vents the ethical concerns that surrounds the use of em bryonic stem
cells. Because of its availability and its reported aptitude to transdifferentiate, stem cells from
bone marrow are thought to serve as an alternative source for other types o f stem cells that
are needed for specific therapeutic approaches.
As with stem cells in general, there is much confusion regarding the correct term inology
and abilities of cells derived from bone marrow. These cells have been referred to as “bone
marrow stromal cells (B M SCs)” or “stromal cells”, because they reside in the stroma o f bone
marrow, or as “bone marrow stem cells” or “bone marrow-derived stem cells”, because a
percentage o f the cells have stem cell abilities. The cells have also been referred to as
“m esenchym al stem cells” or “bone marrow-derived m esenchym al stem cells”, because of
their origin from the mesoderm al germ layer. Due to this confusing term inology it is difficult to
have a clear understanding of the true identity o f the cells used in the various studies. In
32
Review o f literature on Bone Marrow Stromal Cells
addition, their ability to differentiate or transdifferentiate is unclear due m ain ly to the large
variety of induction protocols used by different groups. For exam ple, one group reported that
about 1 % of human and mouse B M SCs can be induced into the neural lineage370, whereas
another group using a different induction protocol reported that 8 0 % of human and rat
B M SCs could become neural cells448. Clearly, different protocols lead to highly variable results
and this makes it difficult to fully understand the abilities of the B M SC and thus its potential
for therapeutic approaches.
The m ajority of groups working with cells derived from the stroma o f bone marrow does
not attem pt to further isolate subpopulations and thus study a heterogeneous cell population
that includes true stem cells as well as precursor and progenitor cells. Therefore, we propose
that “bone marrow stromal cells” is the proper term inology for this collection of cells. We
oppose that they are referred to as stem cells unless proper attem pts have been m ade to
isolate a homogenous subpopulation o f clonally related cells that express known ‘sternness'
genes such as Nanog, Oct-4, and Myc. Moreover, we concur with Parker and co-workers321, that
stem cells in general are best defined operationally. Thus, the term ‘stem cell' can only be
applied when the cells are m ultipotent, able to populate a developing area or repopulate an
ablated or degenerated area with appropriate cell types, able to be serially transplanted, and
able to self-renew. For a more in-depth discussion on this operational definition for stem cells
we refer to a previous publication321.
HARVEST A N D CU LTURING O F BMSC
Although some sm all variations exist, B M SCs are harvested according to largely sim ilar
protocols am ong the m any groups studying these cells for their potential in a variety of
therapeutic approaches. Bone m arrow cells are removed from usually long bones such as the
femurs and tibiae by flushing with cold phosphate-buffered saline with low percentage o f fetal
bovine serum . These cells are washed and cultured in Dulbecco's Modified Eagle's M edium or
Iscove's Modified Dulbecco's M edium with 1 0 -2 0 % fetal bovine a n d /o r horse serum . After 3-5
days in culture, non-adherent cells, m ain ly red blood cells that have a short lifespan o f about
72 h in these culture conditions, are removed, and the rem ain ing cells washed and further
cultured in the sam e m edium . Usually within two weeks after initiatio n, the cultures consist of
spindle-shaped cells with some monocytes and m acrophages present13, 247. The adherent cells
are removed by trypsinizatio n and then replated for further expansion or used experim entally.
These particular cells, i.e., the p lastic adherent cells, are considered to be ‘the BM SCs'.
G en erally these cells are not further phenotypically characterized. However, several groups did
analyze
the
presence
o f a battery o f surface antigens
and with
great consistency
demonstrated the presence on human B M SC s of M H C class I, C D 13, CD 44, CD 63, CD 73,
C D 2 9 , C D 9 0 , C D 10 5, and C D 16 6 and the absence of M H C class II, C D 14 , CD 45, and CD34.
33
Chapter 2
Several other surface antigens (i.e., S H 2, SH3, C D 7 1, C D i2 o a , and C D 124 ) have been
described for rat B M SC s457.
With this in m ind we reviewed the literature on the use of BM SCs (harvested as described
above) for repair of the spinal cord. W e will focus prim arily on the application o f BM SCs and
not only the stem cell fraction thereof. Nevertheless, through transdifferentiation the stem cell
portion and possibly the precursors and progenitors after de-differentiation can give rise to
cells from the neural lineage; neurons, astrocytes, and oligodendrocytes. Especially for sm aller
focal traum atic and dem yelinating lesions it could be beneficial to acquire neural cells from
B M SCs in vitro prior to transp lantatio n into the spinal cord or m anipulate them in vivo such
that they can replenish lost neural cells.
DIFFERENTIATIO N O F BMSC IN TO NEURAL LINEAGE IN VITRO
To get a better understanding o f the true nature of BM SC-derived astrocytes, oligodendrocytes
and neurons it is im perative to define criteria for each of them. Is it acceptable to merely
assum e that cells that express markers specific for a particular neural cell will also have
relevant functional properties or should it be a requirem ent to dem onrtrate this at least io
vitro? The expression
of certain
m olecules
has
been
accepted
as an
ind icatio n of
differentiation into a particular neural cell type. Asttocytes ex press g lial fibrillary acidic protein
(GFAP) and oligodendrocytes express rat insulin promoter (RIP) and m yelin-basic protein
(M BP). Neurons are identified by the prepence of (3 - 3 tubulin (im m ature neurons), neuronal
marker N (N euN ), neuron-specific enolase (NSE), neurofilam ents (NF), and microtudule
associated protein-2 (M A P-2). However, the expression o f cell-specific markers alone is not
adequate and, except for astrocytes, morphologieal fharacterisfics that are in aoparent
agreem ent with a specific cell type can be m islead ing243,448a Indisputably, the dent criteria ior a
BM SC-derived neural cell are its functional properties, which unfortunately is much easier to
dem onstrate in vitro than in vivo. Nevertheless, u nless BM SC-derived cells positive for RI P- or
M BP m yelinate central axons in vitro their designation as oligodendrocytes should be taken
with caution. Sim ilarly, unless BM SC-derived cells positive for n euronal markers have
appropriate electrophysiological properties their designation as neurons should be carefully
considered. In line with this, we propose to use the additive -lik e for cells that express
particular markers but have not been fijn ctio sa ily characterized. We believe that this would
better reflect the uncertainty of the true nature o f the paeticular cell.
Several groups have reported tCat B M SCs can differentiate into cells that; express
neuronal markers or into cells that have a neuron-like morphology47, 270■272, 314, 370 371 387. Figure 1A
dem onstrates rat B M SC s isolated and cultured according to earlier described m ethods13. When
brain-derived neurotrophic factor (BD N F) is added to the culture, the presence o f neuronal-like
cells can be observed (Fig. 1B). To benefit m ost from the ability o f B M SCs to give rise to neural
34
Review o f literature on Bone Marrow Stromal Cells
cell;;, it is im perative to investigate and optim ize the culture conditions that are necessary for
this transdiffeeentiation.
Padovon
and
co-workers3’4 demonstrated
that hum an
B M SCs
proliferated beet and expreeted the highest percentage of7(33-tubulin (about 2 7 % of the total
population) when cultured in the presence o f 2 0 % fetal bovine stru m and 1 o n g /m l basic
fibroblast growth faetor (bFGF or FG F-2). With fibronectin
as a grcwth stbstrate this
percentage was further incteased to approx. 4 8 % 3’4. B D N f rrr fe u ro tro p fin -3 (NT-3) eliciaed
the e x p ^ ^ o n o f (33-tubulin up to ovor 4 0 % oUthe cellst whicU could not f t further in cre ase f
by com bining them with FG F-2 (’4. Witlf these culture conditionst tlfe cells; did not expreet
N euN . In the stm e m edium but without serum, about ’ 0 % o f the cells differentiated into
GFAP-positive astrocytes^4. Unfortunately;, the iautkroirs did n ot further com bine these different
cdture conditions to postibly enhance the induction of BM SCs tee differentiate into neuronal­
like c o IIs ,
AlthoabO thu study of Padovan and colleagubs04 may suggest that serum is necesaary for
neural induction, nestin-positive neural precursor ce1Is were found i n serem-free cuiture
conditions444. These nestin-positive cells differentinted into GFA P-positive ashrocytes atsa:se;cd
on morphology; approx. 4 0 % o f the populatiun) cor N eu N - positive ne u ro n a li ike cellr fa p>prox.
’ 9 % ) ufter 5 days in co-cu l°ure with uerelaell ar granule ceHs445. Tha grcfupr men tioned above
reported elegant and co m p te lie rsive stud ies. U n fo rtu n a te ! a general consensus for culture
cenditions fot neura1 induction of BM SCa has n otyet been esstablished.
B
Fig. 1. Panel A depicts undifferentiated rat BMSCs 7 days in culture expressing green fluorescent protein (GFP). The cells
were isolated and cultured according to a previously described protocol and infected with lentiviral vectors encoding
GFP. Addition olbcain-derived neurotropiic factor- pushes the BMSCa info neurai-like cells as can bn seen in panel B.
35
Chapter 2
A number of studies have s hown that B M SCs can be induced to become neural-like cells in
-
vitro by a dding g rowth factorst ,89, 273 280 3583 4° 5, cJibju'tyiry'l crACM/1 P100 or c hem ical a gents as
p-m ercaptohthanol an d dimhtfyluulfoxide i n coml;)in;a^io n w itf butylatnd fyic^i'oxyianiuol 3444iii449,
Using thess various induction fjro^ocols, 2 -7 6 % of the cells became neural-like cells. Thess
results may indicate that depending on the growth factor the sam e intracellular pathway
resulting i n neural l nduction gets differentiaily activated. However, more Iikely i s that the
different growth factors exert their activity through different intracellular pathways that result
in different degrees u f neural induction.
Padovan and colleagues34 investigated whether am ong B M SC s there is a subpopulation
that can more nasily differentiate i nto the neural Iineage. They compared unsorted BM SCs
and a population o f BM SCs sorted on the pressnce of CD 133 on their m em brane and
demonstrated that it higher percentage of7the latter was able to express neural markera. Thi;;
phenom enon wns n lao demonstnated by com paring gene expression paU erns of7the different
ssbpopulations o f B M SCs cells4’’ 045. Although thi:> particular population of7C D i33-p o sitive cells.
or cells derived thereof, were not further d e ln e d dunctionally, thess results make it c lia r that
the B M SC population wWen obtained as described above is n aeterogeneous cell population.
Interestingly, and stuonglg em p h asizin g that more com plete criteria are im perative to
d e ln e BM SC-derived neuronal c^lls^ Lu and coworkerst43 demonstnated that the neuron-like
cells derived from B M SCs by adding p -m ercaptoethanol to the culture m edium 32 448 14/49 are
actually dying cells! Tim e-lap se m icroscopy revealed that the cellular exXxnsions protruding
from the cells are m ereln a result of cellular shrinkage. Lu and colleagues.43 took thi:;
investigation one level further and demonstnated that these morphological changes of the
B M SCs wem actually due to cellular toxicity, They stowed that cells eeposed to several
stuessors. sucO a s d etnrgentSr chloride a nd exereme p H , exhibited the s am e morphological
cOaracteristics. i.e., neuronal-like cells. as the B M SCs coltured in the presence of
-
m ercaptoethanol. Cleariy, neural cells obtained in vitro from B M SCs need to be functionally
diaracterized.
In
case
of
BM SC-derived
neurons,
dem onstnating
appropriate
electrophysiological behavior is cruci^i.
ELECTRO PHYSIO LO GICAL ACTIVITY O F NEURON-LIKE (CELLS
So far only n lew groups published in vitro evidence that BM SC-derived neuron-like cells have
electrophysiological activity appropriate for neurons185, ,86, 2,3 445. Kohyama and co lleagu es^
demonstnated that s uch cells exhibited a resting m em brane potential (Vrest) o f - 2 ° m V a nd 50 m V a t , 4 a nd 2 8 d ays in vitro, resp ectively Thi;; was nhe l - i t study that idemonstnated 'that
BM SC-derived
neuron-like cells acquite a Vrest resti'ebling that o f neurons, w hidi i s
approxim atele - 70 m V. Jiang and colleagues185, 186 cultured BM SC-derived neuron-like cells
long-term with different m itogens a nd cytokines, then co-cultured them with fetal in- ouse brain
36
Review o f literature on Bone Marrow Stromal Cells
astrocytes and dem onstrated that the neuronal-like cells had a V rest between -8.4 and -55.4
mV. These authors also dem onstrated that prolonged co-culture with the fetal astrocytes
resulted in a further decrease o f the resting m em brane potentials. Moreover, these cells were
then able to fire action potentials’85, ,86. Regrettably, this study did not investigate the potential
of these cells to fire trains o f action potentials, a characteristic o f fully matured neurons62.
Electrophysiologically active cells derived from B M SCs were also described by W isletGendebien and colleagues445. They reported that after 4-6 days in culture some of the cells
demonstrated sensitivity to the neurotransm itters, GABA, glycine, serotonin, and glutam ate,
possessed an outward K+ current but no inward Na+ current, and exhibited a V rest of about -37
mV. These characteristics correspond with those described for neurons in stage , o f their
m aturation445. W islet-Gendebien and colleagues445 further showed that after 7-15 days in
culture the cells were able to fire a single-spike action potential and had acquired Vrest o f -56
m V (characteristics that corresponded to neurons in stage 2 of their m aturation62. These
findings are exciting and dem onstrate that cells within the B M SC population can differentiate
in m aturation stage 2 neurons when cultured under the appropriate conditions. It is
unfortunate that W islet-Gendebien and co-workers445 could not dem onstrate the presence of
fully mature neurons (stage 3)62, which are able to fire trains of spikes and exhibit a normal
V rest o f -70 m V. The results from the studies mentioned above indicate that in vitro the BM SCderived neuronal-like cells acquire a more negative V rest in tim e. Perhaps they could have
succeeded in creating fully matured neurons if they had cultured their cells for longer than ,5
days. The differentiation o f BM SCs into fully m ature neurons in vitro rem ains one o f the more
intriguing challenges in the field of stem cells and C N S repair.
DIFFERENTIATIO N O F BMSC IN TO NEURAL LINEAGE IN V IVO
The first study that provided evidence that B M SCs can differentiate into neural-like cells in vivo
was from M ezey and Chandross270. Using a m ice model, they transplanted m ale bone marrow
cells into the peritoneal cavity o f female recipients. The grafted bone marrow cell preparation
did not contain neuron- or glia-like cells at the tim e o f transp lantatio n, although it should be
noted that about , 8 % of the cells expressed the neural precursor cell marker, nestin when
cultured for several weeks. Using in situ hybridization techniques, Y-chrom osom e co ntaining
neurons were located in the brain o f the host, suggesting that the grafted B M SCs had crossed
the blood-brain-barrier and formed neurons within the C N S.
Interestingly, Cogle and
colleagues87 also dem onstrated Y-chrom osom e co ntaining
neurons that were nicely integrated in the hippocam pus of three fem ale hum ans that had
received transplan ts of m ale bone marrow cells up to 6 years earlier. It should be m entioned
that a fusion between a grafted B M SC and a host cell could result in false-positive results. In
several studies it has been reported that B M SCs can spontaneously fuse with other cells in
37
Chapter 2
vitro4'5’ 458. W hereas this is a real possibility, Cogle and co-workers87 used fluorescence in situ
hybridization techniques to reveal the presence of only one X chromosome, concluding that in
their study the neurons could not have been the result o f cell fusion. The Y-chrom osom econtaining, transgender cells accounted for approxim ately ' % o f all neurons and ' - 2 % of all
astrocytes and microglial cells in the hippocam pus. These studies provide exciting evidence
that B M SC s can m igrate across the blood-brain-barrier and differentiate into neural cells in
the mature C N S , which is prom ising for the use o f BM SCs in C N S reparative approaches.
BMSC FOR SPIN AL CO R D REPAIR
Spinal cord injury results in cell death, axonal damage, progressive loss of tissue, and
im paired motor and sensory functions. Some restoration o f function has been reported
resulting from endogenous self-repair processes or from applied interventions. At present,
due to the lack of repair approaches that cause m eaningful functional restoration, spinal cord
injury results in a w heelchair bound life. In general it is thought that functional recovery can be
achieved
by
addressing
several
key
areas;
prevention
of injury-induced
cell
death
(neuroprotection) close and away from the injury, prom oting axonal regeneration
by
decreasing the inhibitory nature of the environm ent at the injury site or by increasing the
in trin sic ability o f injured neurons to grow their axon, and prom oting m yelination of
regenerated axons and dem yelinated intact axons. It appears that a com bination of these
approaches followed by intensive rehabilitation to develop and stabilize new axonal circuits
will be necessary. Moreover, the interventions need to be applied sim ultaneously and /or
successively thereby creating optim al conditions for morphological and functional repair.
A typical feature o f the injured cord is the progressive loss o f the central gray and
peripheral white m atter creating large fluid-filled cysts. To provide axons with a substrate to
grow across these cavities transp lantatio n o f cells has been widely explored. M any cell types,
alone or in com bination, have been investigated over the last decades55, 354. Over the last years,
the potential beneficial use of B M SCs in restorative approaches of the spinal cord has
attracted am ple attention. Table ' provides an overview of studies in which B M SCs were
applied into the damaged spinal cord and the results that were obtained. Clearly, am ong
these studies some results are confusing and
in disagreem ent with each other. For
optim ization o f B M SC transp lantatio n paradigm s for application in clinical trials several
crucial questions regarding cell survival, m igration, neuroprotection, axonal regeneration and
functional recovery need to be addressed.
38
Review o f literature on Bone Marrow Stromal Cells
Cell survival
An essential aspect for successful cell transp lantatio n approaches is survival o f the grafted
cells. In vitro, B M SCs are cultured in m edium co ntaining 1 0 -2 0 % serum. Factors other than
present in serum are not essential for their survival and proliferation. In fact, addition of
growth factors such as B D N F , FG F-2, or neurotrophin-3 (NT-3) instigates differentiation o f the
B M SCs into neural-like cells rather than affect survival314. In vivo, in a rat contusion injury
model, Hofstetter and colleagues170 showed that more B M SC s survived when transplanted one
week after injury compared to im m ediately after injury. The surviving cells were located within
trabeculae that span the injury site8’ 17°. However, with the one week delay only 1 % o f the cells
(about 3000 total) survived at 4 weeks after grafting and although this is an increase over the
percentage o f cells that survived im m ediate transp lantatio n (< 0 .15 % ) the total num ber of
surviving cells was very low170.
It has been proposed that one o f the m echanism s underlying death o f cells transplanted
into the spinal
cord is injury-induced
inflam m ation409, 455. The cellular and m olecular
com ponents o f the inflam m atory response could initiate cell death28, which would also explain
improved survival with delayed grafting paradigm s. If this were the case, grafting into a
chronically injured cord would further im prove cell survival. Unfortunately, so far there have
been no conclusive results on their survival rate in the chronically injured spinal cord. In one
study, BM SCs grafted into a contusion site at 3 months after injury reportedly survived an
additional 4 weeks but actual numbers were not provided472. From the data so far, it has
become clear that the survival of the B M SC s is com prom ised after transp lan tatio n into the
injured spinal cord.
Several recent publications have reported that in vitro B M SCs produce and secrete a
variety of growth factors such as glial cell line-derived neurotrophic factor (G D N F )143, 457, nerve
growth factor (N G F)143, 219, 247, B D N F 74, 219, 247 and, albeit in sm aller am ounts, FG F-2 247. These
factors may have pronounced effects on repair-related processes such as neuroprotection and
axonal outgrowth, but they may also affect B M SC survival a n d /o r proliferation in vivo through
an autocrine action457. If this is the case why then do B M SCs survive poorly within the injured
spinal cord? It is possible that the grafted B M SCs sim ply do not secrete enough of the
necessary growth factors to positively effect their own survival w ithin an extrem ely harsh injury
m ilieu with m any cells and factors that negatively influence survival. Also, there m ay be batchto-batch differences in the ability to produce growth factors, which was dem onstrated for
human B M SC s291 and that could result in highly variable results m asking the true potential of
B M SCs to survive the spinal injury m ilieu.
Clearly, for the developm ent o f safe and effective clinical application the survival of
B M SCs after transp lan tatio n into an injury has to be improved. Especially when the cells also
function to deliver factors that are most likely necessary to optim ize the neuroprotective and
axonal regeneration response. Future research should concentrate on decreasing death of
39
Chapter 2
B M SCs within the lesion area, possibly by elevating local levels o f growth factors essential for
survival or by preventing the up regulation o f apoptotic m olecules or prom oting the
expression of anti-apoptotic molecules.
In vitro
m od ified
Chopp
et a l, 2000
rat
no
C o ntu sio n at T 9 level. Cells
grafted 7 d p i. Su rvival 5 w eeks.
250,000
H ofstetter
et a l, 2002
rat
no
C o ntu sio n at T 9 level. Cells
grafted im m ed iate o r 7 d p i, 2
m m rostral and caud al.
Survival 5 w eeks.
300,000
A kiyam a
et a l, 2 0 0 2 b
m ice
no
EB-X lesion# T 10 level. C ells
grafted 3 d p i. Rats were
im m u n o su p p ressed .
Survival 3 w e eks.
Corti
et a l, 2002
m ice
no
C ells injected in tail vein after 10 , 000,000
X -irrad iatio n . Su rvival 3 m o nths
Sy stem ically in fused
B M S C s m igrate tow ards
sp in al cord.
Inoue
et a l, 2003
rat
no
EB-X lesion lu m b ar sp in al cord. 10 0,00 0 C ells grafted 3 d p i, sy ste m ic or 10 , 000,000
fo cal. Survival 3 w eeks.
R em yelination of
dem yelinated axons after
B M S C s transp lan tatio n .
Lee
et a l, 2003
m ice
no
C o ntu sio n at T 11 level. Cells
grafted 7 dpi in and 2 mm
rostrally to in ju ry. Su rvival 4
w eeks.
14
rat
no
C o ntu sio n in ju ry T 9 level. Cells
grafted im m ed iately. Survival
i , 2 , 3,4 w e eks.
O hta
et a l, 2004
rat
no
Ankeny
et a l, 2004
rat
no
C o ntu sio n injury T 9 level. 2 dpi
transp lan tatio n o f cells into
lesion cavity. Survival 8 w eeks.
Lu
et a l, 2004
rat
no
B M S C s -N T 3 into lesioned
dorsal co lu m n 5 days after
c A M P in L4 DRG and N T 3
im m ed iate rostral to graft.
Su rvival 3 m o nth s.
Satake et a l, 2004
Rat
No
C o ntu sio n T 9-10 level. 3 , 5,7 dpi
cell injection lu m b ar
sub arachn oid sp ace.
Zurita
et a l, 2004
rat
no
3
0
So urce
te
P ublication
Sig u rjo n sso n et al, hum an
no
2005
Lu
et a l, 2005
rat
Yes§
Injury m odel
Num ber of
cells
5,000
3,000
M ain results
A d d itio n al results
Sig n ificantly im proved U pregulation o f nestin in ependym al
m o tor o utco m e at 2 w ks.
and a ssociated cell layers.
Tra n sp la n te d B M S C s
form guiding stra n d s at
the injury site.
Better BM SC su rvival w ith delayed
grafting (7 d p i). M otor o utcom e
im proved at 5 w eeks.
R em yelination of
dem yelinated axons after
B M S C s tra n sp lan ta tio n .
M ore m yelinated axons with focal
in jection . Focal ap plicatio n m ore
efficient.
N eural differen tiation :
B M S C s differentiate into
n eurons in the brain and
astrocytes in the cord.
1 ,000,000
N europrotective:
signifient reduction o f
cavity vo lum e .
B M S C s su rvival decreased in tim e
up to 3 w eeks. M otor o utco m e
sig nifican tly im proved up to 14 dpi.
C o ntu sio n injury T 9 level. 5,000,000
Im m ed iate cell grafting in C S F
(4 th v e n tricle ). Survival 5
w eeks.
N europrotective:
reduction o f cavity
vo lum e w ith 4 7 % .
Sig n ificant im p ro vem en t o f m o tor
o utco m e after 5 w e e k s. B M S C s
m igrated to cord.
60,000
N euro p rotective: BM SC s
reduce cavity volum e.
Increased spared tissu e
vo lu m e and w h ite m atter.
No sig nifican t im p ro vem en t in
m o tor outco m e a fter 8 w eeks.
200,000
Axonal regeneration:
c A M P and N T -3
com bined prom oted
axonal growth o f sensory
axons.
1 ,000,000
H o m in g o f transplan ted
cells tow ard s lesion area.
Som e B M S C s differentiated into
nestin-p ositive im m a tu re neurons
o r glial cells.
C o ntu sio n at T 7 level. C ells 1 ,000,000
grafted 3 m o post injury at
lesion site. Survival 4 w e eks.
Sig n ificant im provem ent
in m o tor o utco m e (B B B )
after 2 w eeks in the
ch ro n ica lly injured rat.
B M S C s survive and form bridges in
the cavity.
20,000
Grafted cells
in d istin g u ish ab le from
neurons ch ick and also
electrophys. active.
During differentiation lo ss o f C D 34
exp ressio n by B M S C s.
10 0,00 0
Neural induced cells in
vitro do not express
neural m arkers in v ivo .
B D N F secretin g B M S C s led to
higher axon d en sity. No chang e in
m o tor o utco m e at 3 m o nth s.
In ovo surgery and cell
im plantation in chicken embryo
(stage 15 -16 ) . Suvival 4-9 days.
D orsal co lu m n lesion C 3 .
Su rvival up to 3 m o nth s.
§ BMSCs were neurally induced following the procedure described by Woodbury et al., 2002 and modified to express
BDNF.
# EB-X lesion=Ethylbromide injection with X-irradiation to create a demyelinating injury
40
Review o f literature on Bone Marrow Stromal Cells
Cell migration
Are B M SC s able to m igrate towards or away from the site of in ju ry/tra n sp lan ta tio n ? In vitro
studies have shown that B M SCs express CXCR 4, the receptor for the chemokine, C X C L 12 (also
known as stromal derived factor-1, SD F-1 12. C X C L 12 has been im plicated in cell m igration
possibly through the extracellular signal-regulated kinase (ERK) and Akt phosphorylation
pathways39, 134. Interestingly, under some pathological conditions reactive astrocytes produce
C X C L 12 39, 274 It is possible that following spinal cord dam age upregulation of the level of
C X C L 12 attract CXCR 4-positive B M SC towards the injury site. This could be the m echanism at
the basis of the hom ing of BM SCs into spinal cord injury sites. However, at this tim e it is not
known whether this particular chemokine is present within the injured spinal cord.
In vivo, system ically adm inistered BM SCs have been reported to m igrate towards injury
sites in the brain222 240 28°, but the results regarding hom ing towards the injured spinal cord
have been conflicting. Recently, 111In-oxine-labeled B M SCs were shown to m igrate poorly
towards the injured spinal cord following intravenous ad m in istration 96. On the other hand,
B M SCs labeled with iron-oxide microbeads were detected using m agnetic resonance im aging
within a spinal cord com pression injury after intravenous adm inistration406. Satake and co­
workers373 demonstrated that B M SC s grafted into the lum bar subarachnoid space aggregated
onto the cord near a thoracic contusion injury site and that a few migrated into the contusion
injury. Possibly, the m eninges may have prevented more B M SCs to migrate into the spinal
cord parenchym a. Obviously, if B M SCs are able to m igrate towards an injury site in the adult
spinal cord, it would allow for system ic delivery of the cells thereby avoiding invasive
transplantatio n strategies.
M igration of transplanted B M SCs away from an injury site in the spinal cord might be
beneficial for outgrowth o f regenerating axons. It was reported that such m igration did not
occur in a contusion inju ry m odel96. However, at present, the question whether B M SCs are
truly capable of m igration within the injured spinal cord has not explicitly been answered.
Future research should focus on these questions because the outcome is crucial for the design
of B M SC transplantatio n paradigm s for clinical application to repair the injured spinal cord.
Neuroprotection
Although grafting of cells into the injured spinal cord is typically applied to generate a growth
response, a neuroprotection effect can usually also be observed408. Repeatedly, it has been
demonstrated that cellular grafts lim it the loss of nervous tissue in the injured cord338, 4° 8. In
fact, in anim al models o f spinal cord injury and repair im provem ents in motor perform ance
seen after cell transp lantatio n are often contributed to neuroprotection rather than axonal
regeneration. Grafting B M SCs into the contused adult spinal cord also promotes tissue
sparing, which was evidenced by sm aller cavities and preserved host white m atter5, 10 3° 4.
41
Chapter 2
It is likely that the m echanism underlying the neuroprotective effect o f B M SC transplants
is related to the ability of the cells to produce and secrete factors that either arrest a n d /o r
prevent the onset of cell destructive events. B M SC s are known to produce and secrete factor
(G D N F )’43, 457, nerve growth factor (N G F)’43, 2’9, 247, B D N F 74, 2’9, 247 and, albeit in sm aller am ounts,
FG F-2 247. These factors have all been im plicated in neuroprotective effects. N G F and B D N F
increase survival’50 4,8 and decrease apoptotic death o f neurons and oligodendrocytes39’. B D N F
also increases oligodendrocyte proliferation267. G D N F has been im plicated in the rescue of
motor neurons25, 78 possibly by activating M AP kinase and Bcl-2, an anti-apoptotic regulator78.
FG F-2 is known to positively effect tissue sp arin g ’87, ,88, 269 and promote neuronal survival and
angiogenesis269 following spinal cord injury. Another molecule produced by B M SCs that could
positively influence tissue sp aring is V E G F, a potent angiogenic factor’58.
Axonal regeneration
In a few studies the axonal regeneration prom oting abilities of B M SCs have been addressed.
Lu and co-workers244 demonstrated that transp lantatio n of native B M SC s into the contused
spinal cord promoted modest sensory and motor axon regeneration, whereas grafting of
neurally-induced BM SCs did not result in axon growth. O ne explanation for the failure of the
neurally-induced BM SCs to promote axonal regeneration in the injured spinal cord is that
these cells die soon after transp lantatio n. The neural induction of the B M SC s was performed
according to an earlier described method32 448, 449, which, as had been already recognized by Lu
and colleagues245, causes B M SCs to die rather than become neuron-like cells.
An alternative explanation for the lower axonal growth response observed by Lu et al244 is
that
neurally
induced
BM SCs
are
less
effective
in
elicitin g
such
a
response
than
undifferentiated B M SCs, for instance because they produce and secrete less growth-promoting
factors. At present it is known that B M SC s produce and secrete several growth factors.
However, it is unknown whether neurally induced B M SCs actually produce growth factors or
whether they do so but in lower am ounts than undifferentiated BM SCs. Indirect evidence that
the neurally induced B M SCs do not produce enough growth factors to stim ulate axonal
regeneration was provided in two studies dem onstrating that transp lantatio n of neurally
induced B M SCs genetically modified to produce and secrete B D N F did improve the axonal
growth
response244, 248. In
another study,
a multifaceted
and
intriguing
spinal
cord
injury/regeneration model was used to investigate the regeneration-prom oting capacity of
B M SC s244. B M SCs modified to secrete NT-3 were transplanted in a transection injury of the
m id-thoracic dorsal colum ns one week after adm inistration o f cA M P into the L4 dorsal root
ganglion as a preconditioning stim ulus for the sensory neurons. This was then combined with
injection o f NT-3 after injury/grafting within and beyond the injury site244. The com bination of
all interventions resulted in regeneration of ascending sensory axons into and from the B M SC
graft. Either cA M P or NT-3 adm inistration alone did not result in such an axonal response.
42
Review o f literature on Bone Marrow Stromal Cells
These results suggested that a cam binatorial approach that stim ulates both the neural soma
and axon m ight effectively increase the axonal regeneration. Surprisingly, in the study of Lu
and colleagues244 app lication of cAM P alone at the level o f the sensoty neurons did not result
in improved sensory grow/th wUile it had been im plicated in such a response earlier292 as well
as in other types of axonal regeneration responses329. Clearly, as with m any other prom ising
cell types for transplantation in to th e injured spinal cord, more extensive studies need to be
performed before B M SC s can be used effecti vely in rep air strat egies.
Functional recovery
It has been reported that B M SC results in significant im provem ent of hindlim b locomotor
perform ance when transplanted in the acutely45’, sub-ocutely8’ anti chrnninally472 contused
spinal cord. In all three studies hindlim b function was evaluated using the open-field IB B-rent,
which scores for jo in t m ovem ents, paw flace m e n t, weight suppnrt, and fore/hindlim b
coordination23. Although a valid way to test hindlim b function, the BBB test has lim itations; the
scoring is subjective and difficult for fore/hindlim b coordination. This affects the proper
assessm ent o f hindlim b motor performance. Othur sensorim otor tests ruch as foot print, grip and beam walking, and analysis o f gait uoiug the CatW aln® provide a morn com plete
m easurem ent of hindlim b function. In addition, it wns anfortunafe that these particular
studies8’ 45’ 472 did not investigate; wUethor tWe im p ro v rm e n ti in brhanio r vasre associa ted with
an axonal regeneration respanse. Considering that in these atudius th a oUservei functional
im provem ents appeared relatively noon after injury and transplantation, it seem s than
neuroprotective m ech anism s3, r° t54, possibly through secretion op growth fattoas, rather than
axonal regeneration responses242'244 were at th f basis of the im provum ents.
So far studies on behaoioral effects following intrasn inal tran sp lp n taiiu n o f BM SCs have
used a variety of models in differ ent species. In mice or rat different num bers o f B M SCs were
grafted acutely into the cervical244 or thoracic spinal cord’0 ’70, 45’, or sub-acutely8’ or
chronically472 in the thoracic spinal cord. M ost m odels involved a contusion injury’0 8’ ’7° 45’,
others a partial transection model244. The servival p erio t after transp lantatio n as well as thn
studied end points varied am ong fhese s ta lie s . (Hi ven the m ajor differences betwuen th ese
approaches, it is difficult to com pare the respective results and thus to proporly value the
effects of B M SCs on functional recovery and axonal regeneration in the injured spinal cord so
far.
This brings up the question whether there should be one particular model that should be
used uniformly by groups that study the use o f BM SCs in spinal cord repair. Is there a best
model? W e do not support the idea o f only one model. In hum ans, the m orphological and
functional outcome following spinal cord injury is highly variable. Nervous tissue loss, axonal
dieback, neuronal death, and scar formation depend largely on factors such as the site and
degree o f injury and the post-injury care. A p p lyin g one particular model will ultim ately only
43
Chapter 2
benefit a percentage of spinal cord injured patients. However, one particular in vivo model
can be superior to another to answer a particular aspect of spinal cord injury and repair. For
instance, neuroprotective effects of B M SC transp lan ts can best be addressed in contusion
injuries, whereas their effect on axonal regeneration is most reliably assessed in com plete
transection injuries.
The highly variable outcome after human spinal cord injury requires testing o f B M SC
transplantatio n (and of other approaches) in a variety of in vivo injury models. This would
allow proper judgm ent whether following a certain injury B M SC grafting or an alternative
intervention would be best. For understanding the potential o f B M SCs for spinal cord repair it
would be better to have certain studies ind ependently repeated to confirm the results, which if
that would be the case m ay establish B M SC grafting as the type of intervention best suited for
a particular type of injury.
C LIN IC A L APPLICATION O F BMSC
There is considerable experience with the harvesting o f B M SCs from the iliac crests of
patients206. In the clinic, following chem otherapy a n d /o r radiation therapy, the bone marrow
m icroenvironm ent is damaged resulting in dim inished or delayed hem atopoiesis’09, 266.
A llogeneic marrow transp lan ts have been explored for reconstitution of the dam aged marrow
stroma, although at present it seem s that recipients o f such transp lan ts have only host-type
marrow stromal cells after tran sp lan tatio n 390 402. Also, B M SCs are more increasingly used for
surgical approaches for spinal fusion or for degenerated disc disease275, 434.
B M SCs have several features that make them ap p e alin g candidates for transplantation
after spinal cord injury in the human which include the facts that (a) they can be relatively
easily isolated under local anesthetic206, (b) human B M SC s can be rapidly and extensively
expanded in cell culture’3, 47, (c) there is no evidence that they produce tumors in vivo, even
after im m ortalization to ensure an unlim ited source o f self-renewal ex vivo'’ " 5, (d) they have
demonstrated capacity for tissue repair3, 244, and (e) they secrete growth factors in vivo that can
enhance regen eratio n /rep air73, 74. Clearly, BM SCs may be a good candidate for transplantation
into the injured spinal cord. However, a concern is that a considerable variation exists am ong
donors290 29’. M any factors that are difficult to control can influence the variations between
donors, such as gender, genetic background, and general health. Therefore, we agree with
Neuhuber and colleagues290 29’ that specific param eters need to be found that allow rapid and
reliable selection o f B M SCs with therapeutic potential.
Based on the above-m entioned features of BM SCs, there is much excitem ent about the
potential use o f these cells for spinal cord repair. However, it is also clear that B M S C are not
the so-called “silver bullet”, the one therapy that will promote regeneration and restore
function in the injured spinal cord. In fact, it is generally accepted in the field of spinal cord
44
Review o f literature on Bone Marrow Stromal Cells
injury and repair that such a “silver bullet” does not exist. Sp inal cord injury is particularly
complex and involves a variety of histopathological destructive and som etim es constructive
events. A treatm ent for spinal cord injury aim ed at repair of function needs to deal with all of
these events in a tim ely, m ost likely sequential, fashion.
There have already been several clinical trials that have used intravenous adm inistration
of BM SCs for specific diseases such as patients with m align ancie s237, but also efficacy trials in
osteogenesis im perfecta68, ,72, 298, H urler's syndrom e and m etachrom atic leukodystrophy210. Each
of these trials has had various degrees of success depending on the m easured end-points. A
positive outcome from all of these trials is that only one o f the 68 patients that entered in the
studies m entioned has suffered any side effects (i.e., mild urticaria as a reaction to the fetal
bovine serum album in in which the cells were grown), ind icating that B M SCs can be used
safely in clinical settings.
There is considerable debate as to which patients with a spinal cord injury would be the
ideal candidates for testing cell types (including BM SCs) that have shown potential for clinical
applications. H um an trials approved by the United States Food and Drug Adm inistration
(FDA) require safety data prior to efficacy data. Consequently, most cellular transplantation
strategies tested in clinical trials in the United States have focused on patients with
functionally com plete sp inal cord injuries (ASIA A; Am erican Spinal Cord Injury Association).
However, for proper evaluation o f the efficacy o f transplantation
strategies regarding
functional im provem ents patients with incom plete lesions (represented by A SIA B-D) may be
more beneficiary. C lin ical trials tran sp lan tin g B M SCs after spinal cord injury are ongoing in
several countries including Korea, Mexico, Colum bia and Brazil. Other than a Korean study318
results from these trials have not been published yet. In this particular study, six functionally
com plete spinal cord injured (ASIA A) patients received a B M SC transp lan t com bined with
the adm inistration
of granulocyte m acrophage-colony stim ulating factor (G M -CSF). All
patients were operated on in the first two weeks after injury and a total of 1.8 ml with a
density o f 1.1 x 10 6 B M S C s/ I was injected at the epicenter of a contusion lesion at 5 mm
below the dorsal surface. In 5 out of 6 patients motor and sensory function improved, with 4
patients o f them switching from the A SIA A to the A SIA C level. Follow-up evaluations up to 18
months after transp lantatio n revealed no serious com plications. Although these results are
very prom ising, detailed inform ation concerning neurological exam ination is lacking. Further
studies in this arena will need to focus on reproducibility, safety and finally efficacy.
C O N C L U D IN G REMARKS
Studies on the ability o f BM SCs to transdifferentiate in vitro into the neural lineage often use
different protocols370, 448, 449. This leads to a great deal of confusion on the true capacity o f these
cells and thus their therapeutic potential. There clearly is a need for a consensus on how to
45
Chapter 2
induce B M SC s into the neural lineage in vitro. Moreover, this procedure and in particular
alternative ones that claim a higher efficiency need to be repeated independently. Also, there
is a need for standardized criteria (operational definition)32’ that will result in the proper
designation o f BM SC-derived cells as neurons or oligodendrocytes. O n ly then it w ill be
possible to optim ally benefit from the therapeutic potential of BM SCs for spinal cord repair.
In vivo, the use o f BM SCs in spinal cord injury models is still relatively new (see Table ’ )
and m any questions remain unanswered. Some o f these questions are: To what extent do
B M SCs survive when grafted into the contused spinal cord and does tim e o f transplantation
make a difference? Do B M SCs migrate towards and away from the transplantation site and if
so where to? To what extent do B M SCs differentiate when transplanted into the injured cord?
Can grafted BM SCs support spinal cord repair and if so what m echanism s underlie the
biological effects? Based on the reported differences between in vitro and in vivo results, it is
clear that the influence of the m ilieu of the injured spinal cord on B M SCs is not fully
understood. This is a com plicated issue because of the abundance and variety o f factors in
injured
nervous
tissue
all
with
different
effects
on
B M SC
survival,
m igration,
and
differentiation.
For transplantation purposes, it appears that differentiation of B M SCs prior to grafting
would be best for effective repair for sm aller, focal lesions o f the spinal cord or for specific
aspects in larger, more routinely seen, injuries. For instance, BM SC-derived oligodendrocytes
could be used to address specifically a dem yelinating disease such as m ultiple sclerosis or the
effects o f rem yelination on functional restoration o f the spinal cord after injury. Differentiation
into neurons would allow addressing a motor neuron disease such as am yotrophic lateral
sclerosis. However, is ex vivo differentiation o f B M SCs prior to grafting in the spinal cord
necessary. In theory, the C N S environm ent m ay induce the cell to develop into the required
cell type. Im plantatio n paradigm s using undifferentiated B M SCs in vivo have shown prom ise
in the laboratory and clinical trials are ongoing.
46
3
Gene expression pattern and suitability of bone marrow stromal cells.
Early passage Bone Marrow Strom al C s lls express genes involved in nervous system
developm ent supporting their relevance for neural repair.
Submitted Restorative Neurology (2010)
R.D.S. Nandoe Tewarie
K. Bossers
B.
J.A. Grotenhuis
J. Verhaagen
M. Oudega
Blits
Chapter 3
IN TRO D U CTIO N
The application o f mesoderm al bone marrow stromal cells (BM SC) for nervous system repair
has been explored extensively’65, 323. B M SC may promote repair through their ability to
differentiate into neural cells’7, 47, 76, 370 a n d /o r the secretion of growth-promoting m olecules’43,
2’9, 454, 457. The true potential of B M SC to repair nervous tissue is still under investigation.
A more com prehensive understanding o f the neural repair potential o f B M SC and possibly of
the underlying m olecular m echanism s may be obtained by gene expression profiling. B M SC
express genes com m on to cells from the m esoderm al lineage such as bone, cartilage, and
adipose cells’6, ’37, 223. O n the other hand, they also express genes that are typical for epithelial,
endothelial, and neural cells’85, 38°, which may point at their proposed ability to differentiate
across lineages.
Few groups have studied genes involved in nervous system developm ent expressed by
BM SC. It was shown that 3’ 2 genes including neural progenitor genes are co-expressed in
human-derived hippocam pal neural stem cells and undifferentiated B M S C 56. G enes encoding
for the neurotrophins nerve growth factor and brain-derived neurotrophic factor are expressed
in undifferentiated B M S C and in neurally-induced BM SC, with reduced expression in the
latter453, 454.
The expression of genes encoding for neurotrophins and proteins characteristic o f neural
progenitors in cultured B M SC may indicate their ability to support repair after transplantation
into nervous tissue. To further evaluate the overall repair potential we profiled gene
expression of BM SC from passage (P) 3, which are typically used for transp lantatio n studies in
our laboratory, using 44k whole genom e rat microarrays. W e also compared the gene profiles
of P3 and P’ 4 B M SC to investigate effects o f long-term culturing on gene expression.
MATERIAL A N D M ETHODS
BM SC isolation and culture
B M SC were isolated from Sprague-D aw ley rats (8-’ ° weeks, 2 ° ° - 2 5 ° g ; H arlan, In d ian ap o lis,
IN , USA) as described previously’3. Briefly, rats were deeply anaesthetized and decapitated
and their femurs and tibias im m ediately removed. The marrow from these bones was
aspirated with a syringe, plated on p lastic culture dishes, and cultured for 48-72 h in
Dulbecco's m inim al essential m edium (Invitrogen, Carlsbad, CA, USA) with ’ ° % fetal bovine
serum (Hyclone, Logan, UT, USA) and ’ % p enicillin-streptom ycin (Invitrogen) at 3 7°C / 5 %
C O 2. Next, non-adherent cells were removed through w ashing with Hank's buffered salt
solution (Invitrogen) and fresh culture m edium was added to the adherent cells. These were
grown to confluency and then harvested using ° . 2 5 % trypsin, diluted ’ :2 in m edium , plated,
and cultured at 3 7 °C /5 % C O 2. B M SC were obtained from 3 rats and cultured in 4 different
batches to ensure the availability o f biological replicates for microarray analysis.
48
Gene expression pattern and suitability o f bone m arrow strom al cells
RNA isolation and amplification
From P3 and P14 BM SC, total RN A was isolated using Trizol-based and RN easy M ini Kit RNA
isolation methods. Cells were first hom ogenized in ice-cold Trizol (Life Technologies, Grand
Island, New York). After phase separation by addition of chloroform, the aqueous, RNAcontaining, phase was transferred to a new RNAse-free 1.5ml tube and mixed with an equal
volume o f 7 0 %
RNAse-free ethanol. Next, an RN easy M ini colum n (Q iagen, V alen cia,
California) was used to isolate RN A according to the manufacturers' guidelines. RN A yields
and purity were determ ined using a N an o D ro p N D -10 0 0 spectrophotom eter (Nanodrop
Technologies, W ilm ington, Delaw are). RNA integrity was determ ined by the RNA Integrity
Num ber (RIN) as measured by the A gile nt 2 10 0 bioanalyzer (Agilent Technologies, Palo Alto,
California). RNA was am plified and a total o f 500 ng cR N A labelled with Cy3 or Cy5 using the
A gilent Low RN A Input Fluorescent Linear Am plification kit (Agilent Technologies) according
to the m anufacturers' guidelines.
Microarray design and hybridization
For the m icroarray experim ents we used 44K whole genom e rat m icroarrays (Agilent
Technologies) and a design consisting o f direct com parisons between P3 and P14 B M SC (3
biological replicates each). Fragm entation and hybridization was performed according to the
manufacturer's guidelines (Agilent Technologies) using A gilent solutions and buffers. Briefly,
target solution was prepared co ntaining Cy3 and Cy5 labelled cR N A sam ples (500 ng in total).
The cR N A was hydrolyzed for 30 m inutes at 60 °C , followed by hybridization in hybridization
buffer on the m icroarray probes for 17 h in a rotating cham ber at 6 0 °C . After hybridization, the
slides were washed in wash solution 1 (6x SSPE, 0 .0 0 5 % N-Lauroylsarcosine) for 5 min and
then in solution 2 (0.06x SSPE, 0 .0 0 5 % N-Lauroylsarcosine) for 1 m in. The m icroarrays were
then transferred to acetonitrile solution for 1 m in and dried under pressurized nitrogen gas.
Data analysis
M icroarrays were scanned with an A gilent m icroarray scanner. Red (Cy3) and blue (Cy5)
intensity was acquired using Feature Extraction software V7.5.1 (Agilent Technologies). Arrays
were
norm alized
based on
quantile
norm alization.
Differential
gene
expression
was
determ ined by fitting a lin ear model to the norm alized data. N ine m icroarrays were used for
com parisons between P3 and P14 BM SC. Expression of a particular gene was accepted with
an inten sity o f A >6 ,6 , to exclude outliers based on background staining. Oversaturated genes
were also removed from our analyzed set o f genes. In the subset of genes, we looked for the
presence of a clear neural relation in the nam e o f the gene and for this we searched for neur*,
glia*, oligo*, nerv* am ongst others. Furthermore, we searched online in the present gene
database for a known function in any neural processes.
49
Chapter 3
Gene Ontology overrepresentation
Overrepresented
G ene
O ntology
(G O )-classes
were
indentified
using
the web-based
application G O stat (http://gostat.w ehi.edu.au)26. W e first determ ined the num ber of genes
that were expressed in P3 a well as in P14 B M SC (cut-off: > 2 x background intensity) and these
served as a reference for the fraction o f differentially expressed genes. For each top-level G O term (e.g., biological process, cellular com ponent, m olecular function), we determ ined the
overrepresented G O -classes. The overrepresentation o f a G O -class within a set of differentially
expressed genes m eans that this G O -class is statistically higher represented than would be
expected by chance26. Statistical inference was calculated with Fisher's Exact Test. The
B en jam ini and Hochberg False Discovery Rate model was used to correct for m ultiple testing.
The m in im al length of considered G O -p ath s was 3. Significance was set at P < 0.01.
RESULTS
B M SC have emerged as candidate cells for repair o f the brain167, 273, spinal cord6’ 324, and
peripheral nerves17, 356. Profiling gene expression of B M SC may present inform ation on their
repair potential. Here we determ ined the gene expression profile of P3 BM SC, which we
typically em ploy in our transplantation studies. Im portantly, we have focused on genes
associated with neural cells/events (searches for neur*, glia*, oligo*, nerve* in their name)
and on genes with a neural function as indicated in the gene database. In this study we have
also compared gene expression of P3 and P14 B M SC to assess the effects o f long-term
culturing.
Gene expression profile in P3 BM SC
100 highest expressed genes
Am ong the 10 0 highest expressed genes, 55 genes encode for ribosomal proteins, 20 for
proteins involved in protein binding processes, 6 for proteins involved in cell differentiation
and proliferation, 4 for proteins involved in apoptosis, and 3 for proteins involved in enzym e
activity (Fig. 1). The Notchy, Tubb2, and G ja i genes were am ong the top 10 0 highest expressed
genes. These genes are im plicated in central nervous system developm ent. Notchy (Notch
homolog 3) is
involved
in
Notch
sign allin g
in
cell
differentiation1’
183, 194, forebrain
developm ent18’ 256, and neuronal fate co m m itm ent193, 256. Tubb2 is a m em ber of the tubulin
superfam ily which is im plicated in nervous system developm ent8, 3 236. Gja 1 (gap junction
protein 1) is involved in protein and receptor binding in several processes including neuron
m igration and neurite m orphogenesis120 162.
50
Gene expression pattern and suitability o f bone m arrow strom al cells
other
Fig. 1 . Schematic representation o f the top 100 genes with
the highest level o f expression in bone marrow stromal cells
at passage 3 . The numbers in the graph indicate how many
genes out of the top 100 encode for proteins known to be
involved in the indicated respective processes.
Nervous system development
The genes expressed in P3 B M SC were studied for their involvem ent in nervous system
developm ent in general (Table 1A), oligodendroglial developm ent and m yelination (Table 1B),
astroglial differentiation (Table 1C), and neuronal processes (Table 1D ). These genes m ay be
indicative of the potential of B M SC to become neural cells, and may thus be im portant for
their potential to contribute to neural repair. Am ong those involved in nervous tissue
developm ent (Table 1A) were the genes that encode for nestin (Nes)147, 226, insulin-like growth
factor 1 (Igf 1)4° 79, and neural proliferation, differentiation, and control (Npdc 1)117, 396. G enes
im plicated in oligodendroglial developm ent and m yelination (Table 1B) were Cog1, Erbb2,
Mobp and Plp2 97, 171 328, 375. P3 B M SC expressed genes involved in gliagenesis (Table 1C) such as
Igf1, Pdgfra, and Erbb2 77, 230 328 and in glia m igration such as Cspg4 461 462. A m ong those involved
in neuronal processes (Table 1D ) were genes im plicated in cell proliferation and neurite
developm ent (glial cell line-derived neurotrophic factor, Gdnf)124 and synaptic plasticity (Ras
homolog enriched in brain, Rheb)456.
Cell cycle and developmental processes
There were several genes involved in cell cycle and developm ental processes expressed in P3
B M SC (Table 2; not including nervous system developm ent genes as they are listed in Table
1). Am ong these were genes for stem cell factor KL-2 receptor binding (Kitl), which is
im portant for proliferation o f myeloid and lymphoid hem atopoietic progenitors4, 352, insulin-like
growth factor 1 (Igf 1) 40 79, transform ing growth factor beta (Tgfb 3) 43, 268, vascular endothelial
growth factor C (Vegfc)48, 289, and growth arrest and
D N A-dam age-inducible 45 gam m a
(Gadd45g)468. W e also identified the gene for adipose differentiation-related protein (ADRP)
which is indicative for the m esoderm al origin of B M S C 3°2 376.
51
Chapter 3
Growth factors and growth factor signalling
P3 B M SC expressed several genes encoding for growth factors or for proteins involved in
growth factor sign allin g (Table 3). These m ay play a role in cell growth, differentiation and
maturation and are thus potentially im portant for repair. Am ong those encoding for growth
factors were the genes for vascular endothelial growth factor C (Vegfc)48, 289, glial cell linederived neurotrophic factor (Gdnf)'24, 38’, and platelet-derived growth factor alpha (Pdgfa).
G enes involved in growth factor sig n allin g were the gene for fibroblast growth factor receptor­
like 1 (F g fri)398 and for neurotrophin receptor associated death dom ain (Nradd).
Cell death
We found several genes in the P3 B M SC that are involved in cell death (Table 4). These are of
interest as they m ay be targets for im proving B M SC survival after transp lantatio n in the
injured nervous system. Som e of these genes are for members of the tum or necrosis factor
superfam ily, which is involved in cell death113, 411, and others are im plicated in the caspase
pathway (Casp2, Caspy, Casp6, Caspii), which is involved in apoptotic m e ch anism s50 203.
Comparison between Gene expression profiles o f P3 and P14 BM SC
We found that 6687 genes are expressed in P3 as well as in P14 BM SC. O f these genes, 159
were more than 1.5-fold higher expressed in P3 B M SC and 43 were more than 1.5-fold higher
expressed in P14 BM SC. Thus, o f the genes expressed in both P3 and P14 B M SC , 20 2 genes
(3%) were differentially expressed and these genes were considered biologically relevant.
We found that from the 43 genes that were higher expressed in P14 than in P3 BM SC, 6 genes
were over 3 tim es higher expressed (Table 5A). Am ong these were genes involved in cell death
regulation (clusterin; Clu, and growth arrest-specific protein 2; Gas2)22h 384 and cell differentiation
and proliferation (clusterin; Clu, and odd-skipped related 2 protein; Osr2)'96, 384, 423.
From the 159 genes that were higher expressed in P3 than P14 BM SC, 12 genes were over 3
tim es higher expressed (Table 5B). Am ong these were genes involved in cell death (gremlin 1
homolog, Grem i)296, 474, brain developm ent, motor axon guidance, and neuron m igration
(stromal cell-derived factor-1 gamma, S D F-1)K’ 229, 303, and in cell survival (chemokine (C -C motif)
ligand 2, Ccl2) 125.
We used G O stat (http://gostat.w ehi.edu.au) to perform functional data m in in g by G O -an alysis
on the 2 0 2 differentially expressed genes to identify overrepresented G O -classes (Table 6).
The group o f 6687 genes expressed in both P3 and P14 B M SC was used as a reference. From
the 159 genes that were higher expressed in P3 than P14 BM SC, 85 (53%) were annotated in
the G O database. W e found a total of 43 G O -classes that were overrepresented including 29 in
cellular m etabolic processes and
(G O :0048513/
G O :0 0 0 7 2 75 /
9 in developm ental
G O :0 0 4 8 7 3 1/
processes and cell proliferation
G O :0 0 3 2 5 0 2/
G O :0 0 4 8 8 56 /
GO :0035 2 95/
G O :0 0 0 15 6 8 / G O :0 0 0 19 4 4 / G O :0 0 08283). From the 43 genes that were lower expressed in
52
Gene expression pattern and suitability o f bone m arrow strom al cells
P3 than P14 BM SC, 22 genes (5 1% ) were annotated in the G O database. Two G O -classes were
overrepresented and these were involved in organ developm ent (GO: 0048513) and response
to m echanical stim ulus (GO : 0 0 0 9 6 12 ).
DISCU SSION
We found that P3 and P14 BM SC express numerous genes encoding for proteins involved in
oligodendroglial developm ent and
m yelination, astroglial differentiation, and
neuronal
proliferation and neurite form ation. Other genes expressed by these cells play a role in
nervous system developm ent in addition to their functioning in other pathways. These
findings may reflect the described ability of BM SC to differentiate across lineages and become
neural cells. If B M SC possess such an ability it would greatly benefit their use in nervous tissue
repair as grafted B M SC could replace lost a n d /o r damaged neural cells. It was reported that
B M SC can differentiate into neural cells in vivo2'5 or be induced to become one in vitro'00 371 445,
but the evidence is controversial66, 243, 291.
We found that B M SC express genes that encode for proteins involved in the
developm ent o f tissue in general and in growth factor signalling. The expression o f these
genes m ay indicate the potential o f B M SC for cell-based repair as the gene products could
promote tissue formation and functioning372 427. We also found that B M SC express genes
involved in cell death m echanism s. This could be benefited from as these genes can be
targeted to improve survival o f BM SC transplanted into the injured nervous tissue. It has been
demonstrated that survival of B M SC is low after transp lantatio n in for instance the damaged
spinal cord170, 285. Improved survival could enhance the overall repair efficacy o f a B M SC
tran sp lan t322 324, 472.
O ur functional data m in in g revealed that 9 overrepresented G O -classe s in P3 B M SC
(compared to P14 BM SC) were involved in developm ental processes and cell proliferation. O n
the other hand, 1 overrepresented G O -class in P14 (compared to P3 BM SC) was involved in
organ developm ent and m echanical stim ulus. Together, these findings suggest a dim inished
plasticity of B M SC after long-term culturing. Th is idea is corroborated by the finding that the
expression level of the S D F-1 gene was more than 3 tim es higher in P3 than P14 BM SC. S D F-1
gene encodes for a protein involved in brain developm ent, motor axon guidance, and neuron
m igration86, 89, 229, 303 and m ay be indicative of the overrepresentation of developm ental
processes in P3 compared to P14 BM SC. Also supporting the idea is that no genes involved in
neural developm ent were higher expressed in P 14 B M SC than in P3 BM SC.
In the present study, the gene expression profiles of B M SC that were cultured for a shorter
and longer period were com pared to exam ine possible effects after long-term culturing. To our
knowledge the effect of long-term culturing on gene expression in B M SC has not been
investigated previously. In 3 % of the genes that were expressed in P3 and in P14 BM SC,
53
Chapter 3
expression levels had significantly changed after long-term culturing. Previously, it was shown
that, after a sciatic nerve crush, the expression level o f 5 % of the genes in neurons that had
their axon damaged changed significantly’88. This resulted in a crucial phenotypic change as
this group of genes were involved in regenerative events in the dam aged neurons’88. Franssen
and colleagues’39 showed that 7 % of the genes that were differentially expressed between
cultured Schwann cells and cultured olfactory ensheathing glial cells were expressed with more
than 3 tim es difference. Further studies will be necessary to elucidate the relevance of the 3 %
of genes differentially expressed between P3 and P ’ 4 BM SC.
O ur data showed that both P3 and P ’ 4 B M SC express genes involved in developm ental and
differentiation pathways in general, and in nervous system developm ent, m yelination and
gliagenesis in particular. The overrepresentation o f differently expressed genes involved in
these pathways alters significantly between P3 and P ’ 4 and pointed at a decrease in plasticity
in long-term cultured BM SC.
54
Gene expression pattern and suitability o f bone m arrow strom al cells
APPEN DIX
Table 1. Genes expressed in P3 B M SC involved in nervous system pathways.
A: Nervous system development
Gene Name
Exp
Gene
N M _o i2987
XM_239373
731
7 ,69
Nes
Cog1
N M _o i9i 39
11,69
Gdnf
L15011
NM_031357
N M _178866
NM_017003
7,56
6,89
6,9:2
Ctxn
Cln2
lgf1
915
Erbb2
NM_ 1003401
N M _100.4:231
7,74
10,32
NM_012529
7J5
Description
Ontology
Nestin
Predicted: component of oligomeric
golgi complex 1
Glial cell line derived neurotrophic
factor
N eu ron-specific cortexin
Ceroid-lipofuscinosis, neuronal 2
insulin-like growth factor 1
CNS development
CNS development, myelin
formation
anti-apoptosis, cell proliferation,
CNS/PNS, neurite development
CNS development
CNS. development
CNS development
CNS development
Enc1
Npdc1
V-erb-b2 erythroblastic leukemia
viral oncogene horn 2
Ectodermal-neural cortex 1
neural prol / diff and control, 1
Ckb
creatine kinase, brain
CNS development
neural proliferation and
differentiation
CNS development
B: OUgodendroglial development
Gene Name
Exp
Gene
XM_239373
7,69
Cog1
NM_017003
9^47
Erbb2
NM_0 12720
7 >697
Mobp
NM_207601
1:2,61
Plp2
Description
Predicted: component of oligomeric
golg i complex 1
V-erb-b2 erythroblastic leukemia
viral oncogene homolog 2
Myelin-associated oligodendrocyte
basic protein
Proteolipid protein 2
Ontology
-structural component olf myelin
sheath
myelination
myelin formation
myelin protein
C: Astroglial differentiation
Exp
Gene
N M L031022
Gene Name
9,65
Cspg-4
Chondroitin sulfate proteoglycan 4
Insulin-like growth factor" 1
N M L178866
6,9:2
X M L214030
9>72
lgf1
Pdgfra
NM_017003
9.15
Erbb2
Description
Predicted: platelet derived growth
factor" receptor alpha polypeptide
V-erb-b2 erythroblasticleukemia
viral oncogene homolog 2
Ontology
glial cell migration, neuron fate
commitment:, remodelling, etc
glial cell differentiation
gliagenesis
glirl d ll diffe^entiftitn
55
Chapter 3
D : neuronal processes
Gene Name
Exp
Gene
Description
N IVL019139
11,69
Gdnf
N IVL013216
9,43
Rheb
Glial cell line derived neurotrophic
factor
Ras homolog enriched in brain
N M_ 0 12922
7,65
Casp3
Caspase 3
NM_031022
9,64
Cspg4
Chondroitin sulfate proteoglycan
NM_012987
7,31
Nes
Nestin
NM_130740
7,63
Pacsin2
NM_053389
7,09
Sip1
NM_1025400
7,01
Smndc1
XM_233798
10,76
Crim1
NM_031357
L15011
6,89
7,55
Cln2
Ctxn
Protein kinase C and casein kinase
substrate in neurons 2
Survival of motor neuron protein
interacting protein 1
Predicted: survival motor neuron
domain containing 1
Predicted: cysteine-rich motor
neuron 1
Ceroid-lipofuscinosis, neuronal 2
Neuron-specific cortexin
56
Ontology
anti-apoptosis, cell proliferation,
CNS/PNS, neurite development
regulation of neuronal synaptic
plasticity
neuron apoptosis, cell fate
commitment
glial cell migration, neuron fate
commitment, remodelling, etc
neuron differentiation, CNS
development
CNS development
CNS development
Gene expression pattern and suitability o f bone m arrow strom al cells
Table 2. Expression of genes involved in cell cycle and developm ental processes in P3 BM SC.
Gene Name
Exp
Gene
Description
NM_178866
6,92
Igf1
Insulin-like growth factor 1
NM_013174
9.51
T gfb3
Transforming growth factor, beta 3
XM_237999
11 ,51
Gadd45g
NM_053 653
8,68
Vegfc
NM_031022
VO,9
Cspg4
XM_215993
8,74
Edf1
XM_341934
7,56
Tgfb1i1
A B105879
9,03
XM_345649
7,53
XM_216438
7,43
ADRP
XM_224733
7,01
Gdfi
Predicted: growth arrest and DNAdamage-inducible 45 gamma
Vascular endothelial growth factor
C
Chondroitin sulfate proteoglycan 4
(Cspg4)
Predicted: endothelial
differentiation-related factor 1
Predicted: transforming growth
factor beta 1 induced transcript 1
Scf mRNA for stem cell factor KL2 , complete cds]
Similar to development- and
differentiation-enhancing factor 2 ;
PYK2
Adipose differentiation-related
protein (ADRP)
Predicted: growth differentiation
factor 1
Ontology
growth factor activity, antiapoptosis, cell development
protein binding, cell growth,
embryonic development
protein binding, cell differentiation,
apoptosis, regulation of cell cycle
growth factor activity, cell
proliferation, angiogenesis
cell differentiation, cell proliferation,
angiogenesis
transcription, cell differentiation
protein binding, cell differentiation,
cell fate commitment
stem cell factor receptor, regulation
o f apoptosis
growth factor activity
57
Chapter 3
Table 3. Expression o f genes encoding for growth factors or proteins involved in growth
pathways in P3 BM SC.
Gene Name
Exp
Gene
Description
N M _o i9i 39
11,68
Gdnf
NW L139259
7,83
Nradd
N M_053401
9,64
Ngfrap1
NM_053 653
8,68
Vegfc
N M _o i28oi
8,59
Pdgfa
N M _ io ii92i
10,35
Pdgfrl
XM_214030
9,72
Pdgfra
NM_031525
9,14
Pdgfra
NM_199114
8,25
Fgfrl1
N M_0221 82
9,23
Fgf7
Glial cell line derived neurotrophic
factor
Neurotrophin receptor associated
death domain
Nerve growth factor receptor
associated protein 1
Vascular endothelial growth factor
C
Platelet derived growth factor,
alpha
Platelet-derived growth factor
receptor-like
Predicted: platelet derived growth
factor receptor, alpha polypeptide
Platelet derived growth factor
receptor, beta polypeptid
Fibroblast growth factor receptor­
like 1
Fibroblast growth factor 7
NM_178866
6,92
Igf1
Insulin-like growth factor 1
NM_1004274
10,41
Igfbp4
Insulin-like growth factor binding
protein 4
58
Ontology
anti-apoptosis, cell proliferation,
CNS/PNS, neurite development
signal transduction, neurotrophin
p75 binding
induction of apoptosis
growth factor activity, cell
proliferation, angiogenesis
growth factor activity, cell
proliferation, cell migration
cell surface protein
gliagenesis, positive regulation of
cell proliferation and cell migration
anti-apoptosis, positive regulation
of cell proliferation
negative regulation of cell
proliferation
growth factor activity, positive
regulation of cell proliferation
anti-apoptosis, cell development,
glial cell differentiation
regulation of cell growth
Gene expression pattern and suitability o f bone m arrow strom al cells
Table 4. Expression of genes involved in cell death m echanism s in P3 BM SC.
Gene Name
Exp
Gene
Description
Ontology
XM_226833
8,29
C1qtnf3
NM_139194
8,07
Tnfrsf6
NM _013049
7,87
Tnfrsf4
N M _o io o 6952
7,76
Trap1
XM_222503
7,23
Tnfrsfn
a
NM _01010945
7,11
N M _ i8io 86
10,33
NM _013091
9,96
Tnfrsf12
a
Tnfrsf1a
NM _0 1012123
9,61
C1qtnf5
XM_226833
9,49
C1qtnf3
NM _030836
9,48
Arts1
N M _ i8io 86
8,47
Tnfrsf12
a
BC061751
7,96
XM_342470
9,91
X M _2 i 6650
9,14
NM_0535i6
9,0 9
N0I3
Nucleolar protein 3 (apoptosis
repressor with CARD domain)
regulation of apoptosis
XM_342137
8,45
Amid
regulation of apoptosis
X M _23566i
8,34
Predicted: apoptosis-inducing factor
(AIF)-like mitochondrion-associated
inducer of death
Predicted: similar to TGF-beta
induced apoptosis protein 12
NM_053736
8,77
Casp11
Caspase 11
regulation of apoptosis,
inflammatory response
NM _022522
7,69
Casp2
Caspase 2
regulation of apoptosis,
inflammatory response
NM _012922
7,65
CasP3
Caspase 3
regulation of apoptosis, neuron
apoptosis, cell fate commitment
NM_031775
7,291
Casp6
Caspase 6
regulation of apoptosis,
inflammatory response
Predicted: C1q and tumor necrosis
factor related protein 3
Tumor necrosis factor receptor
superfamily, member 6
Tumor necrosis factor receptor
superfamily, member 4
Tumor necrosis factor type 1
receptor associated protein
Predicted: Tumor necrosis factor
receptor superfamily, m emna
Similar to transm protein induced by
tumor necrosis factor alpha
Tumor necrosis factor receptor
superfamily, member 12a
Tumor necrosis factor receptor
superfamily, member 1a
Predicted: C1q and tumor necrosis
factor related protein 5
Predicted: C1q and tumor necrosis
factor related protein 3
Type 1 tnf receptor shedding
aminopeptidase regulator
Tumor necrosis factor receptor
superfamily, member 12a
induction of apoptosis
induction of apoptosis
cell death
positive regulation of angiogenesis
cell death
positive regulation of angiogenesis
cell death
Androgen receptor-related
apoptosis-assoc protein CBL27
APi5
Predicted: apoptosis inhibitor 5
anti-apoptosis
Predicted: similar to apoptosis rel
protein APR-3; p18 protein
induction of apoptosis
59
Chapter 3
NM_139259
7,827
Nradd
Neurotrophin receptor associated
death domain
signal transduction, apoptosis
NM _019139
11,69
Gdnf
Glial cell line derived neurotrophic
factor
anti-apoptosis, cell proliferation
NM_199270
7,79
Bre
Brain and reproductive organexpressed protein
apoptosis
NM _053401
9,64
Ngfrap1
Nerve growth factor receptor
(TN FRSF16) associated protein 1
induction of apoptosis
NM_133546
6,87
M ydn6
Myeloid differentiation primary
response gene 116
apoptosis, response to stress
NM _012588
8,52
Igfbp3
Insulin-like growth factor binding
protein 3
positive regulation of apoptosis
NM _01012154
7,179
Tbrg4
Predicted: Transforming growth
factor beta regulated gene 4
apoptosis
XM_216265
6,842
Ing4
Predicted: Inhibitor of growth family,
member 4
apoptosis, cell cycle arrest, neg
regulation of cell proliferation
XM_237999
1 1 ,51
Predicted: Growth arrest and DNAdamage-inducible 45 gamma
apoptosis, regulation of cell cycle
NM_133544
11,04
Gadd45
g
Usmg5
Upregulated during skeletal muscle
growth 5
apoptosis, regulation of cell cycle
NM _012756
10,76
Igf2 r
Insulin-like growth factor 2 receptor
regulation of apoptosis
NM_013174
9,51
T gfb3
Transforming growth factor, beta 3
regulation of apoptosis, cell growth,
regulation of cell cycle
NM _031525
9,14
Pdgfrb
Platelet derived growth factor
receptor, beta polypeptide
anti-apoptosis, positive regulation of
cell proliferation
NM _017003
9,15
Erbb2
V-erb-b2 erythroblastic leukemia
viral oncogene homolog 2
anti apoptosis, positive regulation of
cell proliferation
60
Gene expression pattern and suitability o f bone m arrow strom al cells
Table 5. D ifferentially expressed genes between P3 and P14 B M SC with A > 3 fold difference.
A: Upregulated at P14
Gene Name
Exp
NM_053021
3,96
U87983
3,66
N M _o i2862
Gene
Description
Clu
Clusterin (Clu)
3,39
Mgp
receptor for hyaluronan-mediated
motility
Matrix Gla protein
XM_574454
3,0 9
NM_0 0 10 1211
8
NM 00 10 118
89
3,08
LOC49
9156
Osr2
3,04
Cldn9
Predicted: similar to growth arrestspecific protein 2 - mouse
Predicted: odd-skipped related 2
(Drosophila)
Predicted: claudin 9
Ontology
protein binding, apoptosis, cell
differentiation /proliferation, etc
regulation of bone mineralisation
cell cycle arrest
positive regulation of cell
proliferation
cell adhesion molecule
B: Upregulated at P3
Gene Name
Exp
Gene
Description
Ontology
NM_012893
N M_080886
2,99
3,19
Actg2
Sc4mol
Actin, gamma 2
Sterol-C4 -methyl oxidase-like
protein binding, cytoskeleton
oxidation reduction
XM_231258
3,25
E-FABP
NM_031776
XM_233037
3,27
3,30
Gda
Pappa
in
d
i
i
b
in
n
o
i
tal
te
m
XM_214778
3,45
TSP-2
similar to Fatty acid-binding protein,
epidermal
Guanine deaminase
Predicted: pregnancy-associated
plasma protein A
Thrombospondin 2
XM_573502
3,78
AF217564
3,79
SD F-1
similar to Fc gamma (IgG) receptor
II alpha precursor
Stromal cell-derived factor-1 gamma
N M _ i0 0 76 i 2
8
,
3
,
Ccl7
Chemokine (C-C motif) ligand 7
N M_030834
NM_031530
4 ,4 0
4 ,44
Slci 6a3
Ccl2
Monocarboxylate transporter
Chemokine (C-C motif) ligand 2
NM_019282
5,0 9
Gremi
Gremlin 1 homolog, cysteine knot
superfamily (Xenopus laevis)
Ppptidase activity, response to
glucocorticoid stimulus
angiogenesis, positive regulation of
synaptogenesis
brain development, motor axon
guidance, neuron migration, etc
chemokine activity, immune
response
transporter activity
anti-apoptosis, immune response,
chemokine activity
apoptosis, cell-cell signalling
61
Chapter 3
Table 6. Overrepresented G O -classes in significantly different expressed genes between P3
and P14 B M SC (A > 1,5 fold difference) com pared to total subset of expressed genes.
GO CLASS
GEN E O NTOLO GY
GROUP
Total
P-value
25
252
3,45e- ° 6
UPREGULATED AT P3
Biological process
organ development
G O :ooo6o66
alcoholic metabolic process
15
75
4 ,10 E-06
G0 :0 0 0 8 2 0 2
steroid metabolic process
11
37
4 ,1°E -o 6
G O :ooo6694
steroid biosynthetic process
9
23
4,84E- o 6
G O :o o i6i 25
sterol metabol ic process
8
19
G0:0032501
multicellular organismal process
38
554
G O :o oo7275
multicellular organismal development
31
4 °9
6,82E-°5
G0 :0 0 0 8 20 3
cholesterol metabolic process
7
17
6,82E-°5
G O :o o i6i 26
sterol biosynthetic process
6
12
1,°5E-°4
G O :oo4873i
system development
lipid biosynthetic process
27
11
345
62
1 ,67E-° 4
G O :ooo86io
G0:0032502
developmental process
38
601
6,29 E- ° 4
G O :ooo6o o7
glucose catabolic process
7
27
0,001
G O :oo46i 64
alcohol catabolic process
7
28
0,001
00:0046365
monosaccharide catabolic process
7
28
0,001
G0 :0 0 19 320
hexose catabolic process
7
28
0,001
00:0048856
anatomical structure development
28
0,002
G0 :0009605
response to external stimulus
14
4 °9
123
G0 :0 0 0 60 9 6
glycolysis
6
21
0,002
G 0:0044275
cellular carbohydrate catabolic process
7
31
0,002
G O :0 0 i 6052
carbohydrate catabolic process
7
31
0,002
G0:0035295
tube development
7
31
0,002
G0:0065008
regulation of biological quality
17
201
0,003
G0:0006936
muscle contraction
6
25
G0 :0 0 0 30 12
muscle system process
6
25
0,005
G0:0006695
cholesterol biosynthetic process
4
9
0,005
G0 :0 009058
biosynthetic process
23
325
0 0 :0 0 4 24 4 6
hormone biosynthetic process
3
4
0,005
G0 :0 0 30 19 9
collagen fibril organization
3
cellular lipid metabolic process
13
4
122
0,005
G0:0044255
00 :0 0 4 8 8 78
chemical homeostasis
9
62
G0:0006935
chemotaxis
5
17
5
-0
3
3
,
6,82E-°5
3,77E-°4
0,002
0
5
0
0
,
0
5
0
0
,
0,005
5
0
00
,
62
L.Ù
00:0048513
0,005
Gene expression pattern and suitability o f bone m arrow strom al cells
G O :0042330
taxis
5
17
0,005
G O :o o o i568
blood vessel development
8
50
0,006
G O :o o o i944
vasculature development
8
51
0,007
G O :ooo6oo6
glucose metabolic process
7
39
0 ,0 0 7
G O :0007267
cell-cell signaling
11
95
0 ,0 0 7
G O :0055082
cellular chemical homeostasis
8
52
0 ,0 0 7
G O :ooo6873
cellular ion homeostasis
8
52
0 ,0 0 7
G O :0030005
cellular di-, trivalent inorganication homeostasis
7
40
0 ,0 0 7
G O :0055066
di-, tri-valent inorganic cation homeostasis
7
0 ,0 0 7
G O :ooo8283
cell proliferation
G O :o o 5o8oi
ion homeostasis
15
8
40
182
55
0,009
G O :0044421
extracellular region part
28
306
3,45E-° 6
G O :o oo56i 5
extracellular space
25
286
3,69e-0 5
G O :o oo5578
proteinaceous extracellular matrix
9
59
0,005
0,008
Cellular component
Molecular function
G O :0008201
heparin binding
5
17
0,005
G O :0005125
cytokine activity
6
29
0,008
G O : oo42379
chemokine receptor binding
3
5
0,008
G O :ooo8oo9
chemokine activity
3
pattern binding
5
5
20
0,008
G O :o o o i871
G O :o oo5539
glycosaminoglycan binding
5
20
0,009
G O :0030247
polysaccharide binding
5
20
0,009
0,009
UPREGULATED AT P14
Biological process
G O :0048513
organ development
10
252
0,005
G O :ooo96i 2
response to mechanical stimulus
3
9
0,008
proteinaceous extracellular matrix
6
59
0,004
Cellular component
G O :0005578
63
I JOHNS HOPKINS
fli M E D I C I N E
Ä
4
4
Kennedy Krieger Institute
4
Survival and neuroprotection.
Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation
into the contused adult rat thoracic spinal cord.
J Neurotrauma 2009; 26 (12): 2313-2322
R.D.S. Nandoe Tewarie
A.Hurtado
G.J. Ritfeld
S.T. Rahiem
D .F. W endel
M .M .S. Barroso
J.A. Grotenhuis
M. Oudega
Chapter 4
IN TRO D U CTIO N
A contusion o f the adult spinal cord causes acute death of neural cells at the lesion epicenter
and sets off a series of events resulting in additional tissue loss and the formation of cystic
cavities’55, 3° 5. Transp lan tatio n o f growth-promoting cells is considered a prom ising approach
for repair of the contused spinal cord3’3 3’3.
Bone marrow stromal cells (BM SC) are am ong the candidates for cell-based spinal cord
repair strategies285. These m esenchym al cells secrete growth factors that could support
repair’43, 247. Their potential was confirmed by the finding that transp lantatio n o f B M SC into
the contused adult rat spinal cord resulted in functional recovery’70 3° 4, 45^ 473 473. The
m echanism s underlying these functional im provem ents observed after B M SC transplantation
are incom pletely understood.
A B M SC graft could contribute to repair by sp arin g spinal cord nervous tissue at the site of
transplantatio n. It is known that B M SC secrete brain-derived neurotrophic factor (B D N F)247,
which has been im plicated in lim itin g spinal cord tissue loss after injury364. A t present, the
evidence for B M SC grafts to elicit tissue sp aring in the injured spinal cord is still am biguous304,
45’, 464 .
Following an insult to the spinal cord, a cytotoxic environm ent develops at the injury
epicenter to d im inish in strength in tim e’55. M ost likely these circum stances decrease the
survival of cells transplanted into the lesion and consequently their effects on sp inal cord
repair. At present, quantitative evidence o f the survival and neuroprotective effects of B M SC
transplanted into a spinal cord contusion is sparse, which obscures their spinal cord repair
potential. W e transplanted B M SC into a m oderately contused adult rat spinal cord at ’ 5 m in
(acutely) and at 3, 7 and 2 ’ days (delayed) post-injury and determ ined tissue sparing and
B M SC survival up to 4 weeks post-transplantation.
MATERIAL A N D M ETHODS
Timeline of the experiment
Rats received a moderate contusion o f the T9 sp inal cord. Some of these rats received a B M SC
injection into the injury epicenter at ’ 5 m in, 3, 7, or 2 ’ days post-contusion. These BM SCtransplanted rats were euthanized at ’ 5 m in, 3, 7, and 28 days post-injection. O ther contused
rats did not receive the B M SC injection (control rats) and, to match survival tim es of the
B M SC-transplanted rats, were euthanized at ’ 5 m in, and 3, 7, ’ 0, ’ 4, 2 ’ , 24, 28, 3’ , 35, and 49
days post-injury. Their spinal cords were removed and prepared for histology to enable
analysis o f tissue sparing and BM SC num bers. D etails on the used techniques are described
below.
66
Survival and neuroprotection
Animals
Adult female Sprague-D aw ley rats (n = 121), 16 0 -18 0 g; H arlan, In d ian ap o lis, IN , USA) were
used in these experim ents. All an im a ls were housed according to the guidelines by the
N ational
Institutes of Health and the United States
D ep artm ent of Agriculture. The
institutional A nim al Care and Use Com m ittees of the University o f M iam i and Johns H opkins
University approved all surgical procedures.
Culture and lentiviral transduction of BM SC
B M SC were obtained from the marrow o f femurs and tibias o f adult fem ale Sprague-D aw ley
rats (n = 8) according to a previously published protocol13. Passage 0 cells were infected with
lentiviral vectors (LV) encoding for green fluorescent protein (GFP) at an M O I of 150. The
vectors were generated using the ViraPow er Lentiviral Expression System (Invitrogen)36.
Expression was under control o f the human cytom egalovirus promoter and the Woodstuck
hepatitis virus Post-transcriptional Regulatory Elem ent284. The titer o f the lentiviral stocks
varied from 1-3 x 10 9 T U /m l. O n ly passage 3 cultures with a transduction rate of about 9 5 %
were used for transplantation.
With enzym e-linked im m une sorbent assays (ELISA) we determ ined whether the B M SC
secreted B D N F and glial cell-derived neurotrophic factor (G D N F ). For this, m edium of nearly
confluent passage 3 cultures (with an average of 2 x 10 6 cells) was refreshed, removed 24 h
later, and used for B D N F ELISA (B D N F Emax Im m unoA ssay System; Promega Corporation,
M adison, WI) and G D N F ELISA (G D N F Emax Im m unoA ssay System; Promega) according to
the m anufacturer's instructions. Standard curves of the ELISA kits were linear between 7.8 and
500 p g /m l for B D N F and 15.6 and 10 0 0 pg/m l for G D N F . ELISAs were analyzed with a
microplate reader (SpectraM ax M5, M olecular Devices, Sunnyvale, CA). The cells in culture
secreted 14.1 p g /m l/2 4 h of B D N F. The am ount of G D N F was below the reliability level o f the
ELISA.
Contusion injury
Rats (n = 108) were anesthetized with 1 - 2 % isoflurane in oxygen and their backs were shaved
and cleaned with Betadine and 7 0 %
alcohol. Lacrilube ophthalm ic ointm ent (Allergen
Pharm aceuticals, Irvine, CA, USA) was applied to the eyes and gentam icin (1.2 mg in 0.03 ml;
Abbott Laboratories, North Chicago, IL, USA) was injected intram uscularly. D uring surgery,
rats were kept on a heating pad to m aintain their body tem perature at 37 ± 0.5 °C . A
lam inectom y was performed at the eighth thoracic vertebra to expose the ninth thoracic spinal
cord segment, which was subsequently m oderately contused (NYU im pactor; 10 g, 12.5 m m.;
G runer 19 9 2). To ensure consistency between contused rats, the contusion im pact velocity
and
com pression
were
monitored
and
all
rats with
more than
5%
error in
these
m easurem ents were removed from the study. Rats that rem ained in the study displayed
67
Chapter 4
h in d lim b p a ra ly sis and scored less than 3 at 1 d ay and less than 7 at 3 days p o st-in ju ry on the
B a sso -B e a ttie -B re sn a h a n
(BBB)
scale 23, 24.
Im p le m e n ta tio n
o f these
crite ria
ensured
a p p ro p ria te n e s s o f the co n tu sio n in ju ry and c o n siste n c y a m o n g the e x p e rim e n ta l a n im a ls . All
la m in e c to m ie s and inj uries w ere perform ed by th e sa m e in ve stig a to r. After in ju ry, o verlying
m u scles w ere sutured in leyers and the al<in w as closad with m etal w ound c lip s. Th e rats
received Ringee’ s solution (10 m < suecutaneoua) and g e n ta m ic in (1.2 m g, in tra m u scu la r). Th e
rats w ere kept in a sm a ll a n im r l ie su b a te r at 3< ° C un til full recovery and then returned to
the ir cages w ith ad Ubhum access to w ater and food. D u rin g the first w eek p o st-in ju ry
g e n t rm ic in
( i.e
m g; Abbott L a b o ra to rie s
IMorth C h icago ,
11_, USA; in tra m u scu la r) w as
a d m in i stored daHy. T h e a n a l gedic, bup ren o rp h 1ne (Bu ferencx®; 0 .0 0 6 m g; R eckitt Benckiser,
Rich m ond, V A , U SA; sub cu taneaus) w as rd m in istered im m e d ia te ly after surgery and o n ce for
t_e next; three days. Tine thadders w ere exprhssecJ m a n u a lly tw ice
a day un til reflex bladder
function retu rned. U rin _ ry tract i n frc tio n did not occur in au y o f tne groups.
BM SC transportation
C o n tu se d tats w ern c n a e sth a tiz e d w ith
a n in tra m u s c u la r in je ctio n o f 25.7 m g /k g ke ta m in e ,
5 .14 m g /k c x y la z in e , and ce85 r u g m
a cep ro m a e in e and tli eir co ntused s p in a l cord w as
expo sedi
A total oC 1 x sd>s B M S C in s jxi D M e M vrais; injectod into the co n tusio n e p ic e n te r
usin g te ch n iq u e s d t s c r iged p re vio u sly’75. B M S C in je c tio n s w ere m ad e at 15 m in , 3, 7, and 21
d a y / p o st-co n tu sio n . In the 15 m in ero up , the re tt re m a in e d eedated w ith the co ntused sp in a l
cord tdvered with a sal in e -m o is ta n ga nze u nti l B M S C in je ctio n . A ll tra ns p la n ta tio n s w ere
perform ed ey the cam e in ve stig a to r. After in je c tio n , m u scles w ere sutured in laye rs and the
skin closed w ith m etal w ound c lip s . go st-surgery caret w as r s dexcribed above.
Assessment o f BM SC viability during nho injection proteVr red
Th e effeete o f t t a in je ctio n procedures o r the n u m b e r c r B M S C w ere d e te rm in e d in vitro u sin g
the sa m e tools and m etho ds a r used So’ aceuai t rc n s p la n ta tio n . First, we asse sse d the v ia b ility
o f 1 x 1 o 6 B M S C in s (el D M E M in an e p e e n d o rf tube after 5 h on ice (the typ ica l le n gth o f a
trd n sp lu n te tio n se ssio e ti A sa m p lu oS tli ese cel Is w es s tr in e d w it i trypan blue (1:1; S igm a)
and t°ie . urce ntage o f lie ing (u n stain ed ) B M S C cal culated u s in g a h em acyto m o te r. S eco nd , we
a sse rse d taMdSCZ v ia b ility iafter in je ctio n . B M S C in D M E M w ere kept on ice for 5 h and then
in je cte d into an e p p e n d o rf tube (1 x i r 6 B M t C in 5 ml D M E M pen tube) n sin g the sa m e pulled
g la ss injeceion n e ed le a tt a c le d to
a H a m ilto n syri ngn as used for tra n s p la n tatio n into the rat
sjuinal cord. T h e se cells w ere then kept at 3 1 ° C / 5 % C O 2 for 1, 3, and 5 h (n = c for each tim e
p o ia t). B M S C tha_ w ere not p assed through the g la ss n fe d le and kept in the in cu b ato r for the
se m c timet perioda served as controlc (n =
3 eon each tim e p o in t). Ait the celected tim e s, and
im m e d iately after pecsi n | tli ro uu t the h la rs n eed le (tim e p o in t 0), ce lls w ere stain e d with
tryp an blue (11:1; S ig m e ) and the p e rc rn (a g e o f livin g (u n stain e d ) B M S C calcu lated u sin g a
68
Survival and neuroprotection
hf;m^cytome:^ei'. The effect o l passing throughi thè glass aendle wes determ ined by thè
differenee i n hhe paraentax as o f Ii\/ingj B M SC betwean control asci Itim e p ain t o celi s. ’SX/1'iethier
the ii'i(^ub)ohl(^n conditions Cad aliTect^sJ d M SC dnath ln tim e wes cjalsulotesd Oroia the differenee
in \zisiSiililty b etwenn contrmi cells a ad ceCs tdet were passed tOron^la tbe glass n endlh a t 1 , 3,
and s Ir. TNird, we determ iaed Iiow m cnd </«sli^ passes tdirongla the needle. A sassipile m l1 x io 6
B M SC in 5 mI D M SM thet were injected into a s ^ppanCerlT^uge usine a pallsd glass needle
attached
e H am ilton tnringe wes otnised v/ithi tri^p>aa blus (1:1; SiCiaie) to calnulste the total
nem der of lihing ^unc1;Esi^ed) BM dC u :singf a SemolytometelP
Histolngical MroceOures
At 15 m in, 3, 3, a isd 28 de^s cfter d M SC injeuèion, rats wern nncosthetized as aSiond (ncte thet
tho in m in d^icus» remhmed anensthstized nnSil fìnatidn). C ostm i rats thet were contused bet
did noS seeeive a B M SC thantcltsst were sin;as;sdOntizzd 15 m in, and a, 7, 10, 4 21 , C8, 28, 31,
355, nnd so dco'd p a st-m isa r Thhcm timo n aiste ooi'r^sf3(3nC with the murvinal tim es efth e BM SCtrantelw nfnd satc. After deap sehetion wes condrm ed, thu heart wes expaced und 0.1 ml
H aporine (500 IU; l-la n s; Sabein, IVInlvllle, N'Y', l_lSA) injedted into /hie l^ft \^€5Sìdricle5. Next, 3 0 ,
mi caline follownd do 50ic:> ml icn-mold 4 % paraformeldehyde in paoupaate buffer (PB; 0.1 IM,
pH n-4 ) vsea pjurtipi^d theongd ithie vasculsr system. Thn sp in si ccnrd wes removed and kept
occrnigat in the eame fìoctlve nt 4 °C . TO m , a 15 m ni long segm ent Cif the sptincl cord
centeted at sbe contusion wes diesectod mut, liispct in paeipaTte-duffnred t ^ <^ sunnaue 0 0 M,
p a 7.4) fai' no
0,
esd Nrooen w itirn Sìliiini^on M
Em eoddin g Mictrix (Theumo Electron
Tc^rp>or;ation, Pistsbergh, fssii, O d A r Frorn thnse blonks, :?o (jrnn-thick eoriodntcl nections were
o it ori a cryostat, sn^nnt^d onto glans elide/, and stored ct -2-8 °C .
Frons innse antr n ln e cd groug, a 1 maa tliicd section wes takan dromo t in epicenter csf 'thie
Icsion for im paratis>n olsnns^t;lEÌn plcsUic seetions fci> Iì^ 1i 1> minrassopy. The j m ei tdieb tissun
ItiI o le were t a p i ih 2 <:8S oluta raldellyde n^i'tl-e s% sunrosc in f3t for at le ast eoi Ir £it 4 °C . In ( %
osmiuns tetraooirJe in e B for S2-16 li, .ehydsatxd, and ^m^edOs!^ io [ifj^ n -^ rii1!:!!^«; (Udectron
|bc^Trou::^f:)^ Sieroinh^, s or1t '^aclcisiJt^on, I^B,. C^nT:-m
tliia1 tsnnsvinrsb e^i:tiont 'Nere cot ssn a
R eid1eo1:-Juno uitra-m icrotom e, utniceed vsi'thi a 1 % toluidino (ili/a^:SX^ m ^thj/len^ Silun-:1^ noliem
Siorat^ uolution assi asad ^o anai;y;se g5ane)ai hlietoloji4: of7the s p in a 1 cord aind trarnspìlant £md
df;^ei'l55id^ ^i^san lous ct tàe iaijur^dtr^istp:>lhsst s^fmi^^nte^r'.
Measdrdmem o f apmol nord tiszue a»a ring
^Co rics g of7 sptincl eors( ti^eu^ vs^s temesse0 in a olin<^ec( foshiien in horioontal (sr^syil isic^e^tsthicesJ cryoutct ss^c^ion^ Lisiing
e (Sovalicri ect;im^^oi> ^anctican of^^tl;rno Incfcstis;at^r®> (i^ S V
Bioucience, ^^llisto n , ^T"). (!ili m^asnrens^n^s wese take!n Io). ^h^ aamn ionnrtigator> F: rom ^^ch
^ n i^ e l, ever}! ^en.h croou^nt; s^c^1© ' (orcionim ivtezvals) lites es^cJ tn dsi^n^m^si^ tne mccium^
a
;. mm Ir^ng1 e^g^m^nt c:en^erf5c( at; t;he contu^ionftrEm tpilantation epi^^nt^r and thet of7apared
69
Chapter 4
tissue within this segm ent. Tissue was considered spared if7 it lacked cavities, areas with high
density of7 in tltra tin g sm all cells, resem bling nsutropails and lympaocytes, and nturons with
darkly-stained cytnnlasm ic N issl bodies409. The volsm e of spared tissue was expressed as a
percentage o f tha average volume of a co mp arable uninjured spinal cond segmdnt.
Measurement o f - he number o f BM SC wWCoo the conSurep spinal cord
From each rat transplantdd w itli E3 INd:^C, every tunth cryoctat section weis coveued with a glass
slip with Vectashield with D A P 1 (4'-6-diam iding-:^-p^l'^a;by'^isictole^ Vector Laboratories, inc.) and
used to dielte;rmine tl-v«; num ber o f l r F^^p>oci^ive n M SC in t h i contu siopia In t l i se sections, in a
blinded fashion, tlm consusion areo with G FP -co citive celln was outlined uncJ^r a 2.5 x
objective. T h in , ijncd^r t 63 n objpctive (oil im m ersion), gride of 250 by 250 | m (coueting iTramv
area, 6 3 p nm 2) we re placed ovsr 'tO/6; outline d a res and each GFP-uor:i^ive ceil with a
secognizdbie n u n le d w st countdd using / h i o ^ ic a l fractionator 'T^ncr'tion o f Stereo
Investigaton® (Kit S I1 B icscience, W illiston, V T, UOA). T h i sam plin g interval was: x = 25 o mm , y
aa ^ 50 (sm . A ) m easurem erts wero ta)en bo the sam e inveFtinarsr. "Thii^ ^euulrtii^jej nunrtjirii^^
wade us ed to c;sl'^ulsit:e; t S i tota l num ber c f GFP-pocitihe B M SC within t lo ^ontusiioni These
totel numbere w /te 1;i^ n x p r e o t e d as a f^ercei^tai^^ o f the totri numbes fomnch Ft 15 m in poctinjection,
Assessment e f BM SC migeaaion
We hound tin^t traesplanted B M SC h id migrated cway from t in injection ¡¡it;«;. Their num bee
location, s n t distance from shy contu^ic^n epicenter were cSe^erminird in one serips ofcryostst
^«j'c'ticsns; oS each o C ^hnen transplanted rats. Because it is peonsible t h it B M S C lind migrated
eeyond 8 mire w hicf wei;:; tfe
I'oximsite; dis^tanc:t; from tine; in"ury epicentat to ihe e d is of
tine; section in rosCral an d cau d d direction, we also sectioned and oxemined the contiguous 10 ­
mm long eoctral and crsiurl^l spinal cord segment. A ) GFP-pocitive CM UC outsida of7 t h i
contu nioc were c:ounted m anually unden a 4 o x oUjective.
Statisti'car analysiv
We uned Sigm srtat® (Systat Software, inc., San Jore, CA) U 55/X) ■fo>r t h i rtatistical analyses. T h i
t-Sest was uned to determ ine differences betw/een groups which; were accepted at po 0.05. T h i
relationship tetw een tissue sparing and B M SC num ber was diteri'nined by the Pearson
correlation coefficient (n).
70
Survival and neuroprotection
RESULTS
B M S C transplants in the contused spinal cord
W ith all tra n s p la n ta tio n p a ra d ig m s, G F P -p o s itiv e B M S C w ere found w ith in the c o n tu sio n at 15
m in after the in je ctio n (Figs. lA -C ) . Th e n u m b e r o f B M S C w ithin the co n tu sio n w as no ticeab ly
d ecreased at 7 days (Figs. lD -F ) and 28 days (Figs. i G - 1) after in je ctio n .
T im e o f In je c t io n
1 5
m in ( a c u t e )
7
d d e la y e d
2 1 d d e la y e d
Fig. l . BM SC transplants within a moderate contusion in the adult rat thoracic spinal cord decrease in size during 28
days post-injection. The transplant is shown at 1 5 min ( A - C ) , 7 days (D-F) and 2 8 days ( G - l ) after an injection at 15 min
(acute), 7 days and 2 1 days, respectively, post-injury.
represents 600 jmin in ( A - 1) .
A ll
microphotographs are from horizontal cryostat sections. Bar in
A
Spared tissue in the contused adult rat spinal cord
W e q u a lita tiv e ly asse sse d the co ntused s p in a l cord se g m e n t o f adult rats w ith ou t (controls)
and w ith a B M S C tra n s p la n t u sin g to lu id in e b lu e -sta in e d tra n sve rse se m ith in p la s tic sectio ns
o f the co n tu sio n
e p ic e n te r and cresyl vio le t-sta in e d
h o rizo n ta l cryostat se ctio n s o f the
co ntused s p in a l cord se g m e n t (Fig. 2). In tra n sv e rse p la s tic sectio n s, a lo o sely o rg a n ize d
t ra n s p la n t that filled up the c o n tu sio n w as found at 3 days after an acute in je ctio n (Fig. 2A ). A t
28 days after a B M S C in je ctio n into the 2 1-d a y old co n tu sio n the s p in a l cord w as not o n ly
d ecreased in size but a lso c o n tain e d one or m ore large cavities (Fig. 2B ). O n h o rizo ntal
sectio n s, after an acute in je ctio n , cavitie s w ere not p re se n t at 3 days (Fig. 2C) but could be
71
Chapter 4
found at 28 days (Fig. 2D ). With a 7 day delayed injection, sm all cavities were found at 3 days
(Fig. 2E) and much larger ones at 28 days (Fig. 2F) post-injection.
Fig. 2. Spared tissue in the contused aduIt- rat spinal coI'd after BMSC transplantation. (A) Loosely organized tissue in
contusion euicenter at a days after sin acute BMSC injection. (B) Cavities present in the contusion epicenter at 28 days
aCtrr a 21 day delayed transplaetation of BMSC. Note that the spinal cord has also decreased in size. (C) At 3 days after
ncute BMSC injection, damaged tissue but not cavities was observed. (D) At 28 days after acute injection, large cavities
were found in the contused segment. (E) WitO a 7 day delayed BMSC injection, damaged tissue and few small cavities
were found at 3 days post-trnnsplantation. (F) /At s 8 days after BMSf injection into the 7-day old contusion, large
cavities were found in the centusfd segmfnt. Images in A and O are from toleidine blue-stained transverse semithin
plastic sections. Images in C-O are from cresyl violet-stainod horizontal cryostat sections (rostral to the left). Bar in A
represents 150 jjLrn in (A-B). Bar in C hepresents 600 |jJin in (C-F).
Using Stereo I n v e s t ig a t o ( software (M BF Bioscience) we determ ined that the volume of
spared tissue in the contused spinal tord segm ent gradually decreased to 10.1 ± 1.7 m m 3 at 49
days post-injury in control ra ts and to 11.5 ± 0.9 m m 3 at 49 days post-injury in B M SC
trainsplanted
rats (Tabln 1). Because narrow ing o f the contused segm ent occurs, we
determ ined th t aolume o f spared tissue relative to th at o f a com parable segm ent from
u nii-s jured an d u u tre ated s ft ina 1 co rd (21.9 ± 3.4 m m 3i SD , n =¡5; Fig. 3A). W e found that with
B M SC injected at 15 m in, 3, or 7 days after injury the; volume o f spared tissue was significantly
higher in tranuplantee ratr than in control rats at the respective endpoints (i.e., 28, 31, and 35
days post-inj ury).
W e then determ ined the relative change in spared tissue volume in transplanted and
control rats between the sam e tim e periods after contusion (Fig. 3B). Im portantly, in B M SC
72
Survival and neuroprotection
t r a n s p l a n t e d rats th e v o l u m e o f s p a r e d t i s s u e a t 15 m in a fter in je c tio n w a s s im il a r to t h a t
fo u n d in con tro l rats o f m a t c h i n g survival t i m e s . T h e resu lts s h o w th a t t i s s u e s p a r i n g d u r in g
t h e 2 8 - d a y s t h a t B M S C w e r e p r e s e n t w a s i n c r e a s e d with a c u t e a n d 3-d ays d e l a y e d but n o t with
7 a n d 2 1 - d a y s d e l a y e d B M S C t r a n s p l a n t a t i o n c o m p a r e d to th e lo ss t h a t o cc u rs in con tro l rats
w ithin th e s a m e t i m e p e r i o d s .
id q 1 2 0 ■
i3 co
:<r +' 1 0 0 80 ■
r*r\ ■
3 ^
W 60
WId
-n
W
Z 8 40 ■
(5 c 2 0 ■
0
15m 3d 7d lOd 14d 21d 24d 28d 31d 35d 48d
A
Time after injury/transplantation
60 -1
50 ■
□
Control
■
BMSC
T3 « 40 ■
(D
ra ffi 30
&.H
<u.!=
-
¡ a s 2 0 -h
0
B
■
J
_
15m-28d
■ ■ ■
3d-31d
7d-35d
21d-49d
Time Intervals
Fig. 3. BM SC transplantation elicits tissue sparing in the contused adult rat spinal cord. (A) Bar graph showing the
volumes of spared tissue in the contused spinal cord segment in rats without (control, open bars) and with (closed bars)
BMSC transplants. Spared tissue volume is expressed as a percentage o f the volume o f a comparable uninjured spinal
cord segment. Time points of the x-axis refer to control rats. The survival times o f BMSC-transplanted rats that matched
these time points were 15 m /15 m, 15 m /3 d, 15 m /7 d, 3 d /7 d, 7 d/7 d, 21 d/15 m, 21 d /3 d, 15 m /28 d, 3 d/28 d, 7 d/28
d, and 21 d/28 d (see also Table 1 ). Asterisks indicate a significant difference control and BMSC transplanted rats (* =
p<0 . 0 5 ; * * = p<0 . 0 3 ; * * * = p<0 .0 1 ). (B) In this graph the decrease in spared tissue volume occurring during the 28 day
time period is shown. The volume at the endpoint is expressed as a percentage o f the volume at the starting point. The
time periods refer to control rats. The matching time points of transplanted rats are listed above.
73
Chapter 4
Loss o f B M S C transplanted into the moderately contused rat spinal cord
W e fo u n d t h a t 9 5 % o f B M S C surviv ed a p e r i o d o f 5 h on ice a n d 9 2 % surviv ed w h e n p a s s e d
th ro u gh a pull ed g l a s s n e e d le . T hu s, our m e t h o d s could h a v e r esu lted in a m a x i m u m o f 1 3 %
d e a th a m o n g B M S C in jec ted into th e c o n t u s io n . F r o m th e i n t e n d e d 1 mill io n c ells , 6 . 7 x 1 0 5
B M S C p a s s e d th ro u gh th e n e e d l e alive. T a k i n g t h e g r e a t e s t p o s s i b l e p e r c e n t a g e o f de a th d u e
to t r a n s p l a n t a t i o n
into ac c o u n t , th is i m p l ie s t h a t 8 x 1 0 5 B M S C w e r e i n je c te d into th e
c o n t u s io n o f wh ic h 1.3 x 1 0 5 c e lls ( 13% ) w e r e d e a d . T o c o r r e ct for v ariabili ty d u e to our
p r o c e d u r e s , B M S C n u m b e r s w e r e e x p r e s s e d a s a p e r c e n t a g e o f th e n u m b e r fo u nd a t 15 m in
p o s t-in je c t io n .
W e fo u nd a t 15 m in p o s t-i n je c t io n t h a t 3 5 % , 2 5 % , 5 8 % , a n d 3 2 % o f th e c a lc u l a t e d a v e r a g e
o f 6 .7 x 1 0 5 (live) B M S C w e r e p r e s e n t a n d a liv e in th e c o n t u s io n w ith th e in je c tio n m a d e a t 15
min, 3, 7 a n d 2 1 - d a y s post-inju ry, r e s p e c t iv e ly . T h e r e w a s no direct r e l a t i o n s h i p b e t w e e n th e
p e r c e n t a g e s a n d th e t i m i n g o f th e in je c tio n ; cell lo ss d u r in g th e first 15 m in p o s t-i n je c t io n w a s
3 5 % with a c u t e a n d 3 8 % with d e l a y e d p a r a d i g m s .
T h e a v e r a g e n u m b e r s o f B M S C a n d c o r r e s p o n d i n g p e r c e n t a g e s (relativ e to th e n u m b e r at
15 m in po s t-in je c t io n ) a r e listed in T a b l e 2 (als o Fig. 4A) a n d revea l th a t th e n u m b e r o f B M S C
a t 7 d a y s p o s t-in je c t io n w a s s ig n i fi c a n tl y h ig h e r afte r a c u t e (p < 0 . 0 1 ) a n d 3-d ays d e l a y e d (p <
0 . 0 0 5 ) in je c ti o n s c o m p a r e d to 7 -d a ys a n d 2 1 - d a y s d e l a y e d in je c t i o n s ; 3 2 % a n d 5 2 % vs. 9 % an d
9 % , r e s p e c t i v e l y (Fig. 4B ) . T h e n u m b e r o f B M S C in th e c o n t u s io n a t 2 8 d a y s p o s t-in je c t io n w a s
c lo s e to 0 in all g r o u p s , in d ic a ti n g r e je c tio n o f th e t r a n s p l a n t e d cells.
Tissue sparing and B M S C survival are strongly associated
The relationship b e tw e e n
tissue sp arin g and
B M S C survival w a s a s s e s s e d
u s i n g th e
P e a r s o n c o r r e la t io n c oeffi cient. W e fo u n d t h a t wit h an a c u t e a n d 3-d ays d e l a y e d in je c tio n , th e
p o s it iv e c o r r e la t io n w a s la r g e with r = 1 . 0 a t 3, 7, a n d 2 8 d a y s p o s t-in je c t io n . With a 7-d ays
d e l a y e d in je c tio n , th e p o s it iv e c o r r e la t io n w a s la r g e with r = 0 . 7 9 a t 3 d a y s a n d wit h r = 1 . 0 at
7 a n d 2 8 d a y s p o s t-in je c t io n . With a 2 1 - d a y s d e l a y e d in je c tio n , t h e re w a s no c o r r e latio n
b e t w e e n B M S C n u m b e r a n d s p a r e d t i s s u e v o l u m e . T hu s, in g r o u p s t h a t r e c e i v e d th e B M S C
t r a n s p l a n t d u r in g th e first w e e k
p o s t-c o n t u s i o n , w e fo u n d
a strong
p o s it iv e a s s o c ia t i o n
b e t w e e n t i s s u e s p a r i n g an d B M S C survival w ith r v a l u e s b e t w e e n 0 . 7 9 a n d 1. 0 .
74
Survival and neuroprotection
Fig. 4. BM SC survivat in a moderate contusion in the
adult rat thoracic spinal cord ts /oio. (A) The number of
BMSC transplanted acutely (15 min post-injury) or
delayed (3 , 7 , and 21 days post-injury) into a moderate
condusion decreases over 28 days post-injection. The
numbers are oxpressed as a percentago of the numbers
Boundat 15 min post-injection. (B)
de:ath veithiin the
moderate contusion at 7 days post-injection is ¡significantly
lower abter ac ute and 3 -day delayed injoctions thnn with 7 and 21 j day delayed injection's. Percentages are relative to
the number at 15 min post-transplantation. Bars represent
standard error o f the mean. * = p<0 .0 1 ; * * = p<0 . 0 0 5 .
Migration o f B M SC into adjacent spinal nervous tissue
T h e n u m b e r o f B M S C in th e t r a n s p l a n t sit e could d e c r e a s e a s B M S C m i g r a t e a w a y fr o m th e
in je c tio n site. W e fo u n d t h a t a f e w d a y s afte r i n je c tio n B M S C s ta r te d to m i g r a t e a w a y m a i n l y
fro m th e rostral a n d c a u d a l a s p e c t s o f th e t r a n s p l a n t (Figs. 5A). O n e w e e k a fter in jec tio n ,
B M S C w e r e fo u n d in th e do r sa l anai v e n t ra l c o l u m n s s o m e a s fa r a s 7 m m a w a y fr o m th e
c o n t u s io n e p i c e n t e e . M o s t of1" t h e s e c s l l s w e r e s p i n d l e - e h a p e d with a s m a ll n u c l e a s a n d several
e x t e n n io n s (Fig. e B). With a 3 d a y f o st-inju ry in je c tio n , a to tal of72 , 1 0 0 ± <349 B M S C a t 7 d a y s
a n d 8 2 0 ± 9c) a t 2 8 d a y s p o s t - t r a n s p l a n t a t i o n had m i g r a t e d a w a y fr o m th e t r a n s p l a n t sita
(Fig. 5C). S i g n if ic a n t ly (p < 0 . 0 0 1 ) m o r e B M S C w e r e f e u a d rostra! (8 0 ± 1 4 % ) t h t n c a a d a l ( f o
± 9 % ) to th e t r a n s p l a a t a t ;i o n site. T h e to tal n u m b e r of7 m i g r a t e d B M S C r e p r e s e a t e d 1 . 2 % of7
t h e n u m b e r o e B M j C in th e c o n t u s i o r f t 15 m in p o s t . n j e c t i o n an d 2 . 4 % o f th e n u m b e r a t 7
d a y s p o s C i n je c t i o e . A t 2 8 da ye p o s t - i n je c t fo a , a l m o s t tw ico a s m a n y B M S C w e r e fo u n d o u t f i d r
o f th e c o n t u s io n c o m p a r e d to within . Thus, B M S C tlrat had m ^ r a t e d aw/ay fr o m th e i n jec tio n
site into th e a d j a c e n t s p i n a l cord t i s s u e a p p e a r e d to be p r o t e c t e d c o m p a r e d to t h o s e t h a t
r e m e r n e d w ith.n th e c o n t u s i o r .
75
Chapter 4
û
C/)
4000
O 3000
CD
CÛ 2000
■0O
)
1000
Œ
V—
g)
"e
0
7 days
28 days
Time after Transplantation
Fig- 5- BM SC transplanted into the moderately contused adult
rat spinal cord contusion migrate into spinal cord white
matter. (A) GFP-positive BMSC (arrow) ‘leaving’ the
transplant (T) at its rostral edge. (B) Several GFP-positive
BMSC that have migrated approximately 5 mm into the rostral
lateral white matter (W M ). (C) Bar graph showing the
number of BMSC that migrated out of a transplant at 7 and
28 days post injection. BMSC were injected at 3 days post­
injury. All microphotographs confocal images from horizontal
2 0 jjm thick cryostat sections. Bar in A represents 15 jjm in
panels A and B.
D ISC U SSIO N
Survival o f B M S C t r a n s p l a n t e d a c u t e ly (15 m in po st-inju ry) or d e l a y e d (3, 7, a n d 2 1 d a y s p o s t ­
injury) into a m o d e r a t e l y c o n t u s e d ad u lt r at s p i n a l cord is lo w with f e w o r n o n e o f th e c e lls left
a t 2 8 d a y s p o s t-in je c t io n . B M S C lo ss a t th e 7 -d a y p o s t-in je c t io n t i m e p o i n t w a s 6 8 % with
a c u t e a n d 4 8 % wit h 3-days d e l a y e d i n je c t i o n s wh ic h w a s s ig n ific a n tly lo w e r th a n with 7- an d
2 1 - d a y s ( 9 1 % both) d e l a y e d in je c ti o n s . This in d ic a te d t h a t th e c o n t u s io n e n v i r o n m e n t is m o r e
d e l e t e r i o u s to t r a n s p l a n t e d B M S C d u r in g th e s e c o n d a n d fourth w e e k a fter i m p a c t t h a n d u rin g
t h e first w e e k a f te r i m p a c t . In con tro l rats, th e a m o u n t o f s p a r e d t i s s u e g r a d u a l ly d e c r e a s e d in
t i m e until 4 6 % o f t h a t in a c o m p a r a b l e u n i n ju r e d s p i n a l cord s e g m e n t a t 4 9 d a y s po st-injury.
T h e p r e s e n c e o f B M S C r esu lted in i n c r e a s e d a m o u n t s o f s p a r e d t i s s u e c o m p a r e d to c on tro ls .
A c u t e a n d 3 -d ays d e l a y e d , but n o t 7- a n d 2 1 - d a y s d e l a y e d , i n jec tio n o f B M S C s ig n ific a n tly
im p r o v e d t i s s u e s p a r i n g . T h e r e w a s a la r g e p o s it iv e c o r r e la t io n b e t w e e n s p a r e d t i s s u e v o l u m e
a n d B M S C n u m b e r with i n je c t i o n s d u r in g th e first w e e k p o s t-c o n t u s i o n .
76
Survival and neuroprotection
Effects o f B M S C on t i s s u e s p a r i n g w e r e a s s e s s e d by d e t e r m i n i n g th e v o l u m e o f s p a r e d
(intact) t i s s u e 409 w ithin th e c o n t u s e d s p i n a l cord s e g m e n t . T his r e v e a le d an i n c r e a s e d v o l u m e
o f s p a r e d t i s s u e a t 7 a n d 2 8 d a y s po s t-in ju ry with an a c u t e B M S C i n jec tio n an d a t 6, 1 0 , a n d 31
d a y s po s t-in ju ry with a 3-d ay d e l a y e d B M S C in je c tio n . T h is p a r t ic u la r a p p r o a c h o f m e a s u r i n g
s p a r e d t i s s u e is e m p l o y e d reg u lar ly but it d o e s n o t t a k e into a c c o u n t n a r r o w i n g o f th e s p i n a l
cord, which t y p ic a lly o cc u rs afte r a c o n t u s io n . T h er efo re , w e a l s o d e t e r m i n e th e v o l u m e o f
s p a r e d t i s s u e r elativ e to t h a t o f a c o m p a r a b l e s e g m e n t fr o m u n in ju r e d a n d u n t r e a t e d s p in a l
cord409. T h is r e v e a le d an i n c r e a s e d v o l u m e o f s p a r e d t i s s u e a t t h e a b o v e m e n t i o n e d t i m e
p o i n t s but a l s o a t 35 d a y s po s t-in ju r y with a 7 -d a ys d e l a y e d B M S C i n jec tio n .
O u r resu lts d e m o n s t r a t e d
t h a t a c u t e ly t r a n s p l a n t e d
B M S C c o n tr ib u t e d to s p a r i n g o f
n e r v o u s t i s s u e fo ll o w in g a m o d e r a t e c o n t u s io n o f th e th o r a c ic rat s p i n a l cord b e t t e r th an
d e l a y e d t r a n s p l a n t e d B M S C . T h e r e d u ce d lo s s o f th e a c u te ly t r a n s p l a n t e d B M S C d u r in g th e
first w e e k a f te r in je c tio n c o r r e la t e d c lo s e l y w ith th e i n c r e a s e d v o l u m e s o f t i s s u e s p a r i n g .
Previo u sly, it w a s r e p o r t e d t h a t a c u te ly t r a n s p l a n t e d B M S C red u ce d th e s iz e o f c a v itie s a t 3
w e e k s a fter an i n jec tio n into th e c o n t u s i o n 451 a n d a t 5 w e e k s a fter an i n je c tio n into th e 4 th
v e n t r i c l e 304. In th e p r e s e n t stu dy , w e h a v e a s s e s s e d s p a r e d t i s s u e v o l u m e r e la t iv e to t h a t o f a
c o m p a r a b l e s e g m e n t fr o m a n u n i n ju r e d a n d u n t r e a t e d s p i n a l cord to a c c o u n t fo r th e n o r m a l ly
o c c u rr in g s h r i n k a g e . D e s p i t e th is d iff e re n c e in a p p r o a c h , our resu lt s a r e in a g r e e m e n t a s th ey
in d ic a te d t h a t a c u te ly t r a n s p l a n t e d B M S C e x e r t n e u r o p r o t e c t i v e a c t i o n s o n th e n e r v o u s t i s s u e
w ithin th e c o n t u s e d s e g m e n t . T h e eff ects o f th e s e n e u r o p r o t e c t i v e a c t i o n s r e m a i n v is ib le until
28
days
p ost-transplantation.
tran sp lan tatio n
of
BM SC
into
Y osh ihara
a
9-day
and
old
c o l la b o r a t o r s 463,
m oderate
464
c o n t u s io n
d em onstrated
did
not
that
r e su lt
in
n e u r o p r o t e c t io n w ithin th e c o n t u s e d s e g m e n t w h ic h is in c o n c u r r e n c e with o ur f i n d i n g s t h a t
d e l a y e d t r a n s p l a n t e d B M S C fail to e x e r t n e u r o p r o t e c t i v e a c t i o n s . O n e p o t e n t i a l m e c h a n i s m
by w h ic h t r a n s p l a n t e d B M S C resu lt in n e u r o p r o t e c t i o n m a y be by s e c r e t i n g B D N F 326, 364 W e
u se d E L IS A to c o n firm t h a t th e B M S C u se d for t r a n s p l a n t a t i o n in o u r s tu d y p r o d u c e d an d
s e c r e t e d B D N F . Fu ture s tu d ie s in w h ic h th e p ro du ctio n o f B D N F by B M S C o r th e a c t i o n s o f
B D N F a r e blo cked will h e lp to e lu c id a te th e role o f B M S C - d e r i v e d B D N F on n e u r o p r o t e c t io n .
O u r d a t a s h o w e d th a t 0 . 0 2 % o f a c u te ly t r a n s p l a n t e d B M S C had surviv ed u p to 2 8 d a y s in a
m o d e r a t e s p i n a l cord c o n t u s io n . In a p r e v io u s study, it w a s s h o w n t h a t 0 . 1 7 % o f B M S C a c u te ly
i n je c te d into a n d n e a r a m o d e r a t e - s e v e r e c o n t u s io n (25 m m , N Y U i m p a c to r ) in th e rat s p i n a l
cord had surviv ed a t 5 w e e k s p o s t - i n ju r y / in je c t i o n 170. W h e n B M S C w e r e g r a ft e d into a 7 -d a y old
c o n t u s io n , 6 t i m e s m o r e c e lls ( 0 . 9 9 % ) had survived a t 5 w e e k s po s t-in ju ry (i.e., 4 w e e k s p o s t ­
in je c tio n ) . In o u r s tu d y w e u s e d a l e s s s e v e r e c o n tu s io n inju ry but fo u n d a lo w e r B M S C survival
a t t i m e p o i n t s th a t w e r e c lo s e (a cu t e i n jec tio n - 2 8 d a y s survival h e r e vs. 35 d a y s s u rv iv al170) or
e x a c t ly m a t c h i n g (7 d a y s d e l a y e d i n je c tio n -
2 8 d a y s survival h e r e vs. 35 d a y s s u rv iv al170),
n a m e l y 0 . 0 2 vs. 0 . 1 7 a n d 0 vs. 0 . 9 9 , r e s p e c t iv e ly . A p o s s i b l e e x p l a n a t i o n fo r this d i s c r e p a n c y
is t h a t in H o f s t e t t e r e t a l . 17° th e B M S C w e r e i n je c t e d 2 m m rostral a n d c a u d a l a s well a s into
77
Chapter 4
t h e c o n t u s io n le s i o n . It w a s n o t s tu d ie d w h e t h e r th e s u rv iv in g B M S C w e r e in fa c t t h o s e
i n je c te d n e a r r a th e r t h a n in th e c o n t u s io n a s h a s b e e n d e s c r i b e d for a s im il a r p a r a d i g m in a 7d a y old m o u s e s p i n a l cord c o n tu s io n m o d e l 222. O t h e r s h a v e r e p o r t e d survival o f B M S C in s p i n a l
cord le s i o n s but w i t h o u t q u a n t i f i c a t i o n ’0' 3° 4, 84, 85 96472' 473.
H o f s t e t t e r e t a l . ’7° i n je c t e d
B M S C into th e c o n t u s e d s p i n a l cord a c u te ly or o n e - w e e k
d e l a y e d a n d e x a m i n e d d u r in g th e e n s u i n g w e e k s lo c o m o t o r fu n ctio n o f th e hind li m bs. Both
g r o u p s w e r e n o t directly c o m p a r e d a n d t e s t e d fo r s ig n i fi c a n c e , but th e d a t a fr o m this s tu d y
i n d ic a te d t h a t im p r o v e d B M S C survival w a s a s s o c i a t e d with i m p r o v e d lo c o m o t o r f u n c t i o n ’70. In
g e n e r a l , this a g r e e s w ith o u r d a t a t h a t im p r o v e d survival o f t r a n s p l a n t e d B M S C elicits p o s itiv e
effects o n s p i n a l cord repair. S t u d i e s t h a t i n v e s t i g a t e th e c o r r e la t io n b e t w e e n B M S C survival
a n d m o t o r a n d s e n s o r y fu n ctio n a r e in p r o g r e s s .
Survival o f graft ed B M S C d u rin g th e first w e e k p o s t-in je c t io n w a s s ig n i fi c a n tl y lo w e r w h e n
t h e c e lls w e r e g r a ft e d 7 a n d 2 ’ d a y s po s t-in ju r y c o m p a r e d to ’ 5 m in a n d 3 d a y s po st-injury.
N o rm a lly , i m m u n e c e lls i n v a d e c o n t u s e d t i s s u e ra p id ly to be fo llo w e d by i n f l a m m a t o r y cells
i n c lu d in g m a c r o p h a g e s . T h e p e a k in m a c r o p h a g e p r e s e n c e is ar o u n d 7 d a y s post-inju ry, to
r e c e d e t h e r e a f t e r 34, ’ 92 34’ . W e fo u n d t h a t B M S C surviv ed b e s t w h e n i n je c te d i m m e d i a t e l y o r 3
d a y s a fte r injury, w h ic h is w h e n th e n u m b e r o f i n f l a m m a t o r y c ells is still i n c r e a s i n g in th e
c o n t u s io n le s io n .
Possibly , m a c r o p h a g e s a r e directly inv olved in t r a n s p l a n t e d
B M S C lo ss
w h ic h w o u ld e x p la i n t h e g r e a t e r lo s s a t in je c tio n t i m e s w h e n m o r e m a c r o p h a g e s c a n b e fo u nd
in th e c o n t u s io n . T h e r e l a t i o n s h i p b e t w e e n m a c r o p h a g e s a n d t r a n s p l a n t e d B M S C w ithin th e
inju red s p i n a l cord is still po o rly u n d e r s to o d .
W e o b s e r v e d t h a t s o m e o f th e t r a n s p l a n t e d B M S C m i g r a t e d fr o m th e c o n t u s io n into th e
rostral a n d c a u d a l w h it e m a t t e r . T o our k n o w l e d g e , this is th e first e v i d e n c e t h a t B M S C
t r a n s p l a n t e d into a c o n t u s io n sit e in t h e ad u lt rat th o r a c ic s p i n a l cord m i g r a t e into th e
a d j a c e n t n e r v o u s t is su e . N e u h u b e r a n d c o l l e a g u e s 29’ d e s c r i b e d th a t h u m a n B M S C g rafted in a
h e m i s e c t i o n in t h e a d u lt r at cervical s p i n a l cord m i g r a t e d into th e n e a r b y s p i n a l ti s s u e . O u r
data
suggested
th at the conditions
at and
p e r m i t t e d m i g r a t i o n o f th e i n je c te d B M S C .
n e a r b y th e 3-d ay old s p i n a l
In th e inju red ad u lt c e n t r a l
cord c o n tu s io n
nervous system ,
c h e m o t a c t i c fa c t o r s a n d c yto k in es such a s m a c r o p h a g e i n f l a m m a t o r y p r o t e i n - ’ ,
m onocyte
c h e m o a t t r a c t a n t p r o t e i n - ’ , a n d in terleu k in -8 h a v e b e e n i m p l i c a t e d in B M S C m i g r a t i o n 433.
In s u m , w e h av e g a t h e r e d e v id e n c e t h a t B M S C t r a n s p l a n t e d i m m e d i a t e l y a n d 3 d a y s after
a c o n t u s io n s urviv e b e t t e r w ithin th e le s io n e n v i r o n m e n t d u r in g t h e first w e e k afte r i n jec tio n
c o m p a r e d to B M S C t r a n s p l a n t e d a t 7 a n d 2 ’ d a y s post-inju ry. T h e im p r o v e d survival is
t r a n s i e n t but th e B M S C t r a n s p l a n t eli cit ed n e u r o p r o t e c t i v e effects t h a t r e su lt e d in im. p r o v e d
t i s s u e s p a r i n g u p to 2 8 d a y s afte r i n jec tio n .
78
JOHNS HOPKINS
i e
D
i
c i
N n
Kennedy Krieger Institute
5
Inflammation and cell survival.
Reducing macrophages to improve bone marrow stromal cell survival in
th e c o n t u s e d s p i n a l cord.
N euroreport 20 10 ; 21 (3): 221-226
G .J. Ritfeld
R .D .S . N a n d o e T e w a r i e
S.T. R a h ie m
A. H u r t a d o
R.A. R o o s
J.A. G r o t e n h u i s
M. O u d e g a
Chapter 5
IN TRO D U CTIO N
A c o n t u s iv e s p i n a l cord inju ry c a u s e s i m m e d i a t e d e a t h o f ne u ral c ells a n d d is ru p t io n o f axo n
c ir cuits’ 55. T h e n u m b e r o f m a c r o p h a g e s in th e inju ry site r a p i d l y i n c r e a s e s o v e r th e e n s u i n g
d a y s a n d c o n tr ib u t e to fu rth er d e s t ru c tio n o f local n e r v o u s t i s s u e 34. T h e r e is no th e r a p y
a v a i la b le t h a t effectiv e ly i m p r o v e s fu n ctio n afte r s p i n a l cord injury.
T r a n s p l a n t a t i o n o f m e s e n c h y m a l b o n e m a r r o w s tr o m a l c ells ( B M S C ) h a s b e e n e x p lo r e d
for s p i n a l cord r e p a i r 285. In d iff e r e n t m o d e l s y s t e m s , B M S C t r a n s p l a n t a t i o n r e su lt e d in t i s s u e
s p a r i n g a n d , in s o m e c a s e s , m o t o r fu n c t i o n ’70, 286, 291 351 473. T h e s e r e su lt s w a r r a n t fu rth er
in v e s t i g a t i o n o f B M S C fo r n e r v o u s s y s t e m re p a ir .
Survival o f a l l o g e n e i c B M S C t r a n s p l a n t e d into th e d a m a g e d s p i n a l cord is lo w ’70, 286, 403. S e v e r a l
s tu d ie s h a v e p o i n t e d a t a role o f m a c r o p h a g e s in th e lo s s o f B M S C a f te r i n je c tio n into th e
inju red n e r v o u s s y s t e m 9’, 403. S o far, q u a n t i t a t i v e e v i d e n c e t h a t w o u ld s u p p o r t such a role is
sparse.
C y c l o s p o r in e
A
(CsA),
m in o c y c li n e
(M C ),
and
m e th ylp red n iso lo n e
(MP)
treatm ent
d e c r e a s e s m a c r o p h a g e infilt ration into a s p i n a l cord le s i o n 22 ’77, 265, 3’ ’ 399, 403. T h e p r e s e n t s tu d y
f o c u s e s o n th e eff ic acy o f t h e s e t h r e e d r u g s to r e d u c e m a c r o p h a g e infi lt ration a n d w h e t h e r this
w o u ld resu lt in i n c r e a s e d survival o f s u b s e q u e n t l y t r a n s p l a n t e d B M S C .
M ATERIAL A N D M ETH O D S
Spinal cord contusion and post-surgery care
Ad u lt f e m a l e S p r a g u e - D a w l e y ra ts ( n = 7 6 , 2 0 0 - 2 3 0 g ;
a n a e s t h e t i z e d w ith an
intrap eriton eal
H a r la n , I n d i a n a p o l i s , IN, USA) w e r e
i n jec tio n o f 6 0
m g / k g o f K e t a m i n e H Cl ( P h o e n ix
P h a r m a c e u t i c a l s , St. J o s e p h , M D , USA) a n d 0 . 4 m g / k g m e d i t o m i d i n e ( D o m it o r , a n alp h a -2 a d r e n e r g i c a g o n i s t ; O rio n C o r p o r a t io n , E s p o o , Fin la n d ) . T h e 1 0 th th o r a c ic s p i n a l cord s e g m e n t
w a s e x p o s e d a n d c o n t u s e d u s i n g th e In fin it e H o r iz o n I m p a c t o r a t a fo r ce o f 2 0 0 k D yn (Fig.
’ A). C o n s i s t e n c y b e t w e e n a n i m a l s w a s g u a r a n t e e d by r e g i s t e r i n g th e i m p a c t fo r ce a n d s p in a l
cord d i s p l a c e m e n t . T h e w o u n d w a s c lo se d a n d th e rats w e r e g iv e n a n t i s e d a n ( a t i p a m e z o l e
hy dr ochloride; ’ .25 m g / k g , in t r a m u s c u l a r ) , an a l p h a 2 - a d r e n e r g i c a n t a g o n i s t t h a t r e v e r s e s
s e d a t i v e a n d a n a l g e s i c effects o f m e d it o m id i n e . All surgical p r o c e d u r e s w e r e p e r f o r m e d by th e
s a m e i n v e s t i g a t o r . P o s t-s u r g e ry m a i n t e n a n c e w a s a s d e s c rib e d p r e v io u sly 286.
80
Inflamm ation and cell survival
Fig. i. Schematic representation o f the experiments and
BM SC harvest and transduction, (a) Rats were contused at
the 1 0 th thoracic spinal cord segment and then divided into 4
groups that received CsA, MC, M P or saline, (b) BMSC were
harvested, transduced to express GFP using lentiviral vectors,
and grown in DMEM. (c) GFP-expressing BMSC in a passage
3 culture which were used for transplantation into the
epicenter o f the 3 -day old contusion. Abbreviations: BMSC,
bone marrow stromal cells; CsA, cyclosporine A; ip,
intraperitoneal; MC, minocylcine; M P, methylprednisolone;
sc, subcutaneous. Bar in C represents 1 0 fxm.
Drug administration
C o n t u s e d rats w e r e di vid ed into 4 g r o u p s t h a t r e c e i v e d CsA , M C , M P , o r s a li n e ( n = i 8 eac h;
Fig. lA)
s t a r t i n g 5 m in afte r th e c o n t u s io n . All i n je c t i o n s w e r e p e r f o r m e d by th e s a m e
i n v e s t i g a t o r . C s A (Bedfo rd Labs, Bedfo rd, O H , USA) w a s a d m i n i s t e r e d s u b c u t a n e o u s l y o n c e
p e r d a y a t a d o s e o f 3 0 m g / k g for th e first t h re e d a y s a n d 15 m g / k g fo r th e n e x t s e v e n d a y s 4“3.
M C (Sigm a-Ald rich, St. Louis, M O , USA) w a s a d m i n i s t e r e d i n t r a p e r i t o n e a l l y a t a d o s e o f 5 0
m g/kg
tw ic e
a
day
fo r
th e
first
tw o
d a y s 399.
MP
(Sigm a-Ald rich)
was
adm inistered
i n t r a p e r i t o n e a l l y o n c e a t a d o s e 3 0 m g / k g 22. S a l i n e w a s giv e n to c o n tr o ls fo llo w in g th e s a m e
r e g i m e a s fo r M P .
B M S C culture and lentiviral transduction
B M S C w e r e o b t a i n e d fro m f e m u r s o f ad u lt f e m a l e S p r a g u e - D a w l e y rats (n =4 ) a s pr e v io u sly
d e s c r i b e d ' 3,286 (Fig. lB ) . B M S C a t p a s s a g e o w e r e t r a n s d u c e d o v e r n i g h t u s i n g len tiv iral v e c t o r s
e n c o d i n g fo r g r e e n f l u o r e s c e n t p r o te in (GFP) a t a n M O I o f 1 5 0 '75, 286 (Fig. lB ) . T r a n s d u c e d
B M S C w e r e c ultu re d in D - 1 0 m e d i u m a t 37 °C/5 % C 0 2. B M S C fro m th e third p a s s a g e (Fig. 1C)
w e r e u sed for th e t r a n s p l a n t a t i o n
e x p e r i m e n t s . T h e tr a n s d u c t io n r ate o f th e B M S C w a s
d e t e r m i n e d u s i n g a F A C S c a n / F A C S o r t e r ( B e c to n D ic k in s o n I m m u n o c y t o m e t r y S y s t e m s (BDIS)
B io s c i e n c e s , S a n J o s e , CA). T h e n u m b e r o f v ia b le ( G F P -p o s itiv e ) c ells r elat iv e to th e total
n u m b e r o f c ells w a s d e t e r m i n e d r e v e a l i n g a t r a n s d u c t io n r a te o f 63 % .
81
Chapter 5
Transplantation o f B M S C
A t t h re e
days
afte r
injury,
24
rats
(6
rats fr o m
each
gr ou p)
w ere
an ae sth e tiz e d
with
i n t r a p e r i t o n e a l i n je c t i o n s o f 6 0 m g / k g o f K e d a m in e HCl ( P h o e n i x P d a r m a c e u t i c a l s ) a n d 0 . 4
m g / k g o S m n d i t o mid ine (D o m it o r ; O rio n C o r p o r a tio n). T h e 1 0 th th o r a c ic s p i n a l cord s e g m e n t
w a s e x p o s e d a n d 5 ml D M E M wit h 1 x 1 0 6 B M S C (Fig. 1A) w a s i n je c te d into th e c o n t u s io n
e p i c e n t e r 13. Four extra c o n t u s e d rats w e r e s im ila rly i n je c t e d a n d p e r fu s e d wit h fixatide (sad
below) 15 m in latnr. T h n s d rats w e r e u s e d to d n r n r m i n e th e n u m b e r o f B M S C in th e c o n t u s io n
a t 15 m in p o s t-in jn c tio n . All B M S C i n je c t i o n s w e r e p e r f o r m e d by th e s a m e i n v e s t i g a t o r . A fts r
t h e inje ctio nn , th e r a ts w e r e m a i n t a i n e b a s S e s c rib n d pr e v io u sly 286.
General histology
Three days
(n=24)
and
ten
days
(n=48)
afte r injury,
rats w e r e
an aesth etized
with
an
in t i a p b r i t o n e o l in je c t i c n o f 9 0 m g / k n o f K s t a m i n e HCl ( P h o e d i x P h a r m a c e u t i t a l f an d 0 . 6
m g / k g of7 m e d i t o m i d i n e
(Orion C o r p o r a t io n ) . A fte r d e e p s e d n tin n w a s o o n f i r m e d , 0 . 1
rrH
H n f a r i n e ( 5 0 0 IU; H e n r y Sgh ein , M elv ille , N's'^ USA) w a s rnjncted inter th e left v e n t r i c l e o f th e
h eart. T h e n , 5013 ml s a li n e fo llo w e d by 5 0 0 ml ice-cold 4 % p a r a e o r m a l d e h y d e in p h o s p h a t e
buffer (PB; 0 . 1
M, pH 7.4) w a s p u m p e d th ro u gh th e v a s c u l a r syn te m . S p i n t l c o r d s w e r e
r e m o v e d w i t h s u t d a m a g i n g thd a n a t o m i c a l id tegrity , post-fix ad fo r 2 4 h is t h e s a m f fixative,
a n d tr a n s f e r r e d to 3 0 % t u c r o s e in p h e s p h a t a - b u f f e r e d s a l i n e ( P B S ; o .i M, p H 7 . f ) 'foi^ 4 8 h. A
12
mm
lo n g s p i n a l cord s e g m e n t c e n t e r o d a t th e c o n t u s io n w a s cut into ;>o juui^
thick
ho riz o n t a l c r y o s t a t s e c t i o n s whlclr w e re m o u n t e d o n g l a s s sli des.
Immunocytochemicnl pronedures
For c h a r a c t e r iz a ti o n o f fd d B M S C , 8 - w e t c h a m b n r g l a r s s lid e r (BD Fa lc o n ; B D B io s c i a n c e s ,
Bedfo rd, MA) w e r e c o a t d d wit h 1 0 0 (jil/ml po ly -D -lys in e For 1 li a t r o o m t s m p e r a t u r e . Atter
w a s h i n g 2 x 5 m i c w i t l d o u fle -d i s ti lle d w a t e ^ 3 0 0 0 B M S C in 2 5 0 jjlI D - 1 0 m e d i o m w e r e p l a t e d
p e r w e ll. A fte r tw o d a y s a t 3 s °C /5 % C O 2 th e c o k u re u w s r n w a s h e d 3 x 5 m in with P B S an d
fixed wit h 4 % p a r a f o r m a l d e h y d e in P B ( 1 0 m i n , roo m t e m p e ratu re. N e x t 1 th e cultu res w e r e
w a s h e d 5 x 5 m in w ith P B S , in c u b a te d with 5 % n o r m a l g o t s s d r u m ( N G S) in P B S for 3 0 m i n u
a n d th e n i n c u b t t e d o v e r n i g h t a t 4 sC with antiloodies Bgainst: C D 9 0 (i:icxo; I m m s n o t e c h ,
B r u s s e l s , B e lg i u m ) , C D 1 0 5 (r: 'd0 0 ; N ^ ,
B e a f o e D i c k e n s o n ) , C D 3 4 (1 : i 0 0 i 8 G 1 2 cl nnn IgC^^
B e c t o n D i c l < a i s t o ) , C D 4 5 (1:10)0; H 1 3 0 c l o n e I g G 1, B e c t o n D i c k e n s o n ) , a n d H U 'l- D R ( 1 : 1 0 0 ;
D ak o , G lo s tr u p , D e n m a r k ) diluted in PB wit h 5 % N G S . S o m e cu ltu res w a r e in c u b a te d with PB
with 5 % N G S only. a n d s e r v e d a s n e g a t i v e (no p r i m a r y an tib od y) c o n tr o ls to e x c lu d e a fa lsep o s it iv e o u t c o m e ° IMext, culturen w e r e w a s h e d 3 x 5 m in w ith PB a n d th en i n t u b a t e d w ith go ata n t i - m o u s e I g G -A l tx a 5 9 s
( 1 : 5 0 0 io PB ; M o k c u ^
P r o b t s , C a r ls b a d , CA) for 2 h a t r oo m
t e m p e r a t u r e . A f t e r w a r d s , cu ltu res w e r e w a s h e d 3 x 5 m in with PB a n d c o v e r e d with a g l a s s slip
82
Inflamm ation and cell survival
with V e c t a s h i e l d a n d
DA PI
(V ector L a b o ra to ri e s ,
Inc., B u r li n g a m e, CA). T h e slide;;; w e r e
e x a m i n e d a n d i m a L e s w e r e t a k e n with a n O l y m p u s Flu o eiew F V 1 0 0 0 c o n fo c a l m i c r o s c o p e .
For i m m u n o s t a i n i n g of7 ac tiv a te d
m acropeages,
e v e ry 1 0 th c r y o s t a t s e c t io n w a s
pre­
in c u b a te d a t r o o m t e m p e r a t u r e for 3 0 m in in 5 % N G S a n d 0 . 3 % T riton X - 1 0 0 in 0 . 0 1 M P B S
(pH 7.4) a n d t h t n in c u b a te d wit h a n t i b o d i e s a g a i n s t E D 1 ( s : 2 0 0 ; S e r o t e c , R a leigh , NC) in 5 %
N G S for 2 h a t r o o m t e m p e r a t u r e fo llow ed by o v e r n i g h t in c u b a tio n a t 4 °C. Afteh w a s h i n g 3 x 5
m in with P B S , s e c t io n s w e r e in c u h a te d with goah a n t i - m o u s o A le x a , 9 4 anti bodi e s ( 1 : 2 0 0 ;
M o l e c u la r P r o f t s ) in P B S ( 0 . 0 1 M; pH 7.4) a t r o o m t r m p t F a t u r e for 2 Is. T h e s e c t i u s s w e r e
th e n w a s h e d r n d c o v e r e d with f g l a s s slips in V e c t a s h i e ld with DAPI (V ectur L ab o sa to ries ,
Inc.). T S e c o v e r s l ip s w e r e s t o l e d w t h nail p o li sh. All s e ca io n s w e r e s to re d a t - 2 0 °C until
e n c ly s is .
Qeantitative a c se s a c r e a ts
For a n a l y s i s o b G F P - p o s ih i e e B M S C , e y e r y 1 0 th c a^ci^tat a ec r io n w a s c o v e r e d wit h a g l a s s slip
with V e c t a s h i eld m o u n t i n g m e d i d m with D A P I (Vectoe Labora doriee, In c) . S t e r e o i n v e s t i g a t o r
(M ic ro B rig h t F ie ld Inc., C o lc h e s te r , V A, USA) w a s u s e d to d e t e r m i n e th e nu m b e rs o f surviv ing
B M S C in th e c o n t u s i o n 168. T h e wect io n r w e r e 2 0 0 m m a p a r t s p a n n i n g t h a wid th o f th e s p i n a l
cord. In e v e r y r e c t io n c o n t a i n i n g G S P p o s itiv e c ells, th e dra n s p l a n t e d a r e a w a s o u tlin ed
m a n u a l l y a t 4 X m a g n i f t a a i o n a n d c o v e r e d hy a 215c) x 2 5 0 fom grid. A t 6 0 X m a d n ifih a t io n with
oil i r o m e r s i c n , G F b - p o s i t i v e c eils with a d i s c e r n a b l e D A P I - p o s it iv e nucieuw w e rn m a r k e d u sin g
t h e o p tic a l f r a c t i o n a ( o r witM a 6 0 x 6 0 fim c^ourrtinsih Urcmh. N u m b e u t w e s e corresSeW fo r th e 63
% tra n s d uction note ob th e tf l\/l Î2C wi th i_^-Cf |tPi For ea cV o i th e g r o u p s , B M S C survival w a s
c nlc u la ted
as
th e
number o f BM SC
rela tiv e to
th e
n u m b e r oh B M S C
at
15
m in
afte r
t r a n s p l a n t a t i o n (wh icF w a s 1 a 8 t 5 9 ± n r 9; S E M , n c 4 ) , Td e effe c t e f t r e a t m e n t on B M S C
survivai w a s a c n e s n e f by e x e r e t a i n g B M S C uurvival f"oa eiachi g r o u p a s a p e r c e n t a g e of7th a t in
c o n tr o ls. For a n r l s s i s o f m a c r o f h w g n s w e e m p l o y e d a m e t h o d p revio u sln e m p l o y e d H a y a sh i
a n d oolle agu htin 3i T his m y t h o d u s e s 'thn-eie s e c t i o n s
p e r rat 'for e x a m i n a t i o n : o n a s e c t io n
tC ro u eL the; c e n t e r c f thw c o n t u n i o n / t r a n s p i a e t (vui'tt t i e d o n s e s t c e lle l a 2 s t a i n i n g ) , an d
s u c tio n s 2 0 0 mm cinsisinl a n d v e n t ra l to tFn c c n t e r . T h r a r e a fractio at o 2 stai n i n g in t h e s e
sactions w a s hoterm inen
u s i n g Sii^d eEFciol< 4 . i ; 0 . i a
henthllloent I m a g i n g
Inn rvatio ns,
Inc,
S a n t a f i o n i c a , CA, USA) a n d n x d 'c n a e d a s a p ^rc:u;ii^t£ijge o f t h a t in co n tr o l a n i m a l s .
Statistica/ aoalysis
S i g m a n t a t ® ( Sys ta t S c ft w a r n , Inc., S a n J o s e , CA, U s a ) w a e u se d tor sta tis tic al a n a l y s e s u sin g
o n e - w a y A N O V A a n d th e B o n fe r ro n i po v t-h oc /est. D i ft e t e n c e s w e r e a c c e p t e d a t p < 0 . 0 5 .
83
Chapter 5
Ethics and surgical approval
All rats u s e d in this s tu d y w e r e h o u sed a c c o r d i n g to th e g u id e li n e s o f th e N a t i o n a l In s titu te o f
H e a lt h a n d U n ite d S t a t e s D e p a r t m e n t o f A gricu lt u re. T h e d e s c r i b e d a n i m a l p r o c e d u r e s w e r e
approved
by th e
I n s titu tio n al
Anim al
Care
an d
Use C o m m itte e
a t th e J o h n s
H o p k in s
University .
RESULTS
Characterization o f B M S C in vitro
Cu lt ured
G F P - p o s i t i v e c e lls
expressed
CD90
(Fig. 2A)
and
C D 10 5
(Fig. 2 B ). T h e s e tw o
e x t r a c e l lu la r m o l e c u l e s a r e bo th w e ll-k n o w n B M S C m a r k e r s 38,98. N o n e o f th e c e lls e x p r e s s e d
t h e blood cell m a r k e r s , C D 3 4 (Oig. 2C) a n d C D 4 5 or th e i m m u n e cell m ar k e r , H L A -D R . No
s t a i n i n g w a s v is ib le i f th e p r i m a r y a n t i b o d y w a s o m i t t e d . T h e d a t a c h a r a c t e r iz e th e c ells u sed
for t r a n s p l a n t a t i o n a s B M S C .
( a
)
—
(
b
)
-
'
( c )
Fig. 2 . Characterization o f
in vitro. Cultured cells expressed the BMSC markers, CD 9 0 (a) and CD 10 5 (b), but
not the blood cell marker, CD34 (c) . Scale lia r=io |xm.
CsA, MC, and M P reduces macrophage infiltration into the spinal cord contusion
Mi o ro s c o p ie a n a ly s i s o f m a c r o p h a g e p i e s e n c e in th e c o n t u s io n r e v e a l e d high n u m b e r s in
c o n tr o ls (Fig. 3a) c o m p a i e d to t r e a t e d rats; (Fig. 3b) a t 3 d a y s a fter injury. T h e n u m b e r s
a p p e a r e d i n c r e a s e d in t r e a t e d rats a t 1 0 d a y s (Fig. 3c) c o m p a r e d to 3 d a y s (Fig. 3b) af ter
c o n t u s io n . Q u a n t i t a t i v e a n a l y s i s d e m o n s t r a t e d t h a t a t t h r e e d a y s
post-inju ry, r elativ e to
c o n trols, m a c r o p h a g e i n S ltr e t ion w e s 4 6 ± 1 0 % with CtA -, 4 7 ± 3 % w ith MC-, a n d 63 ± 3 %
with M P - t r e a t m e n t SFig. 3dS. A N O V A r e v e a le d t h a t th e n u m b e r fo r e a c h o f th e t r e a t m e n t
g r o u p s w a s s ig n i fi c a n tl y s m a l l e r ( p < o . 0 0 1 ) hhan t h a t fo r c o n tr o ls. T h u s t r e a t m e n t - i n d u c e d
reducti a n w a s 5 4 % wrth Cs A, 513 % with M C , a n d 37 % with M P c o m p a r e d to c on tro ls.
8-4
Inflamm ation and cell survival
T r e a t m e n t - i n d u c e d redu ction w a s n o t s ig n i fi c a n t l y d iffe re n t fr o m c o n tr o ls a t ten d a y s p o s t ­
inju ry (Fig. 3d). T h e s e re su lts s h o w e d th a t all t h re e d r u g s w h e n a d m i n i s t e r e d fo ll o w in g th e
t r e a t m e n t r e g i m e d e s c r i b e d a b o v e red u ce d m a c r o p h a g e infilt rati on into th e ad u lt rat s p i n a l
cord c o n t u s io n a t t h re e d a y s post-inju ry.
B M S C survival in contusion is not affected by CsA, MC, or M P treatment
B M S C w e r e p r e s e n t in th e c o n t u s io n a t s e v e n d a y s p o s t-in je c t io n
(Fig. 4 a ). Q u a n t i t a t i v e
a n a l y s i s r e v e a le d t h a t B M S C survival in th e c o n t u s io n a t s e v e n d a y s p o s t-in je c t io n w a s 2 7 ± 4
% with CsA, 2 4 ± 4 % with M C , a n d 33 ± 2 % w ith M P t r e a t e d rats. In s a li n e - in je c t e d contr ol
rats, B M S C survival w a s 2 1 ± 7 % . T o a s s e s s th e effe ct s o f t r e a t m e n t , w e e x p r e s s e d B M S C
survival in e a c h t r e a t m e n t g r o u p r e la t iv e to t h a t in th e con tro l g r o u p (Fig. 4b). W e fo u nd t h a t
B M S C survival w a s 1 2 6 ± 1 7 % with CsA, 1 1 1 ± 1 9 % with M C , a n d 155 ± 1 2 % with M P tr e a t e d
r ats r e la t iv e to con tro l rats (Fig. 4b ). A N O V A r e v e a le d no d iff e r e n c e in B M S C survival b e t w e e n
t r e a t m e n t a n d con tro l g r o u p s ( p = 0 . 1 6 ) .
Fig. 3 . Cyclosporine A (CsA), minocycline (M C), and
methylprednisolone (MP) treatment reduced macrophage
infiltration into the contusion. Photomicrographs o f ED-1 positive cells in control rats (a) and CsA-treated rats (b) at
3 days postcontusion and in CsA-treated rats at 10 days
postcontusion (b). In panel (c) the more intense staining
was found associated with cellular debris. (d) Bar graph
shows that at 3 days postinjury (open bars) relative to
controls (Con), macrophages infiltration in the contusion
was decreased significantly with CsA, MC, and MP
treatment. At 10 days postinjury (solid bars), the decrease
in macrophage presence in the contusion of treated rats
was not statistically different from that in Con. ^Significant
difference between treated and Con groups at 3 days
postinjury with P<0 . 0 0 1 .
85
Chapter 5
(a)
Fig. 4 . Cyclosporine A (CsA), minocycline (M C), and methylprednisolone (MP) treatment did not improve bone
marrow stromal cell (BM SC) survival. (a) Photomicrograph of green fluorescent protein-positive cells within the
contusion at 7 days postinjection. (b) Bar graph showing BMSC survival relative to controls (Con). The differences
were not statistically different, although there was a trend towards higher numbers in CsA-treated rats.
B M S C presence initiates macrophage infiltration into the spinal cord contusion.
W e a s s e s s e d th e e ffe ct o f a B M S C t r a n s p l a n t on m a c r o p h a g e p r e s e n c e in th e c o n t u s io n a t te n
days
po s t-in ju ry
r e lat iv e
to
that
at
t h re e
days
post-inju ry.
With
a
BM SC
tran sp lan t,
m a c r o p h a g e infilt rati on r e la t iv e to c o n tr o ls w a s 2 . 4 fold in Cs A- a n d M P - t r e a t e d rats , a n d 6 .9
fold in M C - t r e a t e d rats. T h e s e d i ff e r e n c e s w e r e s t a t is ti c a ll y s ig n i fi c a n t ( p < o . o o i ) .
In th e
a b s e n c e o f a B M S C t r a n s p l a n t , m a c r o p h a g e p r e s e n c e in tr e a t e d rats r e la t iv e to c o n tr o ls w a s
u n c h a n g e d . O u r d a ta d e m o n s t r a t e d t h a t th e p r e s e n c e o f a B M S C t r a n s p l a n t s ig n ific a n tly
i n c r e a s e d m a c r o p h a g e infi lt ration into th e c o n t u s e d ad u lt r at s p i n a l cord.
D ISC U SSIO N
Q u a n t i t a t i v e i n v e s t i g a t i o n s h a v e d e m o n s t r a t e d t h a t survival o f B M S C t r a n s p l a n t e d into th e
c o n t u s e d a d u lt rat s p i n a l cord is lo w ’70' 286. Previo usly, it w a s p r o p o s e d t h a t m a c r o p h a g e s w h ic h
a r e n a tu r a lly p r e s e n t w ithin an in ju ry sit e a r e inv olved in th e lo s s o f B M S C t r a n s p l a n t e d into
the central
a c ti v a te d
n e r v o u s s y s t e m 9’ . O u r p r e s e n t r e su lt s s h o w e d t h a t a d e c r e a s e d
m a c r o p h a g e s a t th e t i m e o f B M S C
i n jec tio n
presence o f
(th ree d a y s post-in jury) d o e s n o t
i n c r e a s e survival o f g rafte d B M S C . It is p o s s i b l e t h a t o u r t r e a t m e n t r e g i m e n s fa iled to lower
m a c r o p h a g e infi lt rati on to a level w h e r e B M S C survival w o u ld h av e b e e n
im proved. The
m a x i m u m redu ction w h ic h w a s a c h i e v e d h e r e w a s 5 4 % . B e c a u s e m a c r o p h a g e in v a s io n into a
s p i n a l cord inju ry site is ty p i c a lly la r g e 34, ’ 55, 3’ ’ th is red u ction m a y still le a v e m a n y m a c r o p h a g e s
t h a t cou ld p o t e n t i a l ly c o n tr ib u t e to B M S C loss. An ad d itio n al o b s e r v a t i o n is t h a t th e t r e a t m e n t
effe c t wh ic h w a s p r e s e n t a t t h re e d a y s po s t-in ju ry w a s n o t s ig n i fi c a n t a t t e n d a y s post-injury.
86
Inflamm ation and cell survival
Alth o u gh
we
used
treatm en t
p r o to c o ls
known
to
eff ectiv ely
reduce
th e
presence
of
m a c r o p h a g e s 22' ’77, 3"' 399, 403 t h e y m i g h t n o t h a v e b e e n e ffe ct iv e e n o u g h for suffi cient an d
p r o l o n g e d redu ction o f m a c r o p h a g e s .
A n a l t e r n a t i v e e x p l a n a t i o n fo r th e o b s e r v e d lack o f i m p r o v e d B M S C survival w o u ld be t h a t
a n y t r e a t m e n t - i n d u c e d redu ction in m a c r o p h a g e p r e s e n c e w a s m a s k e d by a s u b s e q u e n t
i n c r e a s e in m a c r o p h a g e infilt rati on d u e to th e in tro d u c tion o f B M S C into th e e n v i r o n m e n t .
This n o tio n is s u p p o r t e d by o u r da ta b e c a u s e in a n i m a l s with a B M S C t r a n s p l a n t w e fo u nd
that
m acrophage
infilt rati on
was
d r a s tic a lly
increased.
It
is
likely
that
these
ext ra
m a c r o p h a g e s h a v e e x a c e r b a t e d th e lo ss o f B M S C ’40. It is i m p o r t a n t to k e e p in m i n d t h a t o th er
fa c t o r s t h a n i n v a d e d m a c r o p h a g e s a r e m o s t likely a l s o involved in t r a n s p l a n t e d B M S C loss
such a s th e lack o f o x y g e n a n d / o r n u t r i e n t s within t h e d a m a g e d t i s s u e 470.
Previo u sly,
it w a s
reported
that
BM SC
are
hypo-im m u nogen ic;
th e y
suppress
th e
p r o life r atio n a n d fu n ctio n o f T-cells, B-cells, n a tu ra l killer c ells, an d d e n d r i ti c c e lls 208' 424.
H o w e v e r , t h e s e p u b li c a ti o n s did n o t i n v e s t i g a t e p o s s i b l e eff ects o f B M S C on m a c r o p h a g e
i n v a s io n .
It is p o s s i b le t h a t th e i m m u n o s u p p r e s s i v e p r o p e r t i e s o f B M S C a f fec t o n ly th e
a d a p t i v e i m m u n i t y d u e to th e lo w e x p r e s s i o n level o f h u m a n le u k o cy te a n t i g e n (HLA) m a j o r
h i s t o c o m p a ti b il it y ( M H C ) c la s s I a n d th e a b s e n c e o f c o - st im u la to r y m o l e c u l e s 208, 469. T h is w ould
e x p la i n w h y th e r e c r u it m e n t o f m a c r o p h a g e s (acquired i m m u n ity ) w o u ld n o t be affe c ted by
BMSC.
L o w e r in g t h e n u m b e r o f m a c r o p h a g e s in th e in ju red s p i n a l cord n e e d s to b e a d d r e s s e d
with c a u ti o n . It is well k n o w n t h a t m a c r o p h a g e s c a n s u p p o r t s p i n a l cord r e p a i r by p r o m o t i n g
a x o n r e g e n e r a t i o n a n d m y e l i n a t i o n w h ic h m a y be a c c o m p a n i e d by im p r o v e d f u n c t i o n ’46, 35°.
T h e s e c o n s t r u c ti v e e ffe ct s o c c u r w h ile m a c r o p h a g e s a lso e x e r t d e s t r u c ti v e effects such as
ne u ral cell d e a t h 400. B e c a u s e o f this dual role, d e c r e a s i n g th e n u m b e r o f m a c r o p h a g e s w ithin a
s p i n a l cord inju ry could le a d s i m u l t a n e o u s l y to b en eficial a n d d e t r i m e n t a l e f f e c t s 350. T h u s it is
i m p o r t a n t to aim for a redu ction in m a c r o p h a g e s th a t w o u ld n o t j e o p a r d i z e th eir p o s it iv e
c o n tr ib u t io n s to s p i n a l cord repair.
C O N C LU SIO N
W e h y p o t h e s i z e d th at a d e c r e a s e d m a c r o p h a g e p r e s e n c e in a n a d u lt rat s p i n a l cord c o n tu s io n
w o u ld
support
survival
of a
BMSC
tran sp lan t.
With
CsA,
MC,
and
MP
treatm en t w e
su c c e ss fu lly lo w e re d m a c r o p h a g e infilt ration; ho w e v e r , th is redu ction , w h ic h w a s m a x i m u m 54
% , did n o t i m p r o v e t r a n s p l a n t e d B M S C survival. S u r p r is in g ly , w e fo u n d t h a t B M S C p r e s e n c e
in th e c o n t u s io n in rea lity i n c r e a s e d m a c r o p h a g e p r e s e n c e by a l m o s t 4-fold. B a s e d on our
c u r r e n t k n o w l e d g e it is to be e x p e c t e d t h a t m a c r o p h a g e s a r e inv olved in t h e lo s s o f B M S C
afte r in je c tio n into th e in ju red s p i n a l cord. T h e f i n d i n g t h a t a la r g e d e c r e a s e in m a c r o p h a g e
87
Chapter 5
infilt ration failed to im p r o v e survival o f s u b s e q u e n t l y i n je c te d B M S C , p o i n t a t key r o le s o f o ther
in ju ry-rela te d e v e n t s in th e lo s s o f a n i n t r a s p i n a l B M S C t r a n s p l a n t .
88
6
Functional recovery after transplantation
L o c o m o t o r a n d s e n s o r y fu n ctio n r e c o v e r y a fter a u t o l o g o u s b o n e marrow/ s tr o m a l cell
t r a n s p l a n t a t i o n in th e c o n t u s e d ad u lt rat s p i n a l cord.
Su bm itted Cell Transplantation
G .J. Ritfeld
R .D .S . N a n d o e T e w a r i e
S.T. R a h ie m
A. H u r t a d o
K. V a jn
M. O u d e g a
Chapter 6
IN TRO D U CTIO N
C o n t u s i v e inju ry to th e a d u lt rat th o r a c ic s p i n a l cord c a u s e s i m m e d i a t e l o c o m o t o r a n d s e n s o r y
i m p a i r m e n t s o f th e h i n d li m b s 2’3, 246, 4° 8. L o c o m o to r fu n ctio n g r a d u a l ly i m p r o v e s until a p l a t e a u
is r e a c h e d
after 2-5 w e e k s
d ep en d in g
on
th e
im pact
s e v e r ity 24, 6°
2° 7, 33°. S p o n t a n e o u s
re s t o r a t io n o f s e n s o r y fu n ctio n is m o s tly a b s e n t ’76, 459 but s o m e r e c o v e r y w a s r e p o r t e d to occur
m o n t h s afte r th e i m p a c t 27.
T ransplantation
o f repair-prom oting
e x t e n s i v e l y e x p lo r e d
a s an
intervention
c e lls
into
th e
contused
spinal
cord
has
been
to r e s t o r e fu n ctio n o v e r w h a t is s p o n t a n e o u s l y
o b s e r v e d 29, 122 3’3, 325, 338. O n e o f th e c a n d i d a t e cell t y p e s for s p i n a l cord r e p a i r is th e b o n e m a r r o w
s tr o m a l cell ( B M S C ) . T h e s e c e lls a r e rela t iv e ly e a s y to o b ta in via a b o n e m a r r o w b i o p s y w h ic h
w a r r a n t s th e ir giv e n p r o m i s e fo r clinical a p p l i c a t i o n 285, 325, 43°.
Cu rren tly, th e ability o f B M S C t r a n s p l a n t s to r e s t o r e fu n c t io n a f te r s p i n a l cord c o n t u s io n is
d e b a t e d . S o m e i n v e s t i g a t o r s r e p o r t e d b e n e fic ia l effects on m o t o r f u n c t i o n ’69, ’79, 472 473 but
o th e r s a r e in d i s a g r e e m e n t ’ ^ 385, 463. S p i n a l cord t i s s u e s p a r i n g h a s b e e n p r o p o s e d a s a
p r o b a b le m e c h a n i s m o f B M S C - m e d i a t e d r e p a i r 286, 3° 4. Effects o f a B M S C g r a ft on s e n s o r y
fu n ctio n r e s t o r a t io n afte r s p i n a l cord c o n t u s io n h a s n o t b e e n d e s c ri b e d .
MATERIAL AND METHODS
Ethics and surgical approval
All rats u s e d in th is s tu d y w e r e h o u s e d pre- an d
p o s t- s u r g e r y a c c o r d i n g to th e N a t i o n a l
I n s ti tu te s o f H e a lt h a n d th e U n ite d S t a t e s D e p a r t m e n t o f A gricu ltu re g u id e li n e s . Air in th e
c a g e s w a s c o n t i n u o u s ly r e fr e sh e d a n d w a t e r a n d food w e r e a v a i l a b l e ad libitum. A t all t i m e s
d u rin g th e e x p e r i m e n t , r a ts w e r e k e p t w ithin a do u b le-b a rr ie r facility. All a n i m a l p r o c e d u r e s
w e r e a p p r o v e d by th e In s titu tio n a l A n i m a l C a r e a n d U s e C o m m i t t e e a t th e J o h n s H o p k in s
Univer sity .
B M S C culture and lentiviral transduction
B M S C w e r e h a r v e s t e d fr o m th e m a r r o w o f th e f e m u r s o f ad u lt f e m a l e S p r a g u e - D a w l e y rats
(n=4 , 2 ° ° - 2 3 ° g ; H a r la n , I n d i a n a p o l i s , IN, USA) a c c o r d i n g to p r e v io u s l y d e s c r i b e d p r o t o c o l s ’3,
359.
Len tiv ira l
vectors
encoding
fo r
green
pr e v io u s l y d e s c r i b e d ’75 a n d u s e d a t an
fluorescent
p r o te i n
(GFP )
w ere
prepared
M O I o f ’ 5 ° to t r a n s f e c t B M S C a t p a s s a g e ° .
as
For
t r a n s p l a n t a t i o n , B M S C fr o m p a s s a g e 4 w e r e u s e d o f which 6 3 % e x p r e s s e d G F P a s d e t e r m i n e d
by F A C S c a n / F A C S o r t e r (B e c to n D ic k in s o n I m m u n o c y t o m e t r y S y s t e m s B io s c i e n c e s , S a n J o s e ,
CA, USA ). Previo usly, w e s h o w e d t h a t o v e r 9 5 % o f th e c e lls w e u se d fo r i n t r a s p i n a l in je c tio n
e x p r e s s e d C D 9 ° a n d C D ’ ° 5 , w h ic h a r e ty pica l B M S C m a r k e r s , a n d n o n e o f t h e m e x p r e s s e d
C D 3 4 a n d C D 4 5 , both blood cell m a r k e r s , or H L A -D R , an i m m u n e cell m a r k e r 359.
90
Functional recovery salFter tr;^nsfDls>nts)tion
Pre-surgery procedures
Ad u lt
fem ale
Sp ra gu e-D aw ley
rats
(n=40,
200-230g;
Hadnn)
wi^re;
sedatkd
with
i n t - a p e r i t o n e a l injec1;ions of7 6 0 m g / k g K e t a m i n e HCl ( P h o e n ix O hnrm o ceu tlc als , S 3. J o s e c h ,
M D , USA) a n d 0 . 4 te g / k g rr^ecJi1;ot^icdine ( D o m i t o s ® , a n a l p h a - 2 - a d r e n e r g i c a g o n i s t ; O rio n
C o r o o r a t i o n , E s p o o , F i n la n d ) . A fter d e e p a e d c t io n w a s verified, th e b ac k o f the; r a ts w a s ohav ed
and c l e a n e d with B e e a d i n e a n d 7 0 % alcohol, L acrilu b e B i n t m e n t
0 m g / k g gen tam icin
(Abbot
Laboratories
N o sta
(ilhiicEjjjio,
:s n p p li e d to tli e e ) e s , an d
IL,
USA)
was
adm inistered
in t ra m u s c u la r ly .
Spinal cord contusion
T h e lo w e r th o r a c ic (T) s p in a l c r l u m n w a s e x p o o e d , 1tloe lo m i n a o f th e T 9 w a s r e m o v e d , a n d the;
e x p o s e d u n d e r l y i n g T 1 0 s p i n a l eord o e g m e n t c o n t u s e d u s i n g th e In fin it e H o r iz o n I m p a c t o r a t
a fo rce o f 2 0 0 k D y n e 36, r77. C o e s i c t e n c y n e t w e e n rats w a s g u a r n n S n e d usiir'igt th e r e c o r d s on tg h
comn>j'e;s^ion r a te a n d v elo city b f ta n i m u n c to t; rain fr o m wXieh 1;o ns;e v a l u e s Usviatrrd ovwr r %
w e r e e xc lu d ed fr o m th e sltudy. TOe w o u n d rite w a s rinsmscJ with p h o s e h a t e -b u fp e r e d s a l i n e (XBS)
with 0 . 1 % g e n t a m i c i n (A b bo t L a b o ra to rie s ) a n k th e m u s c l n s w e r e c lo s e d in l^y.'ei'ss u s i n g 5 .0
s u tu res . T h e skin w a s c lo se d wit h M ic hel w o u n d clips.
Cost-contusion procedures
After
c lo s u r e
od th e w o u n d ,
th e
ra ts
received
a subcutrnrous
in je c tio n
of7 1 .
m g/kg
a t i p a m e z o l e h rd ro ch lo rid e ( a n t i s e d a n ® ; a n a l p h a 2 - a d r e n n r r i c n n t a g o n ie t ; tKtzgo inc., N e w
Yo rk , N Y , USA ), to r e v e r s e th e c e d a t iv e a n d u n a l g e s i a eObcts cC m e d it o m id i n x . T e n ml la c t a ted
R i n g u r 'r s o lutio n w a s inju cte d s u b c e t o n e n u s l y a n d 5^ i^g/^lcjsr g e n t a m i c i e (/^laijo'tt; L a b o ta t o r ie s)
iritramr-^culai'ly. T h e rato w e r e k e p t in a s m a ll a n i m c l i n c e b a t o t a t 3 7 ° C untii 'full / e c o c e x an d
w n r e tkiern r e tu r n e d tee th eir c a g e s i Until thuE) s e c o n d srur'j^fesr;!) a t t h r e e d a y s p o s t - c o n t u s i o n , th e
ta ts
r e c e iv e d
d a il y 5 ml
R in ger's
solutio n
(subcutaneour),
(5 m eb k b c e n t a m i c i n (AOOca
L a b o ra to ri e s ; in tram u s c u ln r ly ), an d 0 . 0 5 m g / k g O u p rn n o rp h in (Bu^|i'e;n(r^<r>; S e c S i t t X e nckirh)
P h a r m a c e u t i e a j s Inc., R ic h m o n d , VA; s u b c u t a n e o u s l y b T h e b la d d e r w a s m o e u a l l y empOied
tw ic a frier day.
CsA admicistration
S t a r t i n g 5 m in afte r th e c o n tu a i v e injury, c o n e u e e i rats r e c e iv e d C eA (Bedfo rd Labs; OeObord,
O H , USA) s u b c u t a n e o u e l y o n c e p e r d a y a t a C o s e oC 3 0 m g t k i fit- 1;he Oirst thoee dn y t after
c o n t u s io n in ju ry a n d 15 m g / k g for th e fol lowin g d a y s t n r o u o e s u t th e survival p n d o d r°3.
Q f 0 er e o n f e r e U r afr reeelveU s a i in e inje n tio n s.
C haptsr 6
B M S C transplantation
A t t h re e d a y s a fter c o n t u s io n , rats w e r e s e d a t e d wit h i n t r a p e r i t o n e a l i n je c t i o n s o f 6 0 m g / k g
K e t a m i n e HCl ( P h o e n ix P h a r m a c e u t i c a l s ) a n f 0 . 4 m s / k g m e d i t o m i d i n e (Orion C o r p o r a t i o n )
T h e T 1 0 s f i n e l cord s e / m e n t w a a r e - e x p o s e d a n d 1 x 1 0 6 B M S C in D M E M s e D M E M a l o n e
(total v o l u m t w a s 5 / J if1 both c a s e s ) w a s i n je c te d into th e conUusion e p i c e n t e r u s i n g a
H a m / l o n s y r in g e w ith a pu ll ed g l a s s n e a d l e a t t a c h a d (tip d i a m e t e r : 1 5 0 frm) fixed w ithin a
m i c r o m a n i p u la to r'75,4o8. A fter in je c tio n , t S s w o u n S w a s c lo s e d a s den crib ed a b o ve .
Post-injection procedures
After c lo s u r e o f thn w o u n d , raSs w n r e /ullg r e c o v e r e d in a s m a ll a n i m a l in c u b a to r a t 3C°C b e f t a e
b e i n g r e t v r n e d tn th eir c a g e s . T h e rats r e c e i v e d 5 ml L a c t a t e d R i n g e r 'c s o lutio n d a il y for 2 d a y s
(su b c u t a n e o u s ly ) a n / 6 m g / k g g e n f a m i c i n (AbOoUt C ab o rato ries ) da il y fo r e e v e n d a y s c o s tin je c tio n tin 0r a 72uscularl) 7). T h e rats w e r n i n je c t e d wit h 07.03 m g / k g B a p a e n o r p h i n (Rackitt
B e n c k is e r
P h a n m a c e u ti c a ls
Inc.; r u b c u ta n e o n s ly )
dail y fo r th e first 3 d a y s
p o s t - i n je c t io a .
B la d d e r s w e r e m a n u a l l y e m p t i e d tw ice pier d a y until re flex v o i d i n g s t a i t e d . T h r o u g h o u t th e
r e m a i n d e r o f th e e x p e s i m e n t th e ra ts w o r e m o n i t o r e d daily. In c a s e of7 c s i n o r d i s t r e s s rats
w e r e g iv e n 0 . 0 3 m g / C g e u o r e n s r p h i c (RecCitt B n n ck ixer P h a n m a c e u t i c a ls Inc.) s u b c u t a n e o u s l y
dail y fo r 3 da ys.
Experimental groups
R a ts wit h a T 1 0 c o n t u s i c n r e c e iv e d e i th e r B M S C / D M C M o r D M E M only'’ a n d w e r e t e e v t e d with
C s A c r s a ! i r e . T h u s t h e r e w e r e e e x p e r i m e ntal gr o u p s : B M S C / C s A ( n = 9 ), B M S C / s a l i n e (n = 8 ),
D M E M /C sA (n = i2), and D M E M /s a lin e (n = i0).
Testine o f locomotor function
Ali rets w e r e i e c l u d e d in l o c o m o t o r f u n c t i r e te s t in g i A u t o m a t e d h i n d i i m ! m o v a m e n t s w e r e
a n a l y z e d fo r eig h t w e e k s p o s t-c o n t u s i o n r n i n g t h e B a s e o - B e a t t i e - B r e e n a h a n ( f B B ) t e s t 23. Rats
w e r e fa m il ia r i z e d wit h th e o p e n field befo re injury. T h r e e d a y s a f te r th e c o n t u s io n , j u s t b efo r e
inj ec tio n , ra ts w e r e t e s t e d a n d t h o s e w ith a s co re a b o v e 5 w e r e r e m o v e d fr o m th e stu dy . Rats
w e r e t e s t e d o n c e a w e e k fo r e i g h t w e e k s afte r th e i n jec tio n into th e c o n t u s io n e p i c e n t e r o v e r a
p e riod o f 4 m in by tw o e x a m i n e r s o b li vio us o f th e t r e a t m e n t s .
S p e c i fi c
lo c o m o t io n - r e l a te d
fe a t u r e s
w ere
assessed
u sing
th e
BBB
su b - sc o re 220. P a w
p o sit io n (p arallel initiall y o r a t liftoff) a n d t o e c l e a r a n c e ( o c c a s io n a l, fr e q u e n t, o r c o n s i s t e n t )
for e a c h
hind p a w , a n d th e tail p o s it io n
(up, do w n)
a n d trunk s ta b ili ty (yes,
d e t e r m i n e d . T h e s c o r e s o f both lim b s a n d tail a n d trunk w a s s u m m e d
no) w e r e
( m a x i m u m s co r e
p o s s i b l e w a s 8).
T h e p a t t e r n o f lo c o m o t io n w a s a s s e s s e d u s i n g th e f o o t p r i n t a n a l y s i s which w a s m odified
fro m t h a t o f D e M e d i n a c e l i a n d c o l l e a g u e s 99. H i n d p a w s w e r e inked a n d fo o t p r i n t s w e r e m a d e
92
Functional recovery after transplantation
on p a p e r w ithin a r u n w a y o f a b o u t 1 m l e n g t h a n d 7 c m wid th . The; p r in t s w e r e u s e d to
m e a s u r e s trid e le n g th , b a s e o f s u p p o r t , nnd a n g le o f paw/ r o tatio n . A v e r a g e v a l u e s p e r paw/
w ene c a lc u l a t e d fr o m a t l e a s t 5 s e q u e n t i a l ste p n . V a ln e e fo r both p a w s w e r e a v e r a g e d . Stride;
le n g th w a s d e f in e d h / th e d i s t a n c e b e t w e e n th e c e n t r a l p a d s o f tw o c o n s e c u t i v e p r in t s on
e a c h sid e. B a s e o f s u p p o r t w a s d e t e r m i n e d by m e a s u r i n g t h e c o r e to c o re d i s t a n c e o f th e hind
p a w s c e n t r a l p a d s . Limb) r otation was d e f in e d by t h e a n g l e f o r m e d by th e i n t e r s e c t i o n o f th e
line th rou gh th e prin t o f th e third digit nn d th e p r i n t r e p r e s e n t i n g th e m e t a n a r s o p h a l a n g e a l
i o i n t a n d th e line shro u gh th e c e n t ra i p a d p a ra lle l to th e walki n g d irectio n . F o o t p r i n t t e s t i n g
w a s p e r f o r m e d a t 4 a n d 8 w e e k s afte r t r a n s p l a n t a t i o n a t w h ic h t i m e s all ra ts e xhibited w e i g h t
s u p p o rt .
S e n s o r i m o t o r fu n ctio n o f th e h i n d li m b s w a s a s s e s s e d
a t four a n d
eight w e e k s
post­
c o n t u s io n u s i n g t h e h o riz o n t a l la d d e r t e s t 218. W e u s e d a 1 0 0 c m lo n g h o riz o n t a l la d d e r which
t h e rats c ro v se d t h re e t fm e s e a c h t est. T h e pa s s a g e s w e r e v i d e o t a p e d a nd la ter p l a y e d b ac k for
a c c u r a t e e v a lu a t io n . O nly th e m i d d le 6 0 c m o f th e la d d e v w e s u s e d fo r m e a s u r e m e n ts. S m a ll
(foot: o r p a r t o . foo t), m e d i u m (foot a n d p a r t o f lo wer leg), a n d la rg e (full leg) s lip s w e r e
c o u n t e d a n d e x p r e s s e d a s a p e r c e n t a g e o f th e to tal n u m b e r o f s t e p s .
Testing o f sensory function
M e c h a n i c a l allo d yn ia w a s d e t e r m i n e d by m e a s u r i n g fo o t w it h d r a w a l in r e s p o n s e to a n o r m a lly
i n n o c u o u s m e c h a n ic a l s t i m u lu s a p p l i e d wit h an (ele ctr on ic) v o n Frey a n e a s t h e s i o m e t e r 69, 70.
D u r in g th e t e s t , ra ts w e r e in a p l e x i g la s b o x wit h an e l e v a t e d m e s h floor. T h e rats w e r e
a c c l i m a t e d fo r 5 m in b efo r e t e s t i n g . T h e von Frey tip w a s a p p l i e d p e r p e n d i c u l a r l y to th e midp l a n t a r a r e a o f e a c h hind p a w a n d d e p r e s s e d until p a w w i t h d r a w a l, a t wh ic h t i m e th e p r e s s u r e
(in g) w a s r e c o r d e d (3 t i m e s e a c h te s t ). T h e v a l u e s fo r b o t t p a w s w e r e a v e r a g e d .
W it h d raw al r e s p o n s e to a n o r m a l ly i n n o c u o u s h e a t s o u r ce , a p p l i e d u s i n g a H a r g r e a v e ' s
h e a t s ou rce, w a s u s e d to t e s t th e r m a l h y p e r a l g e s i a 160. T h e rats w e r e k e p t in a p l e x i g la s box
with a n e l e v a t e d m e s h floor for 5 min to a c c l im a t e . T h e r a d i a n t h e a t s o u r c e v^th c o n s t a n t
i n t e n s it y w a s a i m e d a t th e m i d - p l a n t a r a r e a o f e a c h hind pawn. T h e t i m e Sin sec) fro m ¡nitial
h e a t s o u r c e a c tiv a tio n to p a w w i t h d r a w a l w a s reco rd ed . A s e c o n d und third m e a s u r e m e n t w u s
p e r f o r m e d 5 a n d 1 0 m in la ter. T h e v a l u e s fr o m both p v w s w e v e evenn ged.
Statistical analysis
O ne-w ay
an alysis
o f variance
(AN O V A )
fo ll ow ed
by
F i s h e r 's
p r o te o fe d
le u s t - s i g n i n c a n t
d iff e re n c e ( P L S D ) t e s t w a s u s e d to d e t e r m i n e s tatis tic al p iff e r e n c e s b e t w e e n g r o u p s . In c a s e
o f u n e q u a l v a r i a n c e (F te s t ), a n o n p a r a m e t r i c a n a l y s i s (Kruskal-Wallis t e s t fo llo w ed by M a n n ­
W h i tn e y U-test) w a s u se d . S t a ti s ti c a lly s ig n i fi c a n t d if f e r e n c e s w e r e a c c e p t e d a t P < 0 . 0 5 .
93
Chapter 6
RESULTS
Surgery data
Forty -e ight ra ts w e r e inc lu ded in th is study. All r a ts w e r e c o n t u s e d a n d t h re e d a y s la ter
i n je c te d with B M S C / D M E M ( n = 1 7 ) o r D M E M o n ly ( n = 2 2 ) into th e c o n t u s io n e p i c e n t e r . T w o
r ats died p rior to th e t r a n s p l a n t a t i o n
s u rgery. S e v e n
r a ts w e r e r e m o v e d fr o m th e s tu d y
b e c a u s e th eir B B B s c o r e w a s h ig h er th a n 5 a t 3 d a y s p o s t - c o n t u s i o n (n=5) o r th eir i m p a c t
p a r a m e t e r s w e r e o v e r 5 % o f f fro m th e i n t e n d e d v a l u e s ( n = 2 ) . N o n e o f th e i n je c te d rats died
d u rin g th eir survival per io d .
B M S C transplants did not improve open field locomotor ability
A u t o m a t e d o p e n field l o c o m o t o r fu n ctio n w a s e v a lu a t e d u s i n g th e B B B t e s t 23, 24 a n d a n a l y z e d
u s i n g t w o - w a y A N O V A , which did n o t s h o w a n y d i ff e r e n c e s b e t w e e n t r e a t m e n t g r o u p s a n d th e
control g r o u p . T h e a v e r a g e B B B s c o r e o f con tro l ( D M E M / s a l i n e ) rats w a s 1 1 . 0 ± 0 . 0 (SEM ) a t
e i g h t w e e k s after i n jec tio n into th e c o n t u s io n . T h e o th e r g r o u p s had s im il a r s c o r e s ; 1 1 . 4 ± 0 . 2
for th e B M S C / s a l i n e g r o u p , 1 1 . 1
± 0 . 1 fo r th e D M E M / C s A g r o u p , a n d 1 0 . 9 ± 0 . 1 fo r th e
B M S C / C s A g r o u p (Fig. 1A). All rats r e a c h e d th e p l a t e a u s c o r e o f 11 a t 3 -4 w e e k s p o s t - i m p a c t ,
w h ic h w a s c o n s i s t e n t with p r e v io u sly p u b lis h e d d a t a fo r rats wit h a s im il a r c o n t u s i o n o n l y 60.
T h e s c o r e o f 11 refle c ts th e ability to s u p p o r t th eir w e i g h t on th eir h i n d li m b s a n d to m a k e
fr e q u e n t ly to c o n s i s t e n t l y p l a n t a r s t e p s w i t h o u t f o r e lim b -h in d lim b c o o r d in a t io n .
B M S C transplants increased open field locomotion-related features (BBB sub-score).
T h e s e n s itiv ity o f th e B B B s c a l e c a n b e i n c r e a s e d by e v a lu a t in g ind ividual c h a r a c t e r is ti c s o f
lo c o m o t o r fu n ctio n u s i n g th e B B B s u b - sc o rin g s c a l e 220. T w o -w a y A N O V A s h o w e d s ig n i fi c a n t
d iff e r e n c e s a c r o s s t r e a t m e n t g r o u p s (F (3,252) = 55.6; p < 0 . 0 0 0 1 ) . For control rats th e B B B s u b ­
s c o r e w a s 1 . 4 ± 0 . 3 (SEM ) a t e i g h t w e e k s a fter in je c tio n . R a ts with a B M S C t r a n s p l a n t
exhib ited a s c o r e o f 5.7 ± 0 . 3 w h ic h reflec te d a s ig n i fi c a n t 4-fold i n c r e a s e c o m p a r e d to c o n tr o ls
T h e d iff e r e n c e b e t w e e n t h e s e B M S C tr e a t e d rats a n d c o n tr o ls b e c a m e s ig n i fi c a n tl y n o t ic e a b le
s t a r t i n g t h re e w e e k s
po st-transplantation
(Fig 1 B ) .
Betw een
three
and
six w e e k s
af ter
t r a n s p l a n t a t i o n , C s A t r e a t e d rats had h ig h er s u b - sc o re s t h a n c o n tr o ls but this effect w a s no
lo n g e r p r e s e n t a t s e v e n o r a t e i g h t w e e k s . T h e s c o r e s in rats w ith B M S C a n d C s A w e r e not
d iffere n t fr o m co n tr o ls.
94
Functional recovery after transplantation
" B M S C /s a lin e
- DIVlEEM/C:ss^O
- B M S C /C s A
" D M E M /s a lin e
1w
Tim e after Contusion
B
2w
3w
4w
5w
6w
7w
8w
Tim e after C ontusion
Fig. i . BM SC did not improve automated hind limb movements in the open field but enhanced locomotion-related
features. Automated hind limb movements were assessed using the BBB scale and locomotion-related features were
assessed using the BBB Sub-scoring scale. A. None o f the groups demonstrated improved BBB scores over that of the
controls. B. Rats w itha BMSC transplant only (squares) reached significant higher scores than rats with controls with
DMEM injections (circles), rats with DMEM injection and CsA treatment (quadrilaterals), and rats with BMSC
transplants and CsA treatment (triangles). In both graphs the error bars indicate the standard deviation of the mean.
Single asterisks: significant difference between BMSC/saline group and DMEM/saline (control) group (p<0 .0 5 at 3
and 4 weeks; p<o.oi at 5-8 weeks). Double asterisks: significant difference between BMSC/saline group and
DMEM/CsA and BMSC/CsA grou p (p<0 .0 5 at 5 and 6 weeks; p<o.oi at 7 and 8 weeks). Number sign: significant
difference between DMEM*/CsA group and DMEM/saline group) (p<0.05 at 4-8 weeks). Section sign: si gnificant
difference between BMSC/CsA group and DMEM/saline group (p<o.oi a t 4 -8 weeks).
B M S C transplants improved some aspects o f the pattern o f locomotion
F o o t p r i n t a n a l y s i s w a s u s e d to i n v e s t i g a t e c h a n g e s in strid e le n g th , b a s e o f s u p p o r t , an d
a n g l e o f p a w r otatio n w h ic h ar e f e a t u r e s o f th e p a t t e r n o f lo c o m o t io n . .Ait e i g h t w e e k s af ter
in je c tio n into th e c o n t u s io n , o n e - w a y A N O V A s h o w e d no d i ff e r e n c e s a m o n g g r o u p s in str id e
le n g th (F (3.14) = 1 . 1 7 ; p = o . 3 5 ; Fig. 2A) or in b a s e o f s u p p o r t (F (3.17) = 1 . 9 4 ; p = 0 . 1 6 , Fig. 2 B ).
O n e - w a y A N O V A did revea l d i ff e r e n c e s in a n g l e o f hind p a w r o tatio n
(F (3,115) = 8 . 2 9 ;
p = 0 . 0 0 2 , Fig. 2 C ). T u key p o s t - h o c c o m p a r i s o n sOowed t h a t a B M S C t r a n s p l a n t result;, in a
2 6 % s m a l l e r a n g l e o f r otatio n (M = 1 9 . 7 ; 9 5 % CI [18 .7, 2 0 .7 ]) th an (control a n im a lh (M/I = 2 6 .5 ;
9 5 % CI [ 2 6 . 0 , 2 7 .0 ]; Fig. 2 C ). R a ts t h a t r e c e i v e d ChA had a 1 9 % s m a l l e r a n g l e o f r otatio n (M =
2 1 .5 ; 9 5 % CI [20 .5, 22.5]; Fig. 2C) c o m p a r e d to c o n tr o ls an d rats t h a t r e c e i v e d th e c o m b in a t i o n
o f B M S C a n d C s A s h o w e d a 3 6 % s m a l l e r a n g l e o f r otatio n (M = 1 6 . 9 ; 9 5 % CI [1 5 .9, 1 7 .9 ]; Fig.
2C).
95
Chapter 6
140 •
120 ■
: _ 100 ■
UJ 80 ■
? E
E
M
40 ■
20 ■
.
A
BM SC
saline
DMEM
C sA
BM SC
C sA
DMEM
saline
Fig. 2 . (left) BM SC transplants improved some aspects o f
locomotion pattern. We applied the footprint analysis to assess
specific characteristics of locomotion pattern. A. The average
stride length was not different between groups. B. The average
base of support was decreased at 4 and 8 weeks in rats that
received a BMSC transplant compared to rats that rteceived a
DMEM injection (asterisks, p<0 . 05 ). There were no differences
between the other groups. C. The average angle of hind paw
rotation was decreased at 4 and 8 weeks in the BMSC/saline
(asterisks, p<0 . 0 5 ), DEME/CsA (number signs, p<0 . 0 5 ), and
BMSC/CsA (section signs, p<0 . 0 5 ) group. There were no
differences among the groups that received BMSC and/or CsA
treatment. In all three graphs the error bars indicate the
standard deviation o f the mean.
35 ■
c —- 30 ■
.2 2 25 J
+S lil 25
+l 20 15 '«
10 ■
5 -
c
C
BM SC
saline
DMEM
C sA
BM SC
C sA
DMEM
saline
B M S C transplants improved sensorimotor function.
After a c o n t u s io n inju ry to th e s p i n a l cord, rats ex hibit i m p a i r e d i n t e g r a t i o n o f s e n s o r y an d
m o t o r in p u t s. C h a n g e s in s e n s o r i m o t o r fu n ctio n w e r e t e s t e d with a h o riz o n t a l la d d e r 2’8 by
c o u n t i n g th e n u m b e r o f s lip s o f f th e r u n g s by th e r a t's hind p a w ( sm all sli p), hind p a w an d
p a r t o f leg ( m e d i u m slip) or w h o l e hi nd leg ( large slip). T h e n u m b e r o f slip s w e r e q u a n tifie d
a n d e x p r e s s e d a s a p e r c e n t a g e o f th e to tal n u m b e r o f s t e p s . T h e a v e r a g e n u m b e r o f s t e p s w a s
’ 5 .0 ± 0 . 3 a t e i g h t w e e k s . O n e - w a y A N O V A s h o w e d s i g n i f i c a n c e s a c r o s s g r o u p s (F (3,23) =
4 2 . 7 ; p < 0 . 0 0 0 ’ ). C o ntro l rats m a d e s lip s in 7 0 . 3 ± 4 . 7 % o f th eir s t e p s a t e i g h t w e e k s (Fig. 3),
w h ic h
96
was
s ig n i fi c a n tl y
d iffe re n t
fr o m
BMSC
tr e a t e d
an im als,
who
showed
a
70
%
Functional recovery after transplantation
i m p r o v e m e n t (IVI = 2 1 . 4 ; (ill [1 8.8, 2 4 .0 ] ; (Fig. 3). Fiats with B M S C a n d C s A t r e a t m e n t s h o w e d a
52 %
i m p r o v e m e n t Irons c o n tr o ls (M = 33.8; CI [3 1.2, 3 6.4]) a n d
rats t h a t r e c e i v e d C s A
t r e a t m e n t o n ly s h o w e d a 2 6 . 6 % i m p r o v e m e n t fr o m c o n tr o ls (M = 52.3; CI [49 . 6 ,5 5 .0 ^ .
M 120
ÎÈ
[H BM SC/sali ne
W 100
+l
□ DMIEM/CsA
LU
<
/>
Q.
0)
</>
75
•*—1
£o
80
07 BM SC/C s A
■ DMEM/saline
60
40
in
=
w
20
0
s m all
m ed iu m
large
slips/step
F'g. 3. Presence o f a BM SC transplant improved performance on the horizontal ladder. The number o f small, medium,
and large slips was assessed individually. These numbers were also added and expressed as a percentage o f the total
number o f steps necessary to cross the horizontal ladder. The assessment was done at 8 weeks post-transplantation.
Rats with a BMSC transplant (white bar) had a significant better performance (i.e., lower percentage of slips/steps)
than control rats (black bars) (asterisks, p<0 . 0 5 ). This was also the case for rats with CsA treatment (dark gray bars)
and rats with BMSC and CsA (light gray bars). The error bars indicate the standard deviation of the mean.
B M S C transplants improved recovery fro m mechanical ailodynia.
After a th o r a c ic c o n t u s iv e s p i n a l cord inju ry rats d e v e l o p allo d y n ia o f th e hi nd p a w s . Ain
(ele ctronic) v o n Frey a n e a s t h e s i o m e t e r 69, 70 w a s u s e d to t e s t w h e t h e r a B M S C t r a n s p l a n t
w o u ld b e b e n e fic ia l fo r r e c o v e ry fr o m m e c h a n ic a l (tactile) a l l o d y n i a . O n e - w a y A N O V A (F (3,23)
= 24 .4 ; p < o .o o o i )
a n d T u k e y 's post- ho c c o m p a h i s o n s s h o w e d t h a t a t
eigha w e e k s p o s t ­
t r a n s p l a n t a t i o n , a n i m p r o v e m e n t o f 2 1 % in r e c o v e ry fr o m m e c h a n ic a l al l t d y n i a w a s o b s e r v e t
in rats wit h a B M S C t r a n s p l a n t s (M = 8 8 . 0 ; CI [8 5 .8 ,9 0 .2]), co m p a r e d to c o n tr o ls (IV1 =. 5 8.2; CI
[56.5, 59.9]; Fig. 4A ). T r e a t m e n t wit h C s A (M = 7 2.8 ; Cl [7 0 .2 , 75.4]) o r B M S C a c d C s A
c o m b in e d [ M = 7 0 . 7 ; CI [ 6 9 . 6 , 7 1.8 ] did n o t c h a n g e th e w i t h d r a w a l r e s p o n s e c o m p a r e d to
c o n tr o ls (Fig. 4A).
97
Chapter 6
120
.2
100
f
80
<s
* Hi 60
E +l
« £ 40
U(Q
2 3 20
0
BMSC
saline
A
DMEM
CsA
BMSC
CsA
DMEM
saline
9
8
^
7
5^2 6
■^w 6
Ow c
g +l 5
Si I
^O
4
3
h -S 2
1
0
B
BMSC
saline
DMEM
CsA
BMSC
CsA
DMEM
saline
Fig. 4 . Presence o f a BM SC transplant decreases mechanical
and thermal allodynia. Allodynia o f the hind paws was
assessed using a von Frey aneasthesiometer (A) and a
Hargreave’s heat source (B) at 8 weeks post­
transplantation. A. Rats with a BMSC transplant but none of
the other treatment groups had significantly higher response
times (i.e., lowered (improved) hyper-sensitivity) to a
mechanical (tactile) hind paw stimulus compared to the
control rats (asterisks, p<0 . 05 ). B. The response times of
rats with a BMSC transplant was significantly higher than in
controls (asterisks, p<0 . 0 5 ), which indicated lowered
(improved) hyper-sensitivity to a thermal hind paw stimulus.
In both graphs the error bars indicate the standard deviation
o f the mean.
30
B M SC
sa lin e
DMEM
C sA
BM SC
C sA
DMEM
sa lin e
Fig. 5. The presence o f a BM SC transplant improved the amount o f spared tissue. The volume of spared tissue was
assessed at 8 weeks post-transplantation and expressed as a percentage of the volume of an uninjured comparable
spinal cord segment. We found that rats with a BMSC transplant had significantly more spared tissue than control rats
(asterisks, p<0 . 0 5 ) . The other treatment groups were not different from the control group. The error bars indicate the
standard deviation of the mean.
98
Functional recovery ^fte r transfDlsintsition
B M S C transplants improved recovery fro m thermal allodynia
C o n t u s e d rats a lso d e v e l o p th e r m a l allo d y n ia , w h ic h c a n be m e a s u r e d with a H a r g r e a v e ' s h e a t
s o u r c e ’60. O n e - w a y A N O V A s h o w e d d i ff e r e n c e s a c r o s r g r o u p s (F ( 3 . 2 3 = 2 0 .7 ; p < o . o o o ’ ). Fiats;
w it S a s o n t u s i o n o n l y (no t r e a t m e n t ) w i t h d r e w th eir hind p a w s a t 6 . ’ ± 0 . 3 s (Fig. 4B ) . Rats
t h a t r e c e i v e d th e B M S C t r a n s p l a n t w i t h d r e w th e ir hind p a w a t 7 . 6 ± 0 . 3 s wh ic h r e p r e s e n t e d a
2 5 % i m p r o v e m e n t o ver c o n tr o ls (Fig 4 B ). T h e o t h e r g r o u p s w e r e n o t d i ff e re n t fr o m th e control
group) (Fig 4B ).
B M S C transplants elicited tissue sparing.
S p a r e d t i s s u e v o l u m e s a t th e c o n t u s io n sit e were m e a s u r e d a t e i g h t w e e k s after i n jec tio n into
t h e c o n t u s io n using; S t e r e o I n v e s t i g a t o r ® s o f tw a r e ( M B F B io s c i e n c e ) in a b lin d e d f a s h i o n . For
all m e a s u r e m e n t s th e Coefficient; o f Error ( G u n S e r s e n C o eff ic ient) w a s
< 0 .0 5 . T he volum es
w e r e e x p r e s s e d a s a p e r c e n t a g e o f th e v o l u m e of7a c o m p a r a b l e s e g m e n t o f a n a iv e (unin ju re d)
s p i n a l cord. O n e - w a y A N O V A s h o w e d difhere n c e s s c r o s s g r o u p s (F (3,’ 9 ) = 2 0 . 7 ; p = o . o o o 5 ) In
control rats (c o n t u sio n only) th e v o l u m e of7 s p a r e d t i s s u e w a s ’ ¿4.9) ± ’ .6 % (SE M ) o f t h a t o f an
u n in ju r e d s p i n a l cord (Fig. 5). With a B M S C t r a n s p l a n t th e v o l u m e w a s 2 4 . 8 ± ’ . 4 % , wh ich
represented
a s ig n i fi c a n t 6 6
%
i n c r e a s e in s p a r e d t i s s u e v o l u m e c o m p a r e d to c o n tr o ls
(p<o.c>5; Fig. 5). In rats with d a il y C s A t r e a t m e n t t h e s p a r e d t i s s u e v o l u m e w a s ’ 7 .8 ± ’ .7 %
a n d ia ra ts wit h a B M S C t r a n s p l a n t a n d C s A tr e s l m e n t th e v o l u m e w a s ’ 9 . 2 ± ’ .6 % o f t h a t o f
a n u n i n ju r e d s p in a l cord s e g m e o t (Fig. 5). T h e v o l u m e s ie t h e s e t w o g r o e p s w e r e s im il a r a s
t h e v o l u m e in c o n tr o l rats.
CoorelatioEs between tissue sparing and motor and sensory function.
T h e r e l a t i o n s h i p b e t w e e n t i s s u e s p a r i n g e n d m o t o r a n d s e n s o r y fu n ctio n p e r t r e a t m e n t g r o u p
(Tabl e ’ ) an d p e r fu n ctio n (Tab le 2) w a s a s s e s s e d u s i n g th e p e a r s o n c o r r e la t io n c o e f fic ie n t (r).
B M S C t r a n s p l a n t a t i o n r esu lte d in s s t r o n g a s s o b a S.on (r > 0 .7 5) b e t w e e n t i s s u e o p a r i n g an d
BIBB sub-sco re, h a r i z o n ta l la d d e r w a lk in g , a n g l e erf7 r otatio n, a n d m e c h a n i c a l allod yn ia . A
m o d e r r te associa tion
(a > 0 . 5 0 , < 0 .7 5) w a s fo u n d b e t w e e n t i s s u e s p a r i n g a n d th e rm a l
allo d yn ia (Tnble ’ ). With C s A t f e a t m e n t t i s s u e s p a t i n g w a s s t r o n g ly a s s a c i a t e d wit h th e a n g l e
o f r otatio n, m o d e r a t e l y wit h B B B sub-sc ore , a n d w e a k l y (s < 0.5(0) with h o rizo ntal la d d e r
w a lk in g ,
m ech an ical
allo d y n ia ,
an d
th e r m a l
allo d y n ia
(Table
1).
With
both
BMSC
t r a n s p l a n t a t i o e a n d C s A t r e a t m e n t t i s s u e s p a r i n g w a s s . r o e g l y a s s o c i a t e d with th e a n g l e o f
ro tatio n , m o d e r a t e l n w i t h th e B B B sun-sco re, h o rizo n tal la d d e r w a lk in g , a n d th ern fal allo d yn ia ,
and w eak ly
w XE
m e n h a n ic a l allo d y n ia (Table 1). In o u r control rats 'tine r e ln ti o n s h ip w a s s t r o n g
b e t w e a n t i s s u e s p a e i n g a d d th e r m a l alln d yn ia bot w e a k fo r all o th e r f n n c t i o n s l
If7 all fo ur t r e a t m e n t g r o u p s w e r e p o o le d t o g e t h e r p e r s p e c if ic hunction (Tabln 2), t i s s u e
s p ar i n g w a s s tr o n g ly a s s o c i a t e d .with BIBB s x b - s c o r e (Fig. 6A), m o d e r a t e ln a s s o c i a t e d with
99
Chapter 6
ho riz o n t a l la d d e r w a lk in g (Fig. 6 B ) , a n g l e of7 r otatio n (Fig. 6C), a n d th e rm a l a l lo d y n ia (Fig.
6 D ) , a n d w e a k l y a s s o c i a t e d with m e c h a n i c a l allo d yn ia (Fig. 6 E ) .
35
r = 0.85
30
♦
♦
= 25
8 20
: ♦ I
♦
♦
Ϋ
3 10
H 5
0
0
1 2
3 4 5 6
B B B su b -score
8
7
0
20
40
60
80
100
Horizontal ladder (% slips/steps)
35
r = -0.73
30
r = 0.57
.E 25
| 20
♦
♦
♦ ♦♦ ♦
<D 15
13
« 10
V)
F
♦
♦ ♦
H+t
♦
♦
5
0
0
5
15
10
20
25
30
A ngle o f rotation (degrees)
35
30
Fig. 6. The amount o f tissue sparing correlated with motor and
r = 0.39
♦
♦
.E 25
8 20
Q.
</> 15
0)
3 10
♦ ♦♦
♦
♦
%
♦
♦
I-- 5c
0
0
20
40
60
80 100
M echanical allodynia (grams)
100
0 1 2 3 4 5 6 7 8 9
Thermal allodynia (sec)
sensory outcomes. The relationship between tissue sparing and
motor and sensory function (with all four treatment groups
pooled) was assessed using the Pearson correlation coefficient
(r). Correlations between tissue sparing and individual groups
are described in text. Scattergrams show tissue sparing was
strongly associated with (A) BBB sub-score, moderately
associated with (B) horizontal ladder walking, (C) angle of
rotation and (D) thermal allodynia, and weakly associated with
(E) mechanical allodynia.
Funct iona l r e c o v e r y afte r
nspl antation
Fu n ction
Treatm ent
BBB
B M S C / saline
0.01
DMEM / CsA
0.35
DMEM / CsA
B M S C / CsA
-
B M S C / CsA
DMEM / saline
-
DMEM / saline
-
B M S C / saline
0.86
B M SC / saline
0.78
BBB Subsfore
DMEM / CsA
B M S C / CsA
Horizontal Ladder
slips/step
r
-
B M S C / saline
- 0.85
- 0.04
- 0.74
- 0.40
B M S C / CsA
DMEM;/ / saiine
Treatm ent
Foot Print (anale)
B M S C / saline
MechanicaV Allodrnia
0.73
0.67
DMEM / saiine
DMEM / CsA
F u n c tio n
DMEM / CsA
B M S C / CsA
DMEM / ¡saline;
Thermal Allodrnia
B M S C / saline
DMEM / CsA
B M S C / CsA
DMEM / saline
r
-0.92
-0.96
-0.95
0.20
0.10
0.10
0.70
0.30
0.73
0.99
Table 1 . Correlation between tissue sparing motor and sensory function. The relationship between the different
outcomes pertreatment group was determined by the Pearson correlation coefficient (r).
Abbreviations: * r = Pearson correlation coefficient; BBB = Basso-Beattie-Bresnahan open field locomotor scale; BMSC
= bone marrow stromal cells; CsA = cyclosporine; DMEM = Dulbecco’s minimal essential medium.
*
F u n ctio n
r
BB B
B B E S u b s c o ro
0.85
Horizontal L a d d e r
- 0.70
Fo o t Pric( (an gle)
- 0.73
M ech an ica l Allodynia
0.39
T herm al Allodynia
0.57
Table 2 . Correlation between tissue sparing motor a n f sensoryfunction.
The relationship between the different outcomes was determined per specific
function by the Pearson correlation coefSicitnt (r). Abbreviafons: * f =
Pearson correlation coeffitient; BBB = Batso-Beattie-Brestchan lecomotot
scale.
101
Chapter 6
D ISC U SSIO N
W e e v a lu a t e d l o c o m o t o r a n d s e n s o r y r eco v e ry in rats with a c o n t u s e d t h o r a c ic s p i n a l cord th at
re c e iv e d a B M S C t r a n s p l a n t into th e e p i c e n t e r o f th e inju ry a t t h r e e d a y s p o s t - i m p a c t . T h e
m a i n f i n d i n g in o u r s tu d y is t h a t th e p r e s e n c e o f a B M S C t r a n s p l a n t r esu lts in im p r o v e d m o to r
a n d s e n s o r y fu n ctio n . Ra ts t h a t r e c e iv e d a B M S C t r a n s p l a n t had i m p r o v e d B B B s u b -sc o res,
s m a l l e r a n g l e o f hind p a w r o t a tio n , im p r o v e d p e r f o r m a n c e on th e h o riz o n t a l la dder, a n d le s s
h y p e r s e n s i t iv it y to m e c h a n ic a l a n d th e r m a l s tim u li . B M S C - t r a n s p l a n t e d rats did n o t b e n e fit
fu n c t io n a lly fr o m d a il y C s A t r e a t m e n t . T h e a m o u n t o f s p a r e d t i s s u e a t th e c o n t u s io n sit e in
B M S C -tran sp lan ted
rats, but n o t in a n y o f th e o th e r g r o u p s , w a s i n c r e a s e d s ig n ific a n tly
c o m p a r e d to c o n tr o ls.
M o r e o v e r , rats wit h a B M S C t r a n s p l a n t s h o w e d a s t r o n g c o r r e la t io n
b e t w e e n th e a m o u n t o f t i s s u e s p a r i n g a n d th e fu n c t io n a l o u t c o m e . O u r d a ta s u g g e s t th a t
B M S C - m e d i a t e d t i s s u e s p a r i n g in th e c o n t u s e d ad u lt r at s p i n a l cord is i n t im a t e ly inv olved in
r e c o v e r o f m o t o r an d s e n s o r y fu n ctio n.
S o far, th e b e n e f i t s o f B M S C for m o t o r fu n ctio n r e s t o r a t io n had b e e n u n c l e a r a s se v e r a l
s t u d i e s ,0, 385 463 w e r e
at
varian ce
with
e a r lie r
reports
th a t
described
m otor
fu n ctio n
i m p r o v e m e n t s ’ 69, 170 472 473. W e n o w d e m o n s t r a t e t h a t B M S C t r a n s p l a n t a t i o n h a s w i d e s p r e a d
b e n e fi ts
fo r
m otor
fu n ctio n
r e s t o r a t io n
a fter
spinal
cord
c o n t u s iv e
injury.
A
p o s s i b le
m e c h a n i s m t h a t m a y u n d e r l ie t h e s e m o t o r fu n ctio n i m p r o v e m e n t s is B M S C -e l ic it e d n e rv o u s
t i s s u e s p a r i n g 286, 304. In d e e d w e fo u n d t h a t rats with B M S C had i n c r e a s e d a m o u n t s o f s p a r e d
tissue
a t th e
c o n t u s io n
site
and,
in t e r e s t i n g ly ,
th is
s t r o n g ly c o r r e la t e d
wit h
fu n ctio n a l
outcom es.
W e did n o t o b s e r v e a b e n e fic ia l eff ect o f t r a n s p l a n t e d B M S C o n a u t o m a t e d hind limb
m o v e m e n t s a s a s s e s s e d in th e o p e n field (B B B - te s t) . A lso , B M S C t r a n s p l a n t s had no eff ect on
str id e le n g th a n d b a s e o f s u p p o r t . A n a l y s i s o f o p e n field lo c o m o t io n with th e B B B - t e s t r e v e a le d
t h a t all ra ts r e a c h e d a p l a t e a u o f a b o u t , , . Such a s c o r e fo r rats t h a t r e c e i v e d o n ly th e
c o n t u s io n a n d n o t a B M S C t r a n s p l a n t a n d / o r C s A t r e a t m e n t is in a g r e e m e n t w ith a p r e v io u s
p u b li c a t i o n 60. O u r o b s e r v a t i o n t h a t B M S C - t r a n s p l a n t e d rats did n o t exhibit im p r o v e d BBBs c o r e s o v e r co n tro l rats is in full c o n c u r r e n c e w ith p r e v io u s r e p o r t s ’0, 385 463 bu t in con flict with
o t h e r s ’69, ’ 7°, 472, 473.
T h e lack o f i m p r o v e m e n t s o f a u t o m a t e d w a lk in g in th e o p e n field is s u r p r i s i n g in light o f
the observed
im provem ents
in
s p e c if ic fe a t u r e s
o f lo c o m o t io n ,
locom otor
pattern,
an d
s e n s o r i m o t o r p e r f o r m a n c e . It a p p e a r s likely t h a t B M S C - i n i t i a t e d m e c h a n i s m s t h a t c a u s e th e
o b s e r v e d i m p r o v e m e n t s w o u ld a l s o b e f u n d a m e n t a l to o p e n field a u t o m a t e d lo c o m o t io n .
Future
research
will
need
to fo c u s o n
th is
u nexpected
finding
and
e lu c id a te
possible
underlying m e c h an ism .
W e fo u n d t h a t t h e p r e s e n c e o f a B M S C t r a n s p l a n t r e su lt e d in im p r o v e d r e s p o n s e s to
m e c h a n ic a l a n d th e r m a l s tim u li to th e hind p a w s a t 8 w e e k s a fter t r a n s p l a n t a t i o n . T o our
k n o w l e d g e this is th e first r e p o r t t h a t d e s c r i b e s e ffe ct s o f B M S C t r a n s p l a n t s in th e inju red
102
Functional recovery after transplantation
ad u lt r at s p i n a l cord on s e n s o r y fu n ctio n r e s t o r a t io n . A p o s s i b l e m e c h a n i s m t h a t u n d e r l ie s the;
i n c r e a s e d s e n s o r y fu n c t io n is t i s s u e s p a r i n g elicited by th e B M S C t e a n s p l a n t 286,3°4. W e did finp
t h a t rata with B M S C had i n c r t a e e d a m o u n t s oh s p a r e d t i s s u e a t t h e c n n tu s i o n eite a n d ,
in teres ain gly, th is s t r o n g ly c o r r e la t e d w ith f u n c t io n a l o u t c o m e s .
R a ts t h a t r e c e i v e d da ily C s A exhib ited im p r o v e d o u t c o m e s in s o m e o f th e m o t o r t e s t s but
n o t in th e s e n s o r y t e s t s . O ve r a ll, the: C s A t r e a t m e n t did n o t b e n e f i t th e o u t c o m e in a n y o f th e
fu n ctio n a l pests op B M S C - t r a n s p l a n t h d rats. P rev io u rly, it haa O c e t r e p o r t e d t h a t t e e a t m e n t
with C s A s u p p o r t s c eilu lar a n d f u n c t io n a l r e p a i r a fter s f i n a l cord inju ry’77. H o w e v e r , w e did not
o b s e r v e a n y such fu n c t io n a l b e n e f i t s o f C s A t r e a t m e n t in t r a n s p l a n t e d rats. A t p r e s e n t it is
u n c le a r th ro u gh w h ic h m e c h a n i s m C s A t r e a t m e n t w o u ld in flu e n c e fu n c t io n a l g a i n s m e d i a t e d
by t r a n s p l a n t e d B M S C .
The
presence
fontu sion
o f a B M S C tra n sp la n t increased
site a t 8 w n e k s
po st-transplantation
th e a m o u n t o f s p a r e d t i s s u e a t th e
cem pared
to con tro l
rats . This w a r
not
o b s e r v e d in t;he o t h t r t r e a t m e n t g r o u p s . Important;ly, c o r r e la t iv e a n a t y s i s r e v e a le d t h a t th e
a m o u n t o f tisoue s p a r i n g w a s s t r o n g ly a s s o c i a t e d with fu n c t io n a l o u t c o m e in r a ts wit h a B M S C
t r a n s p l a n t . T h e s e f i n d i n g s r e v e a le d t h a t B M S C - m e d i a t e d t i s s u e s p a r i n g in th e c o n t u s e d adult
ra t s p i n a l cord is in t im a t e ly inv olved in re c o v e r o f m o t o r a n d s e n s o r y fu n ctio n.
103
7
Summary and general discussion.
Chapter 7
S p in a l cord inju ry (SCI) resu lts in n e r v o u s t i s s u e lo ss a n d i m m e d i a t e f u n c t io n a l i m p a i r m e n t s .
E n d o g e n o u s r e p a r a t i v e e v e n t s o cc u r w ithin th e d a m a g e d s p i n a l cord but g e n e r a l l y t h e y do not
r esu lt in m e a n i n g f u l r e s t o r a t io n o f fu n ctio n . T hus, S C I - m e d i a t e d lo s s o f fu n ctio n is p e r m a n e n t
a n d p e o p l e w h o e x p e r i e n c e SCI m a y b e d e s t i n e d to s p e n d th e r e m a i n d e r o f th eir lives in a
w h e e lc h a i r . A p p r o a c h e s a i m e d a t r e p a i r i n g th e s p i n a l cord a n a t o m i c a l l y a n d fu n c t io n a lly ar e
b e i n g i n v e s t i g a t e d in la b o r a to r ie s a r o u n d th e w o rld . S o m e o f th e m o r e p r o m i s i n g a p p r o a c h e s
a r e b e i n g t e s t e d in th e c linic but s o fa r n o n e o f t h e s e h a v e e m e r g e d a s o n e t h a t r e v e r s e s th e
d e v a s t a t i n g f u n c t io n a l c o n s e q u e n c e s o f SCI.
One
of
th e
poten tial
treatm en ts
that
c ould
support
spinal
cord
repair
is
th e
t r a n s p l a n t a t i o n o f cells t h a t a r e k n o w n to c o n tr ib u t e to cellu la r, a n a t o m i c a l , a n d fu n ctio n a l
r e s t o r a t io n . B o n e M a r r o w S t r o m a l C e lls ( B M S C )
h av e r e c e i v e d a m p l e a t t e n t i o n fo r their
p r e s u m e d p o t e n t i a l to r e p a i r c e n t r a l n e r v o u s s y s t e m (CN S) le s i o n s . An i m p o r t a n t a d v a n t a g e
o v e r o th e r ce llu la r c a n d i d a t e s fo r s p i n a l cord r e p a i r is t h a t B M S C c a n b e o b t a i n e d fro m rou ti n e
b o n e m a r r o w b i o p s i e s fr o m th e p a t i e n t . T his a d v a n t a g e a l lo w s fo r a u t o lo g o u s t r a n s p l a n t a t i o n
in w h ic h reje c tio n o f th e t r a n s p l a n t e d c ells m a y b e m i n i m a l.
T h e overall g o a l o f th is th e s i s w a s to i n v e s t i g a t e th e p o t e n t i a l a n d suita bil ity o f B M S C to
r e p a i r th e in ju red s p i n a l cord. T his w a s a d d r e s s e d in a s e r i e s o f e x p e r i m e n t s t h a t fo c u s e d on
ra t B M S C g e n e profi li ng, B M S C survival a fter t r a n s p l a n t a t i o n , a n d th e ir eff ects on t i s s u e
s p a r i n g a n d o n l o c o m o t o r a n d s e n s o r y fu n ctio n r e s t o r a t io n . For th e in vivo e x p e r i m e n t s a rat
s p i n a l cord c o n t u s io n m o d e l s y s t e m w a s e m p l o y e d to m i m i c th e m o s t fr e q u e n t ly o cc u rr in g
t y p e o f h u m a n s p i n a l cord injury. In th e fo ll o w in g s e c t io n , th e m a i n r e su lt s fr o m t h e s e s tu d ies
a r e s u m m a r i z e d a n d d i s c u s s e d in light o f w h a t is k n o w n fr o m th e c u r r e n t li te r a t u r e a n d w h a t
w o u ld be n e c e s s a r y to a c h i e v e a n a t o m i c a l a n d fu n c t io n a l r e p a i r o f th e s p i n a l cord.
In C h a p t e r 1, SCI a n d a v a rie t y o f re la t e d a s p e c t s a r e in tro d u c ed . In th e U n ited S t a t e s th e
i n c id e n c e a n d p r e v a l e n c e o f SCI h a s b e e n stu died in d e p t h . A n n u a l ly , a p p r o x i m a t e l y 1 1 , 0 0 0
n e w c a s e s o f SCI o cc u r in th e US. T h e s iz e o f th e g r o u p o f s p i n a l cord inju red p e o p l e in th e US
h a s c o n s e r v a t i v e l y b e e n e s t i m a t e d b e t w e e n 2 5 0 , 0 0 0 a n d 4 0 0 , 0 0 0 . L e s s is k n o w n a b o u t th e
i n c id e n c e o f SCI in E u r o p e a n d T h e N e t h e r l a n d s .
M a i n l y b e c a u s e o f th e la r g e v ariab ili ty a m o n g SCI it is difficult to d e f in e t h e b e s t clinical
p r a c tic e . D u r in g th e e ar ly p h a s e , t r e a t m e n t s t h a t s t a b i li z e th e p a t i e n t ' s health a n d a t t e m p t to
limit th e overal l lo s s o f tis s u e / f u n c t i o n n e e d to be i m p l e m e n t e d . O p t i m a l t r e a t m e n t n e e d s to
be d e t e r m i n e d o n a c a s e - t o - c a s e b a s i s . D e c o m p r e s s i o n s u r g e r y w ith o r w i t h o u t fixation o f th e
s p i n a l c o l u m n m a y a c c e l e r a t e f u n c t io n a l i m p r o v e m e n t s a n d resu lt in s h o r t e r h o sp ital an d
reh a b ilita t io n p e r i o d s . A s lo n g a s p r o p e r clinical trials h a v e n o t b e e n e x e c u t e d th e effects o f
t h e t i m i n g o f d e c o m p r e s s i o n s u r g e r y will r e m a i n elu siv e.
106
Sum m ary and general discussion
P h a r m a c o lo g i c a l t r e a t m e n t s to limit s e c o n d a r y injury a f te r th e initial d a m a g e h a v e b e e n
stu died in t e n s iv e ly . B e s t -k n o w n is t r e a t m e n t with high d o s e m e t h y l p r e d n i s o l o n e ( M P ) . T h e
effects o f M P in p a t i e n t s wit h SCI w e r e i n v e s t i g a t e d in 3 c o n s e c u t i v e N a t i o n a l A c u t e S p i n a l
Cord Injury S t u d i e s ( N A S C I S ) . T h e r esu lts d e m o n s t r a t e d t h a t a c u t e M P t r e a t m e n t r e su lt e d in
n e u r o lo g ic a l i m p r o v e m e n t s u p to 6 m o n t h s a f te r injury a n d , a s a resu lt o f t h e s e fi n d i n g s , M P
w a s th e s t a n d a r d o f c a r e for m a n y y e a r s . H o w e v e r , a fter a th o r o u g h re v ie w o f th e resu lts from
t h e N A S C I S s tu d ie s a n d a m o r e c o m p r e h e n s i v e a s s e s s m e n t o f th e b e n e fi t s a n d risks involved
in high d o s e M P - t r e a t m e n t , th e t h e r a p e u t i c b e n e fi ts o f M P t r e a t m e n t h a v e b e e n d is cr ed ited .
E s p e c i a lly in p a t i e n t s with c o m p l e t e SCI high d o s e ster o id t r e a t m e n t c a n le a d to a d v e r s e
effects such a s
m yopathy and w ound
infe ctio n t h a t m a y n e g a t i v e l y i n fl u e n c e fu n ctio n a l
o u t c o m e a n d in s o m e c a s e s m a y b e life - th r e a t e n i n g . Cu rren tly, m o s t cli nics h av e d i s c o n t i n u e d
t h e ‘ s t a n d a r d ' a c u t e a d m i n i s t r a t i o n o f M P a f te r SCI.
C h ap ter 1 a l s o in t ro d u c e s s t e m c e lls (SC) a s a p o t e n t i a l t h e r a p y fo r SC I. O v e r th e la st
y e a r s , S C h a v e g a i n e d a t t e n t i o n in th e field o f o r g a n r e p a i r a n d o r g a n o g e n e s i s . E m b ry o n ic
s t e m c e lls (ESC) ar e th e c e lls within th e i n n e r cell m a s s o f th e b la st o c y s t. D u r in g d e v e l o p m e n t
t h e s e c e lls a r e r e s t r a i n e d to g e r m la y e r s w h e r e
th eir fa t e is d ir e c t e d . U n d i ffe r e n t i a t e d SC-like
c ells c a n b e fo u n d a m o n g d iff e r e n ti a t e d c e lls o f a s p e c if ic t i s s u e a fte r birth. T h e s e c e lls ar e
kn o w n a s ad u lt SC , alth o u g h a b e tt e r te r m w o u ld be ‘ s o m a t i c S C ' s i n c e th e y a r e a l s o p r e s e n t in
children a n d umbili cal cords. It h a s b e e n r e p o r t e d t h a t S C a r e a b le to c r o s s g e r m la y e r s in vitro
given th e right ‘ in d uctio n cocktail' o f grow th fa ctors. Even th o u g h this is still pro fo u n d ly
debated
am ong
s c i e n t i s t s , th is p r e s u m e d
potential
has open ed
m any
n e w a v e n u e s for
r e s e a r c h in d iffe re n t d is c i p l in e s .
O n e o f th e i s s u e s t h a t s u r r o u n d th e u s e o f E S C is th e t i m e p o i n t a t w h ic h w e h a r v e s t th e
c ells fr o m th e e m b r y o . C a n a t t h a t t i m e th e e m b r y o in fa c t b e c o n s i d e r e d to be alive, to be a
p e r s o n ? D i s c u s s i o n s on w h a t c o n s t i t u t e s ‘ life' a n d w h e n ‘ life' s ta r ts a r e o ften i n t e n s e a s th ey
a r e driven by m o r a l c o n c e r n s fu e le d by re lig io u s a n d politic al id e a s . T h e s e i s s u e s n e e d to be
a d d r e s s e d w ith r e s p e c t to all o p p o n e n t s . Ethical i s s u e s t h a t s urrou n d th e u s e o f S C involve
p o s s i b l e m i s u s e , such a s with t h e r a p e u t i c c lo n i n g a n d g e n e t i c m a n i p u l a t i o n . A d u lt S C h av e
le s s ethical b a r r ie r s a n d a r e t h e r e f o r e i n t e r e s t i n g c a n d i d a t e s fo r i n t e r v e n t i o n p a r a d i g m s .
C h ap ter 2 r e v i e w s th e u s e o f B M S C s in n e u r o s c i e n c e in g e n e r a l a n d fo r r e p a i r o f th e inju red
s p i n a l cord s p ecific all y. B M S C a r e m e s e n c h y m a l SC-lik e c ells t h a t r e s i d e in th e b o n e m a r r o w .
This p a rt ic u la r lo c atio n
a l lo w s fo r e a s y h a r v e s t fro m th e p a t i e n t a n d , c o n s e q u e n t l y , for
a u t o lo g o u s t r a n s p l a n t a t i o n w ith m i n i m a l r e je c tio n . Ethical i s s u e s a r e a l s o u n d e r m i n e d a s
B M S C c a n b e h a r v e s t e d fr o m b o n e m a r r o w fr o m ad ults. W h e t h e r B M S C c a n t r a n s d i f f e r e n t i a t e
into c e lls fr o m th e ne u ral cell l i n e a g e , o r o th er l i n e a g e s for t h a t m a t t e r , is still u n c l e a r an d
d e b a t e d a m o n g s c i e n t i s t s . H o w e v e r , th e e a r ly r e p o r t s t h a t B M S C a r e in fa c t c a p a b l e o f
t r a n s d i f f e r e n t i a t i o n h a v e la u n c h e d a p l e th o r a o f in v e s t i g a t i o n e x p l o r i n g th eir r e p a i r p o t e n t i a l .
107
Chapter 7
S o m e e x a m p l e s a r e th e s tu d ie s on th e u s e o f B M S C fo r r e p a i r o f th e h e a r t m u s c l e after
m y o c a r d ia l infa rc tio n in c ard iology , o s t e o g e n e s i s i m p e r f e c t a in o r t h o p e d i c s , o r g a n o g e n e s i s in
in t e rn a l m e d ic i n e , in t e r v e r te b r a l d i s c d i s e a s e in n e u r o s u r g e r y , a n d s trok e / n e u r o d e g e n e r a t i v e
d i s e a s e s in n e u r o lo g y .
A s th e d e b a t e o n th e p o t e n t i a l o f B M S C to t r a n s d i f f e r e n t i a t e c o n t i n u e s , th e y c ould a l s o be
u se d fo r r e p a i r fo r th eir n a tu ra l ability to p r o d u c e a n d s e c r e t e m a n y r e p a i r - r e la t e d m o le c u le s .
T his ability cou ld be b e n e f i t t e d fro m in for i n s t a n c e th e in ju red s p i n a l cord a s t h e s e m o le c u le s
could s u p p o r t ce llu la r a n d a n a t o m i c a l re p a ir . H o w e v e r , w h e n B M S C a r e u se d fo r s p i n a l cord
r e p a i r w i t h o u t d iff e re n tia t io n into n e u r a l c e lls p rior to t r a n s p l a n t a t i o n , it is p o s s i b l e t h a t th e
i n je c te d c ells d iff e r e n t i a t e in viv o into m e s e n c h y m a l cell t y p e s such a s fat, m u s c le , c a rt ila g e , or
b o n e c e lls . This w o u ld be a real c o n c e r n a s t h e s e c e lls w o u ld i m p a i r th e o verall r e p a i r o f th e
s p i n a l cord. S o far, r e p o r t s t h a t th is in fa c t o c c u rs h a v e n o t b e e n p u b lis h e d . A lso , in th e in vivo
s tu d ie s d e s c r i b e d in th is t h e s i s w e h a v e n o t fo u n d a n y s i g n s t h a t this t a k e s p lace .
C h ap ter 3 d e s c r i b e s th e g e n e e x p r e s s i o n profile o f ad u lt rat B M S C for wh ic h 44k w h o l e g e n o m e
rat
m icroarrays
w ere
em p lo yed .
The
m ajor
go al
o f this
s tu d y
was
to
increase
our
u n d e r s t a n d i n g o f th e p o t e n t i a l a n d suita bil ity o f B M S C fo r s p i n a l cord r epair. In ad d itio n , w e
a i m e d to a s s e s s th e effects o f lo n g -ter m c u ltu rin g o n g e n e e x p r e s s i o n by c o m p a r i n g B M S C
fro m th e 3 rd (P3) a n d 1 4 th (P14) p a s s a g e in cultu re. Both P3 a n d P 1 4 B M S C e x p r e s s e d g e n e s
inv olved in n e u r a l d e v e l o p m e n t a l e v e n t s such a s glial d iff e re n tia t io n a n d m y e li n a t io n , an d
n e u r o n a l p r o life r a tio n a n d n e u r ite f o r m a t io n , i n d ic a t i n g th e p o t e n t i a l o f B M S C to d iff e re n tia t e
into n e ural l i n e a g e . B M S C a l s o e x p r e s s e d g e n e s e n c o d i n g fo r grow th fa c t o r s a n d for p r o t e i n s
inv olved in g r o w th fa c t o r s ig n a l i n g . A to tal o f 6 6 8 7 g e n e s w e r e e x p r e s s e d in P3 a n d in P 1 4
B M S C , with a 9 7 % o v e r l a p o f g e n e s e x p r e s s e d a t a s im il a r i n t e n s i t y a n d 3 % ( 2 0 2 g e n e s )
e i th e r h ig h e r in P3 B M S C (15 9 g e n e s ) o r higher in P 1 4 B M S C (43 g e n e s ) . F u n ctio n a l d a ta
m i n i n g by G e n e O n t o l o g y ( G O ) -a n a ly s i s r e v e a le d t h a t 8 5 / 1 5 9 an d 2 2 / 4 3 w e r e a n n o t a t e d in
t h e G O d a t a b a s e . In P3 B M S C , 4 3 G O - c l a s s e s w e r e o v e r r e p r e s e n t e d with 9 inv olved in o rg a n
d e v e l o p m e n t a n d cell p r o life r a tio n . In P 1 4 B M S C , 2 G O - c l a s s e s w e r e o v e r r e p r e s e n t e d wit h 1
inv olved in o r g a n d e v e l o p m e n t . O ve r all, o u r g e n e pro fili n g d a t a s u p p o r t th e u s e o f B M S C for
ne u ral r e p a ir . A n u m b e r o f g e n e s a r e e x p r e s s e d in B M S C fr o m which th e p r o d u c t could
s u p p o r t c e llu la r r e p a i r o f th e s p i n a l cord. A lso , w e fo u n d e v id e n c e t h a t lo n g -te r m c u ltu rin g o f
BM SC
may decrease
th eir
p l a s t i c ab ili ties
a fter t r a n s p l a n t a t i o n .
Furth er g e n e
pro fili ng
r e s e a r c h will b e n e e d e d to im p r o v e o u r u n d e r s t a n d i n g o f th e n e r v o u s s y s t e m r e p a i r - p o t e n t i a l
o f B M S C a n d to p o s s i b ly p r o v id e a b a s i s for m a n i p u l a t i o n o f th e B M S C to i n c r e a s e th eir
eff ic acy p rior to t r a n s p l a n t a t i o n .
One
p o s s i b l e w a y by w h ic h
B M S C cou ld s u p p o r t s p i n a l
cord
r e p a i r is by li m itin g th e
p r o g r e s s i v e t i s s u e lo s s (e.g., e li c it in g t i s s u e s p a r i n g ) t h a t n o r m a l ly o cc u rs afte r th e initial
108
Sum m ary and general discussion
d a m a g e . Im p o r t a n t ly , to profi t fr o m this ability p o o r survival o f B M S C afte r t r a n s p l a n t a t i o n
into th e c o n t u s i o n e n v i r o n m e n t is a p o t e n t i a l li m itin g fa ctor. In C h ap ter 4 th e survival o f
B M S C a n d th e e ffe ct s o n t i s s u e s p a r i n g afte r t r a n s p l a n t a t i o n into th e c o n t u s e d rat s p i n a l cord
a r e d e s c r i b e d . W e i n je c t e d B M S C into th e m o d e r a t e l y c o n t u s e d ad u lt rat th o r a c ic s p i n a l cord
a t 15 m in (acute) a n d a t 3, 7 a n d 2 1 d a y s (dela yed) po s t-in ju r y a n d q u a n tifie d t i s s u e s p a r i n g
a n d B M S C survival u p to 4 w e e k s p o s t - t r a n s p l a n t a t i o n . B M S C survival w ithin th e c o n t u s io n at
7 d a y s p o s t - t r a n s p l a n t a t i o n w a s s ig n ific a n tly h ig h e r with a n a c u t e in je c tio n ( 3 2% ) a n d 3-days
delayed
i n jec tio n
U n fo rt u n a t e ly ,
(52 % ) th an w ith a 7- or 2 1 - d a y s d e l a y e d
BM SC
presence
at 28
days
i n je c tio n
po st-transplantation
was
(9%
both; p < o . o i ) .
c lo s e
to
0
in
all
p a r a d i g m s , in d ic a ti n g m a s s i v e cell d e a th .
In c o n t u s e d rats w it h o u t a B M S C t r a n s p l a n t (contro ls), th e v o l u m e o f s p a r e d t i s s u e
g r a d u a l ly d e c r e a s e d until 4 6 % ( p < o . o o i ) o f th e v o l u m e o f a c o m p a r a b l e u n i n ju r e d s p in a l cord
s e g m e n t a t 4 9 d a y s post-inju ry. A c u t e a n d 3-d ays d e l a y e d but n o t 7- a n d 2 1 - d a y s d e l a y e d
in je c tio n o f B M S C s ig n ific a n tly im p r o v e d t i s s u e s p a r i n g , w h ic h w a s s t r o n g ly c o r r e la t e d (r =
0 . 7 9 - 1 .0 ) to B M S C survival in th e first w e e k a f te r i n je c tio n into th e c o n t u s io n . This s e t o f
resu lts d e m o n s t r a t e d t h a t B M S C t r a n s p l a n t e d into th e c o n t u s e d s p i n a l cord o f an ad u lt rat
surviv e p o o rly a n d B M S C d e a th w a s g r e a t e r w h e n c e lls w e r e i n je c te d d e l a y e d c o m p a r e d to
e a r ly a f te r c o n t u s io n . T h e d a ta fu rth er s h o w e d t h a t th e n e u r o p r o t e c t i v e e ff e c t s o f B M S C
t r a n s p l a n t e d into a m o d e r a t e rat s p i n a l cord c o n t u s io n d e p e n d s tr o n g ly on th eir survival
d u rin g th e first w e e k p o s t-in je c t io n ; a c u t e ly i n je c te d B M S C elicit m o r e t i s s u e s p a r i n g th an
d e l a y e d i n je c te d B M S C .
T h e resu lts o b t a i n e d in th is s tu d y to s o m e d e g r e e c h a n g e s o m e i d e a s on c e ll- b a s e d r e p a i r
a p p r o a c h e s for th e in ju r e d s p i n a l cord. For lo n g it w a s th o u g h t t h a t d e l a y e d t r a n s p l a n t a t i o n o f
c ells w ith in a c o n t u s io n e n v i r o n m e n t w o u ld be b en eficial fo r t r a n s p l a n t e d cell survival a n d ,
c o n s e q u e n t l y , fo r th e e ffe ct s m e d i a t e d by th e cells, a s it w o u ld c ir c u m v e n t th e h e ig h t o f th e
inju ry-i nitia ted e n d o g e n o u s i n f l a m m a t o r y a n d i m m u n e r e s p o n s e s . This w i s d o m w a s g e n e r a l l y
a c c e p t e d w ith in th e field o f s p i n a l cord in ju r y / r e p a ir m a i n l y b e c a u s e o f its logical r a t i o n a l e an d
a s lo n g a s th e a c tu al d e l a y o f cell in tro du c tion w ithin th e inju ry w o u ld n o t p a s s th e t i m e p o i n t
w h e r e g e n e r a l d e t e r io r a ti o n o f th e s p i n a l cord w o u ld limit o r e v e n p r e v e n t a n y b e n e fi t s fro m
cell t r a n s p l a n t s .
Even
th o u g h this
idea w a s
gen era lly acknow ledged,
strong
sup po rtin g
q u a n t i t a t i v e e v i d e n c e h a s b e e n la ck ing. O u r d a ta in d ic a te t h a t it m a y b e m o r e b e n e fic ia l to
t r a n s p l a n t B M S C e a r ly r a th e r th a n la te a t l e a s t to b e n e fi t fr o m th eir t i s s u e s p a r i n g abili ties.
Furth er r e s e a r c h will b e n e c e s s a r y to d e t e r m i n e th e u n d e r l y i n g m e c h a n i s m s o f th e i m p r o v e d
B M S C survival w ith ea r ly a s o p p o s e d to d e l a y e d t r a n s p l a n t a t i o n . A lso , it r e m a i n s to be
d e t e r m i n e d w h e t h e r th is w o u ld a l s o p e r t a in o t h e r c a n d i d a t e cell t y p e s fo r s p i n a l cord repai r.
109
Chapter 7
Tli e go al o f C h ap ter 5 w a s to im p r o v e B M S C survival a fter t r a n s p l a n t a t i o n into a c o n tu s io n
e n v i r o n m e n t . W e h y p o t h e s i z e d t h a t r e d u c i n g m a c r o p h a g e infi lt ration p rio r to in tro du cti on o f
B M S C w o u ld i m p r o v e th eir survival in ^ e c o n t u s e d ^ o r a c i c ad u lt ra t s p i n a l cord. Quickly af ter
a c o n t u s iv e i m p a c t to th e s p i n a l cord a n i o v a s io n o f m a c r o p h a g e r c a n b e o b s e r v e d . W e
f em onstrated
that
m e th ylp red n iso lo n e
tre a rm e n t
(M P)
regim es
r esu lted
in
of
a
c y c lo s p o r i n e
s ig n i fi c a n t
(CsA),
decrease
m in o c y c li n e
(p< o .o o i)
in
( M C ),
or
m acrophage
infilt ration a t t h r e e d a y s post-inju ry. D e s p i t e th is p r o m i s i n g result , survival o f B M S C ( i x i o 6/5
ml) in jr c t e d into th a c o n t u s io n e p i c e n t e r nt th is 3-day t i m e point; w n s not; s ig n ific a n tly d iffere n t
fro m t f a t o f B M S C i n je c te d into a n im a l s wUli o u t th e m a c r o p h a g e - r e d u c in g t r e a t m e n t s . In
fact, t h t fer e s t n c e of" E3M S C resulteri in r s ig n i fi c a n t a v e r a g e f .9-fold i n c r e t s o ( p < o . o o i ) in
m a c r o p h a g e infi lt ration into th e c o n t u s i o n o f treateri rats to la tive to c o n tr o ls. O n e c o n c lu s io n
fro m tlie sh re su lts is th a t B M S C in je c te d w ithin th e c o e t u s i o n e p i c e n t e r a t t r a c t ad ri;tional
i n f l a m m a t o r y c e lls e v e n wit h c o n c u r r e n t a d m i n i s t r a t i o n of7 m a c r o p h a g e - r e d u c i n g dr u gs . It m a y
be t h a t t h o s e n M S C t h a t die s o o n a f te r i n jec tio n recruit t h e s e extra m a c r o p l i a g e s . D e a t h o f
Isn ahn lan ted
IBM/ISC
is
a
multi-factorial
.oroblem
w h ic h
shotld
be
addressed
in
a
m u lt id ir c ip li n a r y m a n n e r . Fu ture s tu d ies should Ire d ir e ct e d t o w a r d s f i n d i n g a n d s u b s e q u e n t l y
m a n i p u l a t i n g o th e r c u e s fo r coN d e a t h t e c h a s p a t h w a y s in n e c r o s i s o r a p o p t o s i s o f th e
t r a n s p l a n t e d cells.
In C h ap ter Or w e i n v e s t i g a t e d th e e ffe ct s o f B M S C grofts con hin d l i m ° lo c o m o t o r a n f s e n sory
l u e c t i o r in a d u lt rate witd a c o n t u s e d t h o r a c ic s p i n a l cord. R a ts w e r e c o n t u s e d u s i n g th e
In fin it e H o r i z o n s i m c a c t o r a t a fo r ce o f 2 n n kDyn a n d n ^i D M E M w i t t w r o 6 B M S C s or
D M E M n lo n e w a s i n je c te d into th e inju ry c p i c e n t e r th reo d a y s la t e r. S o m e rats r e c e iv a d C s A
dail y th rou g h o s t th e survival p e riod. Even t h o u g t it w a s s h o w n t h a t C s A t r c a t m n n r did not
im p r o v e B M S C survival, its a S m i n i s t r a t i o n nould support; r e p a i r - r e la t e d e v e n t s t h a t t o f e t h e r
with
tfcse
of
OMSC
could
resu lt
in
im prnv nd
outcom ea.
Two
m onths
afte r
dM SC
t r a n s p l a n t a t i o n r a ts had i n c r e a s e d B a s o c - l e a t t i c - B r e s n a h a n (BIBB) sub-scGres, a 1 7 % s m a ll e r
l a s a o f o u p p a r t d f th e hinh lim b s, a 3O % s m a l l e r a n g l e o f hind p a w r o t a tio n , a n d p e r fo r m e d
38 % b e tt e r o n th e h o r i z o n t a l la dder. In ad d itio n , a t 4 w e e k s p o r t - t r a n s p l a n t a t i n n , t h e s e rats
exhib ited a 31 % a nd 5 0 % i m p r o v e m e n t in th eir r e s e o e s e to a n r e c h a n ic a l an d th e rm a l
s tim u lu s, r esp eceiv e ly , to th e n io d p a w s . A t 8 w e e k s
p o s t - t r a n r p l a n t e t i o n , t h e rats still
exM b ited a 2 2 % i m p r o v e m e n t in tCeir r o s p o n s e to a m e c h n n i c a l s t i m u lu s but n o t a n y Io n g e r
ter n th e r m a l s tim u lu s. R a tr th o t rnceiv ed C s A t s e a t m e n t a l o n n did n o t d e m o n s t r a t n fu n ctio n a l
c h a n g e s c o m p a r e d to c o n tr o ls e x c e p t fo r a 2 9 % s m a l l e r a n g l e o f hind p a w r o tatio n . T h e r e
w a s no overall ad d itive e ffect o f C s A t r e a t m e n t o n B M S C - m e d i a t e d fu n c t io n a l i m p r o v e m e n t s .
O u r d a ta r e v e a le d t h a t t r a n s p l a n t a t i o n o f B M S C into a 3-d ay old c o n tu s io n in th e ad u lt rat
s p i n a l cord i m p r o v e d s o m e a s p e c t s o f lo c o m o t o r a n d s e n s o r y fu n ctio n. O u r resu lts s u p p o r t
furth er e x p lo r a t i o n o f B M S C fo r th e d e v e l o p m e n t o f s p i n a l cord r e p a i r s t r a t e g i e s .
110
Sum m ary and general discussion
This t h e s i s m a y s e r v e a s a s t a r t i n g p o i n t o f m a n y s t u d i e s th a t fo c u s on u n d e r s t a n d i n g an d
im p r o v i n g th e e ffic ac y o f B M S C to r e p a i r th e in ju red s p i n a l cord. T h e s tu d ie s in th is th e s i s
d e m o n s t r a t e t h a t B M S C h av e th e g e n e t i c ability to be e ffect iv e in n e r v o u s s y s t e m r epair, t h a t
t h e y elicit t i s s u e lo ss w h ic h g r e a t l y d e p e n d s on th e ir survival, a n d t h a t th e y d e s p i t e th eir
s h o r t c o m i n g s r e su lt in b e h a v io r a l i m p r o v e m e n t s a f te r s p i n a l cord c o n t u s iv e injury. It is c lear
fro m o ur resu lt s t h a t B M S C survival is a n i m p o r t a n t i s s u e t h a t n e e d s to be a d d r e s s e d such th at
th eir e ffe ct s on r e p a i r c a n b e o p t i m i z e d .
111
8
Nederlandse samenvatting.
Chapte r 8
L etsel v a n h et r u g g e n m e r g kan r e s u lt e r e n in fu n c t ie v e r li e s v a n d e e x t r e m i t e i t e n . In d ie n het
v e rlie s v a n fu n c t ie h e r m u h e n t is, k u u t e n p a ti U n t e n v o o r d e r e s t v a n hun le v en
r o l s t o e l g e b u n h e n z ijn . I n t e r v e n t i e s t r a t e g i e n zi jn gerin ht o p f h u n t i o u t t l t e p u ptom is clc
herste l v a n h e t h e s c p a d i g h e r u g g e n m e r g , m p a r h e b h e n to t h u td e n ie t g e le id t o f fun(rtiuhel e
v e rb eteu iu g . CunP anks d e v e le u n d e r z h e k e n p p b a s a a l w e te n s c p a p h e r lij k g e b i e d is t r a n s l a t i e
pp a r h e klinie k v o o r a l s o o g n iet m u g e li jk .
hen v a n h e m g g e lij k h e d e n mm h et b e s c P a d i g d r u g g e n m e r g te h e r s t e ll e n is d o o r m iddel v a n h e
t r a n s p l u n t a t i e v a n c e lle n . o u h e M a r r o w S t r n m a l C e l le n ( B M S C ) z:ijn s t a m c e l l e n die
g e m a n k e lijk te v erk rij gen ^ijn m i n h e ls b d e n m e r p h u n (r tie s . H ie r d o o r is a u t o l g g e
c e l t r a n s p l a n t a t i e m o g e li jk en is h et nisico p p a f s t o t i g g d o o r h e t lic h a a m m i n i m a a l .
In dit p r o efs ch r ift is h e ge s c h ik th e id vun B M S C v o o r h erstel v a n h e t h e s c P a d ig d
r u g g e n m e r g h e s tu h d e rd . hen sehie e x d e r i m e n t e n z ijn u it g e v o e r d , w aau in h e g e n e x p r e s s i e v an
B M S C , o v e h e v i g g v a n h e c e lle n pa t r a n s p l a n t a t i e e n hun in v lo e d p p d e f u n c O u d e le u itk n m s t
is b e s tn h o e rd in h en r u g g e n m e r g l e t s e l m o h e l in d e rat.
In H oofdstuk 1 w o r d ^ e n a l g e m o e n o v e r z i c h t v a n r u g g e n m e r g l e t s e l ge u ntro d ucherd. In h e
V e r e n i g d e S t a t e n z ijn h e i n c ih e n t i e e n p r e v a l e n t i e vun r u g g e n m e r g l e t s e l u itgeb re id
u n d e rz o c h o m e t h e n i n c ih e n t i e vun u n g e v h e r 1 1 , 0 0 0 p a t i ë n t e n h e r jp ar. D e m l e s t
v o o r k n m e n h e o o r z a k e n zi jn ^ g e v a l l e n , g e w e l d , s o o r l e n zie k te .
W e t e n s c P a p l e i n k bewij s v o o r h e b e s t e b e P a n h e l m e t o o h e i s e r niet, m e h e v a o w e g e h e
g r o t e v a ria b ilite it v a n d e u n g e v a l l e n . In h e v r o e g e f a s e is d e h e h a n h e l r n g ger icho p p
O e m o d u p a m is c h e s t a b i li s a t i e v a n h e p a t i ë n t . H i e r r a k o m r d e h e h a n h e i r n g v a n h e ( in stabiele )
w e r v e lk o lo m pan h e o rd e, w p arbij h en h e c o m p r e s s i e v e l a m i o e c t o m i e al p a n n iet
g e c o m b i o o e r d m e t fixatie vun h e t i n s t a b i e l e s e o m e n t v a n h e w e r v e l k o l o m h e f fu n cO un o el
herste l ka n b e v o r d e r e n e n ka n r e s u lt e r e n in he n kort et verb>lijf in h e t z i e k e h huis e n
reva lip atiek lin iek . In h e c h r o n is c h e f a s e stpu n t> reventie v a n infe(rties un d o o rlig p le k k e n
ce tl trp£il, w p a r b ij a s h e c t e n a l s pijn e n in fertilite it o o k in g g e n s c h o o w g e o o m e n d ie h e n te
w o r h e n . h e t e r e k iin is c h e s t u d i e s zi jn o o o Z z a k e i ij k o m h et v o o r h l e l v a n h en p p e r a t i e in h e
vroege fa se adequpat te h e stu d e re n .
D e lp a t s t e j a r e n is h e t g e b r u ik v a n s t a m c e l l e n (SC) in h e w e t e n s c p a p ste rk t o e g e o o m e n .
n m b r y u p a le s t a m c e l l e n (ESC) zijn c e lle n in h e zi ch u n t w i n k e le n h e b la s t o c y s te . Al v r o e g in de
untw inkelr n g b e v in d e n h e z e S C zi ch in h e k i e m l a g e n e c t o - / m e s o - en e n d o h e r m . N a he
g e b o o r t e z ijn s t a m c e l l e n o o k p a o w e z i g in v e rs c h il le n h e o p a l e n . V e r s c h i lle n h e h u b lic a tie s
h e b b e n p u g g e t o u n d dan m e t hen j u i s t ^ d u c l e pro to co l S C in s t p a t z ijn zich te u n t w in k e le n tot
cel lij h e fh uip a n h e r e k i e m l a g e n , h o e w e l hieroveu o o g g h e n c u n s e n s u s is bereikt. H ie r d o o r is er
hen n i e o w e g o l f v a n i n t e r m s e v o o r heg gebru i k v an S C u n t s t p a n .
114
Nederlandse sam envatting
H e t b e la n g r ij k s t e a r g u m e n t t e g e n h e t g e b r u ik v an s t a m c e l l e n is d e d i s c u s s i e o v e r
w a n n e e r e e n e m b r y o e e n m e n s e l i j k p e r s o o n g e n o e m d kan w o r d e n . Is h e t b e g in v a n h e t le v e n
bij d e b e v ru c h tin g v a n d e eicel o f bij le v e n s v a t b a a r h e i d v a n d e fo e t u s ? Dit is e e n d i s c u s s i e vol
e m o t i e . P o t e n t i e e l m is b ru ik v a n s t a m c e l l e n is d e v o o r n a a m s t e o o r z a a k v a n d e v e l e eth is c h e
discussies.
In H oofdstuk 2 is d e b e s c h i k b a r e li t e r a t u u r k e n n is o v e r d e m o g e li jk h e d e n v a n B M S C v o o r
h erste l v a n h et b e s c h a d i g d r u g g e n m e r g b e s t u d e e r d . B M S C zi jn m e s e n c h y m a l e S C die in het
beenm erg
aan w ez ig
zijn .
H ie r d o o r
zi jn
ze
g e m a k k e l ijk
te
v erk rij gen
m i d d e ls
b e e n m e r g p u n c t i e . D o o r d e m o g e lijk h e id v a n a u t o g r a f t in g is h et risico o p e e n a f s t o t i n g s r e a c t i e
g e m i n i m a l i s e e r d . T e n s l o t t e zi jn er bij h et g e b r u ik v a n v o l w a s s e n s t a m c e l l e n g e e n e t h is c h e
bezw aren, aan g ezien
er g e e n
e m b r y o n a a l w e e f s e l w o r d t gebruikt.
D o o r d a t v e r s c h il le n d e
pu b li c a ti e s h e b b e n a a n g e t o o n d d a t B M S C k u n n e n t r a n s d i f f e r e n t i ë r e n , is er d e l a a t s t e j a r e n
toenem end
a a n d a c h t h ier vo o r o n t s t a a n
o p h et g e b i e d v a n
h e rstel v a n
d e h a r t s p i e r na
m y o c a r d i n f a r c t e n in d e c a rd io lo g ie , o s t e o g e n e s i s i m p e r f e c t a in d e o r t h o p e d i e , o r g a n o g e n e s e
in
de
interne
geneeskunde,
n euro degen eratieve
a a n d o en in g en
in
de
n e u r o lo g ie
en
g e h e r n i e e r d e d i s c u s in t e r v e r t e b r a li s in d e n e uro chirurgie.
D o o r hun m e s o d e r m a l e o r i g i n e z ijn B M S C e e r d e r g e n e i g d te d i ff e r e n ti ë r e n in c e lt y p e n
van
d e b e t r e f f e n d e k i e m la a g .
ruggenm erg
n iet
z u ll e n
H e t is o n z e k e r o f d e z e c e lle n
d iff e r e n ti ë r e n
in
vet,
bot,
in e e n
kraakbeen
o m g e v in g van
o f spierw eefsel.
het
Het
is
n o o d z a k e li jk d e ge s ch ik th e id v a n B M S C v o o r herste l v a n h e t b e s c h a d i g d r u g g e n m e r g g o e d te
o n d e r z o e k e n e e r dit in e e n k lin is ch e s e t t i n g t e t e s t e n .
In H oofdstuk 3 w o r d e n d e r e s u lt a t e n b e s c h r e v e n v a n o n d e r z o e k n a a r d e g e n e x p r e s s i e v an
B M S C m e t b e h u lp v a n 4 4 k to t a a l r a t g e n o o m m ic ro a r ra y s . T o t n o g t o e is d iff e re n tia t ie v an
celtypen ,
waaronder
m em braanm arkers.
o ok
D eze
BMSC,
z ijn
echter
b e o o r d e e ld
aan
n i e t s p e c if ie k
en
de
hand
som s
van
z e l fs
m o r fo lo g ie
en
onbetrouw baar.
Het
b e p a l e n v a n h et g e n e x p r e s s i e p a t r o o n v a n B M S C is e e n b e t e r e m a n i e r o m d e g esch ik th eid
van
dit c e l t y p e v o o r n e u r a le d iff e re n tia t ie t e
bepalen .
D a a r n a a s t is o ok h et e ffect v an
l a n g d u r i g e c e lk w e k e n o p d e g e n e x p r e s s i e v a n B M S C b e s t u d e e r d d o o r p a s s a g e (P) 3 en P 1 4 te
v e rg e lijk e n . Z o w e l P3 a ls P 1 4 B M S C b r e n g e n v e r s c h e i d e n e g e n e n t o t e x p r e s s i e die e e n rol
sp elen
in
neur(on )ale
processen
zoals
g lia le
d i ff e re n ti a t ie
en
m y e li n i s a t i e ,
n euronale
p r o life r a tie en n e u r i e t fo r m a t ie . D e g e n e x p r e s s i e v a n d e z e g e n e n b e v e s t ig d d e p o t e n t i e v a n
B M S C o m te t r a n s d i f f e r e n t i ë r e n
in d e n e u r a l e cellijn .
In t o t a a l k o m e n
6687 genen
to t
e x p r e s s i e m e t e e n 9 7 % o v e r la p in g e n e x p r e s s i e . V a n d e o v e r i g e 3 % k o m e n 1 5 9 g e n e n h o g e r
to t e x p r e s s i e in P3 e n 4 3 g e n e n
h o g e r in P 1 4 .
F u n c t io n e le d a ta
s t r u c tu r e r in g l a a t e e n
o v e r r e p r e s e n t a t i e v a n 4 3 G O - k la s s e n in P3 B M S C z i e n , w a a r v a n 9 e e n rol s p e l e n in o r g a a n
o n t w ik k e lin g en c e lp r o life r a tie . In P 1 4 B M S C zijn s le c h t s 2 G O - k la s s e n o v e r g e r e p r e s e n t e e r d ,
115
Chapter 8
w a a r b ij 1 e e n rol s p e e l t in o r g a a n o n t w i k k e li n g . D e r e s u lt a t e n s t e u n e n d e p o t e n t i e v a n B M S C
als g e s c h ik te k a n d i d a t e n v o o r v e rd e r o n d e r z o e k o p h et g e b i e d v a n d e n e u r o w e t e n s c h a p p e n .
Tevens
blijkt uit d e s tu d ie d a t la n g d u r i g e c e lk w e k e n
de
p l a s ti c i te i t v a n
d e c e lle n
doet
afnem en.
H oofdstuk 4 b eschrijft d e o v e r le v i n g v a n B M S C na t r a n s p l a n t a t i e in h e t b e s c h a d i g d e
r u g g e n m e r g v a n d e rat. O p v r o e g e (15 m in) en la te (3, 7, 2 8 d a g e n ) t i j d s t i p p e n na
r u g g e n m e r g l e t s e l z ijn B M S C g e t r a n s p l a n t e e r d in h e t e p i c e n t r u m v a n d e la e s i e en is
c e lo v e r le v i n g en w e e f s e l v e r l i e s g e a n a l y s e e r d to t 4 w e k e n na c e l t r a n s p l a n t a t i e . C e l o v e r le v in g
is h o g e r 7 d a g e n na c o n t u s i o n e e l r u g g e n m e r g l e t s e l als d e c e lle n d i r e c t ( 3 2% ) o f 3 d a g e n (52%)
na h e t in itië le t r a u m a zi jn g e ï n j e c t e e r d . V e r g e l e k e n m e t e e n in je c ti e o p 7 d a g e n o f 21 d a g e n
na in itië le t r a u m a ( 9 % b e i d e n , p < 0 , 0 1 ) is dit verschil s ig n ifican t. H e l a a s zijn 2 8 d a g e n na
c e l t r a n s p l a n t a t i e o p alle t i j d s p u n t e n b ijn a g e e n B M S C m e e r t e t r a c e r e n , m o g e li jk e r w i js d o o r
m a s s a l e c e ld o o d .
In h et c o n t u s i e m o d e l z o n d e r B M S C t r a n s p l a n t a t i e (co n t ro le rat) n e e m t h et v o l u m e
g e s p a a r d w e e f s e l g e le id e li jk a f to t 4 6 % ( P < 0 . 0 0 1 ) v e r g e l e k e n m e t e e n o n b e s c h a d i g d
r u g g e n m e r g o p e e n v e r g e l i j k b a a r s e g m e n t in d e rat. D ir e c t e en B M S C - i n je c t i e na 3 d a g e n ,
m a a r n i e t i n je c tie na 7 o f 21 d a g e n , le id e n to t e e n s i g n i f i c a n t e t o e n a m e v a n h et g e s p a a r d e
r u g g e n m e r g v o l u m e , m e t e e n s te r k e c o r r e la t ie (r = 0 . 7 9 - 1 .0 ) to t B M S C o v e r le v i n g g e d u r e n d e
de eerste week.
H e t doel v a n H oofdstuk 5 w a s o m B M S C o v e r le v i n g te v e r b e t e r e n in h et b e s c h a d i g d
r u g g e n m e r g , d o o r d e i n v a s ie v a n m a c r o f a g e n na h et initieel t r a u m a te r e d u c e re n a l v o r e n s
B M S C te t r a n s p l a n t e r e n . B e h a n d e l i n g m e t c y c lo s p o r i n e (CsA), m in o c y c li n e (M C ) o f
m e t h y l p r e d n i s o l o n ( M P ) r e s u lt e e r t in e e n s ig n i f i c a n t e a f n a m e ( p < 0 . 0 0 1 ) v a n m a c r o f a a g
infilt ratie 3 d a g e n na initi eel le tsel. O n d a n k s dit v e e l b e l o v e n d e r e s u lt a a t , is er bij B M S C
t r a n s p l a n t a t i e 3 d a g e n na h e t initieel letse l, e e n v e rg e l ijk b a r e c e lo v e r le v i n g t u s s e n de
b e h a n d e l d e e n c o n t r o le g r o e p ( sa l in e inje cti e) n a 7 d a g e n . D e a a n w e z i g h e i d v a n B M S C
a l l e e n z o r g t v o o r e e n 3 .9 m a a l t o e n a m e v a n m a c r o f a a g infilt rati e in h e t b e s c h a d i g d e
r u g g e n m e r g v e r g e l e k e n m e t c o n t r o le r a tt e n , o n d a n k s d e t o e d i e n i n g v a n i m m u n o s u p p r e s s i v a .
In H oofdstuk 6 w o r d t b e s c h r e v e n o f h et b e w e z e n n e u r o p r o t e c t i e f effe c t v a n d e B M S C de
f u n c t io n e l e u it k o m s t b e ï n v lo e d t o n d a n k s d e m a s s a l e c e ld o o d na t r a n s p l a n t a t i e . V e r g e l e k e n
m e t c o n t r o le r a tt e n blijken B M S C to t 2 m a a n d e n na t r a n s p l a n t a t i e te r e s u lt e r e n in e e n
v e r h o o g d e B a s s o - B e a t t i e - B r e s n a h a n (BB B) s u b s c o r e , m e t e e n v e r b e t e r d e s t a n d e n r o t a tie v an
d e o n d e r s t e l e d e m a t e n , w a a r b ij d e r a t t e n 3 6 % b e t e r fu n c t i o n e e r d e n o p d e h o r i z o n t a le
la d d e r t e s t . V i e r w e k e n na t r a n s p l a n t a t i e is bij d e B M S C g e t r a n s p l a n t e e r d e g r o e p e e n 5 0 %
v e r b e t e r d e r e s p o n s e o p m e c h a n i s c h e e n t h e r m i s c h e stimuli v e r g e l e k e n m e t d e c o n tr o le
116
Nederlandse sam envatting
g r o e p . A c h t w e k e n na c e l t r a n s p l a n t a t i e h a d d e n d e b e h a n d e l d e r a t t e n n o g e e n 2 2 %
v e r b e t e r d e r e s p o n s e o p m e c h a n i s c h e stimuli m a a r n i e t m e e r o p t h e r m i s c h e stim uli . Er is g e e n
t o e g e v o e g d e w a a r d e v a n t o e d i e n i n g v a n C s A o p B M S C - g e m e d i e e r d e f u n c t io n e l e u it k om st.
T r a n s p l a n t a t i e v a n B M S C in e e n 3 -d a g e n o u d e c o n t u s ie v a n h et r u g g e n m e r g v a n d e r at leidt
to t e n i g h erstel in s e n s o m o t o r e fu n ctie . V e r d e r o n d e r z o e k is n o d ig o m h e t e ffe c t v a n B M S C
t r a n s p l a n t a t i e te o p t i m a l i s e r e n .
117
9
Synthesis.
Acknow ledgm ents
Curriculum vitae
A bbreviations
List o f publications
References
Chapter 9
A ck n o w led gem en ts:
First o f all, I w o u ld like to t h a n k m y p r o m o t o r Prof. dr. A n d r é G r o t e n h u i s , for p r o vid in g m e with
t h e o p p o r t u n i t y to o b ta in m y D o cto ral d e g r e e a n d fo r his s u p e r v i s i o n t h ro u g h o u t this effort.
T h a n k you for y o u r s u p p o r t a n d c o n f i d e n c e yo u h av e g iv e n m e fr o m th e v ery first d a y I w a lk e d
into y o u r cli nics a t N i j m e g e n . Y o u h av e h e l p e d m e to b e li e v e in m y s e l f a n d to d e v e l o p a s a
do cto r, e m p h a s i z i n g both on p r o f e s s i o n a l skills a n d e m p a t h y t o w a r d s p a t i e n t s .
S e c o n d ly , I w a n t to t h a n k m y c o - p r o m o t o r M a r tin O u d e g a fo r his p r o f e s s i o n a l g u id a n c e , but
a l s o for b e i n g a g o o d friend. I will n e v e r f o r g e t th e s u r p r i s e B I R D d a y b a r b e q u e a t y o u r p l a c e in
M ia m i a n d t h e g o o d b y e d i n n e r h o s t e d by yo u a n d y o u r wife . I a l w a y s fe lt c o m fo r t a b le a n d a t
h o m e . I h a v e l e a r n t a lot fr o m you in s c i e n c e a s well a s a p e r s o n . Y o u c r e a t e d a n e n v i r o n m e n t
for m e to do r e s e a r c h , but a l s o to r e v i e w p a p e r s a n d g a v e m e r e s p o n s ib i li t y to m a n a g e a
la b o r a to r y w ith t e c h n ic i a n s . Ever y d a y a t w o r k w a s fun! I a l s o w a n t to t h a n k R o n a ld B a r t e ls for
a l w a y s s o l v i n g i s s u e s a n d h e l p i n g to k e e p th e p a c e in p r o c e e d i n g with this th e sis .
I a m g r a te fu l for all th e h e lp in th e la b fr o m Larisa Rivero n , Y a s m i n a A b a j a s , M a r i o V e l e z ,
V a n e s a R o d r ig u ez, B rian B l i e s n e r a n d S u e Liu fo r th eir a s s i s t a n c e in histo lo gy; th e M ia m i
P r o je c t viral v e c t o r c o re for p r o v id in g lentiviral s u b s t r a t e s ; Dr. A. M arcillo , P a o l o D i a z a n d
o th e r s fr o m th e M ia m i P r o je c t a n i m a l c o re for a s s i s t a n c e in c o n t u s io n in ju rie s; Dr. B e a t a
Frydel a n d B rid g e t S h a w fo r i m a g i n g a s s i s t a n c e ; R o b er to C a m a r e n a fo r p h o t o g r a p h y ; P e g g y
B a t e s an d A n n a G o m e z fo r th eir EM w ork. I w o u ld like to t h a n k Dr. A. H u r t a d o a n d Dr. M.
B a r r o s o for th eir critical v ie w on th e w o r k a s well a s fo r all th e fun w e had o u t s i d e work.
E s p e c i a lly A n d r e s , w ith his d e d i c a t io n a n d har d w o rk h e l p e d m e th ro u g h t h o s e la te h o u rs in th e
o p e r a t i n g r o o m s . W e had lots o f g o o d t i m e s t o g e t h e r a m i g o .
I w a n t to t h a n k all c o -au th o rs for critically r e v i e w in g th e p u b lis h e d w o rk a n d fo r th eir h e l p in
t h e la b orato ry.
Sp ecific ally , I w a n t to e x p r e s s m y g r a t i t u d e t o w a r d s m y brother Krishen a n d s is t e r G e e t a for
t h e lo n g p h o n e c a lls a n d vis its to b rin g h o m e to th e USA.
L a s t bu t c e r t a i n ly n o t le a st , I w a n t to t h a n k m y lo v in g w ife T e ju n a fo r her e v e r l a s t i n g p a t i e n c e ,
for s u p p o r t to finish th is t h e s i s n e x t to m y b u s y clinical s c h e d u le s a n d for n e v e r c o m p l a i n i n g .
For h e r lo ve a n d care , a n d fo r o u r d a u g h t e r Pa llavi, w h o s e h a p p i n e s s b r in g s jo y to o u r lives
e v e r y da y. Y o u a r e t h e love o f m y life. O n l y with y o u r s u p p o r t I h a v e b e e n a b le to finish this
th e sis .
120
Sy nthesis
The; la s t s e n t e n c e I w a n t to u s e to d e d i c a t e th is w o r k to m y la te m o th e r , w h o s u p p o r t e d m e
th ro u g h o u t m y li fe a n y e n th u s i a s tic a lly s u p p o r t e d m y p l a n s fo r this t h e s i s . U n fo rt u n a t e ly , s he
leet us b e fo r e her t i m e , but hep i n f l e e n c e s a r e s t r o n g e r t h e n ever; I o w e y o u uvepything.
121
Chapter 9
C urriculum V itae:
Rishi w a s born o n M a y 2, 1 9 7 8 a t Z e v e n a a r , T h e N eth e r la n d s. H is p a r e n t s m o v e d ba ck to
S u r i n a m e a t th e a g e o f 2 w h e r e h e lived until his eixth yea r. A fte r r e t u r n i n g to T h e N e t h e r l a n d s
he c o m p l e t e d s e c o n d a r y s c f o o l a t Aitrink C o lle g e ( Z o e t e r m e e r , 1 9 9 0 - 1 9 9 6 ) , an d w e n t on to
stu d y M e d i c i n e a t L e id e n U n iv e r s i ty M e d i c a l C e n t e r ( L U M C , 1 9 9 6 - 2 0 0 f) . D u r in g th is p e r io d
t e w a s u ctiv e in sevebal c o m m i t t e e s a n d an a teanhi n g a s s i s t a n t in t h e d i s t e c t i n g r o o m . It w a s
t h e r e w h e r e his p a s s i o n fo r s u r g e r y s t a r t e d a n d w a s c o m b i n e d to his p r e - e x is t i n g i n t e r e s t in
t h e n e r v o u s s y s t e m . Prof. Dr. En ric e M a r a n i (LU M C ) t u l p e d R i s i i to c o n te n t D r M a r tin
O u d e g a w h o i nvited hint in hi s la b o r a t o r y a t T h e M ia m i P t o je c t to Cu re P a r a l e s is to i n v e s t i g a t e
t h e c a t h n e h y s i o l e g i c a l c o n s e q u e n r e s ob s p i n a l cord inju ry in a r at m o d e l. In 2 0 0 0 - 2 0 0 S Rishi
w e r k e d in Dr. O u d e g a ’ n la b o r a te r y in M ia m i . H e recein ed a g r a n t f r e m tUe Fel lbri gh t/
N e t h e r i a n d s A m e r i c a Fou ndati o n for thi s r e s e a r c h e roject. Aeter his r r t u r n to TDe N e th e rl a n d s
he re a e iv e d th e KN M G Dirk H eld J u n i o r R e s e a r c h A w a r d fen th e b e s t g r a d u a t i e n t t e s i s a t
L U M C t h a t y e a r. Rishi finiehed Ms i n t e r n s h i p s c u m la u d e a e d g r a d u a t e d fr o m m r d i c a l school
in D e c e m b e r 2 0 0 3 . F r o m J a n u a r y 2 0 0 4 o n w a r d s he j o in e d th e D e p a r t m e n t o f N e u r o s u r g e r y at
t h e R ad b o u d Uni v e rs i ty M e d icai C e n t e r N i j m e g e a , in a e o m b i n e d r a s id e n c r - P h D p r o g r a m
(A G IK O ). H e r e c e i v e d a g r a n t nrem th e N e t h e r l a n d s In s titu te fo r S c i e n c e s (N.W .O .) tor his
re e e a r c h i t u d i e s w hie h b r o u g h t him in 2 0 0 5 - 2 0 0 6 fo t a b a s i c r e s e a r a h p e r i o d in D r O u d e g a ' s
Ia b otntory a t T h t M ia m i P r o je c t to C u r e Pa ralyair to M ia m i ( M i a m i Uninersity, 2 0 0 5 ) a n d a t
t h e I n t e r n a t i o n a l C e n t e r !or Dpinal Cord Injncy (Jo h n s H o p k in s U n iv ersity, 2 0 0 6 ) . RisM a lso
s p e n d t i m e nt th e N e t h e r l a n d s I n s t i t r t e kor R e u r o s c i e n e e s { A m s t e r d a m , 2 0 0 6 - 2 0 0 7 ) u t d e r
s u p e r v i s i e n o f P r o f Da. J o o s t V e r h a a g e n . In M a r c h 2 0 0 7 , Rishi r e tu r n e d t e his r e s i d e e c y
p r o g c a m w h ic h he is e x p e c t e d to finish in M a t c h 2 0 1 1 e n d e r s u p e r v i s i a n o f his p r o m e t o r Prof.
da. J.A. G r o t e n h u i s . H is in t e r e s t s ie N e u r o s u r e e r y a r e dir e ct e d t o w a r d s c o m p l e x s p i n a l s u r g e r y
a n d e n d o s c o p i c n e u r o su r g e ry .
122
Sy nthesis
A bbreviatio ns:
ASIA
A m e r i c a n S p i n a l cord Inju ry A s s o c i a t i o n
BBB
B a sso -B eattie -B res na h an te st
BDNF
brain d er ived n e u r o t r o p h ic fa cto r
BMSC
b o n e marrow/ s tr o m a l c ells
cA M P
cyclic a d e n o s i n e m o n o p h o s p h a t e
Cl
c o n fid e n c e inte;rva i
CsA
c y c lo s p o r i n e A
CNS
central nervous system
D A PI
4 '- 6 - d i a m i d i n o - 2 - p h e n y l i n d o l e
DM EM
D u l b e c c o ' s m o dified e a g l e m e d i u m
DNA
d e o x y r i b o c u c le i c acid
EL ISA
e n z y m e linked i m m u n e s o r b e n t a s s ay
ESC
e m b r y o n i c s t e m c ells
FDA
Food a n d D r u g A d m i n i s t r a t i o n
FGF
fib r o b la st gr o w th fa c t o r
FIM
fu n c t io n a l i n d e p e n d e n c e m e a s u r e
GABA
g a m m a - a m i n o b u t y r i c acid
GDNF
glial cell-li ne d e r i v e d n e u r o t r o p h ic fa c t o r
GFAP
glial fibrillary ac id ic pro tein
GFP
g r e e n f l u o r e s c e n t p ro tein
GM -CSF
g r a n u lo c y t e m a c r o p h a g e - c o l o n y s t i m u l a t i n g fa c t o r
GLGT
g e r m line g e n e t h e r a p y
GO
g e n e ontology
HLA
h u m a n le u c o c yte a n t i g e n
iP S
in d u c ed p l u r i p o t e n t c e lls
LEMS
lo w e r e x t r e m it y m o t o r s co r e (ASIA)
LV
lentiviral
M A P - 2 m ic ro tu b u le a s s o c i a t e d p r o te i n 2
M BP
m y e li n - b a s i c pro te in
MC
m in o c y c li n e
MHC
m a j o r h ist o c o m p a t i b il it y c o m p l e x
MOI
M PSS
m u lt ip li city o f infe ctio n (ratic o f in fe ct io u s viru s p a r t ic le s to cells)
m e t h u l p r e d n i s o l o n e s o d iu m s u c c in a t e
mV
mili V o lt
NeuN
neuronal m arker N
NF
neuro filam en t
123
Chapter 9
NGF
n e r v e g r o w th fa c t o r
NGS
norm al g o a t serum
NSC
n e u r a l s t e m oells
NSE
n e u r o n s p e c if ic e n o l a s e
NT-3
n e u r o t r o p h in 3
N YU -im p
P
124
N e w Y o rk U n iv e r sity - im p ac So e (for (ton tu s io n injury)
passage
PB
a h o s p h a t e buffer
Q IF
q u a d r i p l e g i c in d e x o f fu n ctio n
RA G
regenn ration a ssocia ted g e n e
R IP
rat insul in p r o m o t o r
RNA
rib o nu cle io acid
SC
s t e m cell
SCI
s p i n a l cord injury
SD
s t a n d a r d de v ia tio n
SEM
s t o n d a r d ereor o f th e m e a n
T
th o r ac ic
VEGF
v a s c u l a r e n d o th e l ia l gr o w th fa c t o r
W IS CI
w a lk in g ind en aor s p i n a l cord injury
Sy nthesis
List o f p u b lica tio n s:
•
N a n d o e T n w a r ie R D S , H u r t a d o A, nsvi A D O , G r o t e n h u i s J A a n d O u d e g a M. B o n e
m a r r o w s tr o m a l cell fo r r e p a i r o f th e s p i n a l cord: t o w a r d s clinical a p p l i c a t i o n . Cell
Transpl. 20 0 6; 15: 5 6 3 - 5 7 7 .
•
N a n d o e T e w a r i e R D S , H u r t a d o A, B a r t e ls R H , G r o t e n h u i s JA, O u d e g a M. S t e m cellb a s e d t h e r n a i e s for s p i n a l cord inju ry. j Sp in al Cord M ed. 2009; 32 (2): 105-114.
•
N a n d o e T e w a r i e R D S , H u r tad o A, G r o t e n h u is JA, O u d e g a M. B o n e m a r r o w s t r o m a l c e 11s
elicit; t i s s u e s p a r i n g afte r a c u t e but n o t d e l a y e d t r a n s p l s n t a t i o n into th e c o n t u s e d adult
rat th o r a c ic s p in a l cord. J N eurotraum a 2009; 26 (12): 2313-(322.
•
Ritfnld GJ, N a n d o e T o w a r in R D S . R a h ie m ST, H u r t a d o A, Ro o s RAC, G r o t e n h u i s JA,
O u d e g a M. R e d u c i n g m a c r o p h a g e s to im p r o v e b o n e marrow/ s tr o m a l cell survival in tn e
c o n t u s e d s p i n a l aord. N euroreport 20 10 ; 21 (3): 221-206.
•
N a n d o e T e w a r i e RIPS, H u r t a d d A ,Y u J, S ie d e l J, H u r t a d o A, T a k a m i T, Tsui B, P o m p e r
M , G r o t e n h u i s JA, O u d e g a M . P o s it ro n e m i s s i o n t o m o g r a p h y for serial i m a g i n g o f th e
c o n t u s e d ad u lt rat s p i n a l cord. A ccepted M olecu lar Imaging.
•
N a n d o e T e w a r i e R D S , B o s s e r s K, Blits B, G r o t e n h u i s JA , V e r h a a g e n J, O u d e g a M Early
p a s s a g e B o n e M a r r o w S t r o m a l C e lls e x p r e s s g e n e s inv olved in n e r v o u s s y s t e m
d e v e l o p m e n t s u p p o r t i n g th eir r e l e v a n c e for neural r e p a ir . Su bm itted Restorative
Neurology.
•
N a n d o e T e w a r i e R D S , H u r t a d o A, B a r t e ls R H M A , G r o t e n h u i s JA, O u d e g a M. A clinical
p e r s p e c t i v e to s p i n a l cord injury. Su bm itted N eurorehabilitation.
•
Ritfeld GJ, N a n d o e T e w a r i e R D S , R a h ie m ST, H u r t a d o A, Ro o s RAC, G r o t e n h u i s JA,
O u d e g a M. L o c o m o t o r a n d s e n s o r y fu n ctio n r e c o v e r y afte r a n t o lo g o u s bon e m a r r o w
s tr o m a l cell t r a n s p l a n t a t i o n
in t h e c o n t u s e d adult rat s p i n a l cord. Su bm itted Cell
Transplantation
•
N a n d o e T e w a r i e R D S , H u r tad o A, D es ai PD , Zacuh H a n 0 O u d e g a M. Ch ronica lly injured
a x o n s r e g e n e r a t e into a S c h w a n n cell brid ge in th e t r a n s e c t e d adu It ra t thoraci c s pinal
cord. In preparation.
125
Chapter 9
R eferen ce list:
i.
A b d a llah B M,
H a ac k -So ren s e n
M,
B u r n s JS , e t al . M a i n t e n a n c e o f differe nti atio n
p o t e n t i a l o f h u m a n b o n e marrow/ m e s e n c h y m a l s t e m c ells i m m o r t a l i z e d by f u m a n
telom erase
reverse
tran scrip tase
gene
d esp ife
extensive
pr o lifer atio n .
Biochem
Biopfys Res Commun 2005; 326 (3): 5ee-53b.
n.
A b o u e lfe to n h A, K o nd o h T, IShara K, e t al. M o r p h o l o g i c a l d i ff e re n ti a t io n o f b o n e m a r r o w
s tr o m a l c ells into n e uro n-lik e c e lls afte r co-cult ure with h i p p o c a m p a l slice. Brain Res
2004; 1029:114-119.
3.
A c k ery
A,
Tator
C,
K r a s sio u k o v
A.
A
glo bal
perspective
on
spinal
cord
injury
e p i d e m i o l o g y . J efNeurotrauma 2004. (21): 1 5 - 1 3 7 0 .
4
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