Title: Treatment of brain
United States Patent: 7,247,298
Issued: July 24, 2007
Inventors: Hodges; Helen
Assignee: ReNeuron Limited
Appl. No.: 10/342,692
Filed: January 14, 2003
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The present invention relates to the
treatment of brain damage by cellular transplantation. According to one
aspect of the invention, a method for treating a motor, sensory and/or
cognitive deficit comprises administering a composition comprising
pluripotent cells into the damaged brain in a region contra-lateral to
that containing the site of damage. The cells are preferably conditionally
SUMMARY OF THE
It has now been realised that pluripotent
cells can successfully repair damage when administered into the side of
the brain contra-lateral to that containing the site of damage.
Therefore, according to one aspect of the invention there is a method for
treating brain damage comprising administering a composition comprising
pluripotent cells into the damaged brain, wherein administration is into
the brain hemisphere contra-lateral to that containing the site of damage.
Preferably, the pluripotent cells are neuroepithelial stem cells, in
particular, those from the MHP36 clonal cell line, defined herein.
The cells are preferably conditionally immortal. Immortalisation may be
achieved by the transduction of a temperature-sensitive oncogene into the
cells as disclosed in WO-A-97/10329.
The advantage of administering the cells contra-laterally is that the
intact (contra-lateral) region may provide a more tolerant environment for
cell grafts, avoiding the inflammatory response at the site of damage
which might cause cell rejection.
DESCRIPTION OF THE
The cells of the present invention are
capable of correcting a sensory, motor and/or cognitive deficit when
implanted into the brain hemisphere contra-lateral to that of the damaged
part of the human brain. The term "damage" used herein includes reduction
or loss of function caused by cell loss. Damage may be caused by any of a
variety of means including physical trauma, hypoxia (lack of oxygen),
chemical agents, for example, damage may be caused by drug abuse, and
disease. The following diseases and pathological conditions are examples
of diseases or conditions which result in deficits which may be treated in
accordance with the present invention: traumatic brain injury, stroke,
perinatal ischaemia, including cerebral palsy, Alzheimer's, Pick's and
related dementing neurodegenerative diseases, multiple sclerosis,
multi-infarct dementia, Parkinson's and Parkinson's-type diseases,
Huntington's disease, Korsakoff's disease and Creuzfeld-Jacob disease.
Amnesia, particularly following transitory global ischaemia such as after
cardiac arrest or coronary bypass surgery, may also be treated in
accordance with the present invention.
The present invention is particularly suited to the treatment of stroke
where damage occurs primarily in one brain hemisphere e.g. due to an
occlusion in the middle cerebral artery.
By "contra-lateral" it is intended that this refers to the hemisphere of
the brain that does not contain the site of damage. Therefore, if there is
an occlusion in the left hemisphere, then, obviously, the contra-lateral
region is the right, undamaged, hemisphere.
Of course, in some instances damage may occur in both hemispheres, and in
these cases the contra-lateral region is understood to be the hemisphere
which exhibits least damage.
The term "pluripotent" is used herein to denote an undifferentiated cell
that has the potential to differentiate into different types or different
phenotypes of cell, in particular into cells having the appropriate
phenotype for the intended use. The cell type or phenotype into which such
a pluripotent cell finally differentiates is at least partly dependent on
the conditions in which the cell exists or finds itself.
For use in the present invention the cells should be capable of
differentiating into cells appropriate to repair or compensate for damage
or disease in the target area of the brain. It will be appreciated that
cells for transplantation need not be capable of differentiating into all
types or phenotypes of neural cells. The cells may, for example, be
bipotent. However, a high degree of potency is generally preferred as this
gives greater flexibility and potential for transplantation into different
areas of the brain.
Suitable pluripotent cells include those known in the art as "stem cells"
and those called or known as "precursor cells". In particular,
neuroepithelial stem cells are suitable for use in the present invention.
However, other cells may also be used. Alternative cells may be those
defined as haematopoietic stem cells which may be capable of
differentiating into neural cells.
The pluripotent neuroepithelial cells are advantageously, and will
generally be, conditionally immortal and may be prepared as disclosed in
The treatment may be carried out on any mammal but the present invention
is especially concerned with the treatment of humans, especially treatment
with human cells, and with human cells and cell lines.
To treat a patient it is necessary to establish where damage has occurred
in the brain. This may be carried out by any method known in the art, e.g.
magnetic resonance imaging (MRI). Once the existence of damage has been
established, whether it be in one isolated area or in several areas,
treatment by implantation of cells into the contra-lateral region to that
of the damaged area may be carried out, again by conventional means. The
pluripotent cells may be transplanted at a single site, or preferably at
multiple sites, and may be able to migrate to the site(s) of damage and,
once there, differentiate in response to the local microenvironment, into
the necessary phenotype or phenotypes to improve or restore function.
In addition to administering the cells into the contra-lateral region, it
may also be desirable to co-administer the cells into the damaged
hemisphere (ipsi-lateral region). Treatment in this manner may promote the
improvement or restoration of brain function by different mechanisms.
Without wishing to be bound by theory, it may be that repair following
transplantation into the contra-lateral region results from migration of
the pluripotent cells into the area of damage, with the reconstitution of
local circuits to restore or sustain function. It may also be that the
transplanted cells augment spontaneous processes within the intact
(contra-lateral) side which attempt to compensate for the damage. If the
latter is correct, then it may be unnecessary for the transplanted cells
to cross to the side of damage to exert an effect.
It may be possible to promote repair by encouraging the activity of
particular regions of the brain. By using passive or active exercise of
certain regions, it may be possible to augment the spontaneous processes
occurring after transplantation. For example, it should be possible to
stimulate particular brain regions by requiring certain tasks to be
performed. In doing so, the brain region may generate biological signals
that aid repair.
The stimulation of the brain may be visualised using detection techniques
such as magnetic resonance imaging (MRI). These techniques can be adapted
to permit the patient to visualise the active brain regions, so that,
through the process of biofeedback, the patient can stimulate particular
regions that may encourage repair.
Preferably, treatment will substantially correct a motor, sensory and/or
cognitive deficit. However, that may not always be possible. Treatment
according to the present invention and with the cells, medicaments and
pharmaceutical preparations of the invention, may lead to improvement in
function without complete correction. Such improvement will nevertheless
be worthwhile and of value.
The number of cells to be used will vary depending on the nature and
extent of the damaged tissue. Typically, the number of cells used in
transplantation will be in the range of about one hundred thousand to
several million. Treatment need not be restricted to a single transplant.
Additional transplants may be carried out to further improve function.
Methods for transplantation of cells into humans and animals are known to
those in the art and are described in the literature in the art. The term
"transplantation" used herein includes the transplantation of cells which
have been grown in vitro, and may have been genetically modified, as well
as the transplantation of material extracted from another organism. Cells
may be transplanted by implantation by means of microsyringe infusion of a
known quantity of cells in the target area where they would normally
disperse around the injection site. Suitable excipients and diluents will
be apparent to the skilled person, based on formulations used in
conventional cell transplantation.
The following non-limiting example illustrates the invention.
Conditionally immortal pluripotent neuroepithelial cells from the MHP 36
clonal cell line were prepared as disclosed in WO-A-97/10329.
21 Wistar rats were subjected to left intraluminal occlusion of the middle
cerebral artery (MCAo-IL) under halothane anaesthesia as disclosed in
Ginsberg et al, Cerebrovascular disease, 1998; Volume 1: 14-35.
Following exposure of the left internal carotid artery, a 3.0 mm proline
filament coated at the tip with silicon was inserted 18-20 mm up to the
junction of the circle of Willis and tied in place for 60 min. Anaesthetic
was discontinued after insertion of the filament, and the rat tested for
neurologic deficit (contra-lateral paw flexion and circling) to establish
the presence of ischaemia. Anaesthesia was resumed after 60 min for
retraction of the filament to the external carotid stump, where it was
left in place, the excess filament trimmed off, and the wound sutured.
Neurologic and health status were monitored for a week, until normal
feeding was seen and post-operative weight regained. Control rats (n=11)
were sham operated by exposure of the left internal carotid artery only.
Transplant and sham graft surgery was undertaken 2-3 weeks after occlusion
or sham surgery. Rats were anaesthetised with Immobilon (0.01 ml/100 g, im)
after pre-treatment with Hypnovel (midazolam: 0.03 ml/100, im), and placed
in a stereotaxic frame. Holes were drilled in the right side of the skull
to allow the penetration of a 10 .mu.l Hamilton syringe at the following
coordinates (mm) derived from Bregma, with the skull in the flat position
(-3.2 mm). AP represents "anterior-posterior", L represents "lateral" and
V represents "ventral".
TABLE-US-00001 AP: -0.3 L: -3.5 V: -4.5, -6.0 L: -5.5 V: -4.0, -5.5 AP:
-1.3 L: -3.0 V: -5.0, -6.5 L: -5.5 V: -5.0, -6.5
3.0 .mu.l of suspension (25,000 cells/.mu.l) were infused over 2 min at
each of the 8 sites, and the cannula was left in place for a further 2 min
to allow diffusion from the tip. Medial descents were aimed for striatum
and lateral descents were aimed for cortex at the estimated anterior and
posterior extent of the area of infarct on the opposite side, to target
potential regions of reorganisation. Controls received infusions of
Rats were tested from 4-6 weeks after transplantation over a period of 10
months. During this time the performance on the repeated measure test
(Bilateral Asymmetry Test) remained stable.
Bilateral Asymmetry Test
The bilateral asymmetry test (BAT) has been used to access a variety of
lesions (Schallert et al, Pharmacol. Biochem. Behaviour, 1982;
16:455-462). Strips of tape 1 cm wide and 5 cm long were wound round each
of the two forepaws, in random order. Animals were placed in an
observation cage and timed for latency to contact and to remove each tape.
The side first contacted was also noted by the Experimenter blind as to
the experimental groups. Random checks on reliability were included by
comparing the Experimenter's scores with those of a second observer;
typically inter-rater reliability was above 90%. Rats were tested before
surgery, and during the week before grafting, to establish pre- and
post-operative baselines. One session of 4 tests of 3 min was carried out
each week, for 18 weeks, commencing 4-6 weeks after transplantation to
assess long term recovery.
This is a test for measuring rotational bias. When challenged with
amphetamine, MCAo animals show a clear bias to the direction of the
Spontaneous and drug-induced rotation was measured approximately 38 weeks
after transplantation in an 8-bowl rotameter (TSE GmbH, Bad Hamburg) in
which rats were harnessed for 30 min and swivels recorded in either left
or right direction. Rats were tested for response to saline (baseline).
They were then tested once a week, on alternate weeks, with either
amphetamine (Sigma: 2.5 mg/kg) or apomorpine (Sigma: 0.5 mg/kg) on three
occasions over a testing time of 6 weeks. All injections were given in a
volume of 1.0 ml/kg, ip.
Before occlusion, rats did not show a mean difference in paw use as judged
by the latency to contact and removal of tapes from the left and right
paw. After sham surgery the controls continued to show no preference.
However, rats subjected to MCAo showed a marked disparity between the two
paws, with the right paw contacted and the tape removed significantly more
slowly than the left, indicative of contra-lateral sensorimotor
impairment. This robust and stable deficit persisted throughout the 18
weeks of testing. In rats that received MHP36 grafts, the stroke-induced
forepaw disparity was non-significant by 8 weeks after transplantation,
and this improvement persisted through the 18 weeks of testing, so that
there was no difference between paws. Hence grafted rats did not differ
from controls as both groups showed that the two paws were equivalent in
latency to contact and remove the tape, and that the right paw was
contacted first as often as the left. In a group of rats that were
subjected to 60 min MCAo and which did not receive sham transplants, the
paw disparity was comparable to that in the group injected on the intact
side with a large volume of vehicle. This result suggests that injection
damage on the intact side neither exacerbated nor reduced the extent of
sensorimotor deficit induced by MCAo.
Baseline (spontaneous) rotation was mildly asymmetrical in stroke rats
without grafts, in that there was more turning to the right than the left,
whereas control and grafted rats showed comparable turning in both
directions. However, in response to amphetamine on weeks 2, 4, and 6 of
testing, stroke rats without grafts showed marked turning to the left,
towards the lesioned side, indicative of asymmetric dopamine release on
the intact side. Group differences were very substantial and the non
grafted group differed significantly from the grafted and control groups,
which did not differ in response to amphetamine. A similar, but less
marked effect, was seen with the postsynaptic dopamine agonist apomorphine.
Groups differed on weeks 3, 5 and 7 with the non-grafted stroke group
showing more marked leftwards rotation than the grafted and control groups
which did not show a turning bias. In all groups the number of turns was
lower in response to saline than to the dopamine agonists. However all
groups showed a similar activation in response to drug, so that drug
induced changes in bias in the non-grafted group were not associated with
differences in activity.
At the end of behavioural testing (approximately 11 months
post-transplantation) histology studies were undertaken. Rats were
perfused with 4% paraformaldehyde, flushed through the upper body
vasculature via a cannula inserted through the heart and into the aorta,
which was attached to a pump. 50 .mu.m coronal sections were cut through
the brain, placed on gelatine-coated slides and frozen to prevent
dehydration. Serial sections were stained for the presence of .beta.-Gal
labelled cells, and cells reactive to glial fibrillary acidic protein (GFAP)
and tyrosine hydroxylase (TH) to identify glial and neuronal cell types
within the graft.
In a first study, only one brain from the grafted and non-grafted groups
subjected to MCAo was examined. In both animals subjected to MCAo,
cavitation was severe, amounting to approximately 75% of the hemisphere
volume. Ventricles on the infarct side were enlarged, so that only a thin
strip of striatal tissue separated the lateral from the infarct.
Ventricles were also enlarged to a lesser extent on the intact side, and
distortion, possible via oedema soon after occlusion, had pushed the
midline towards the intact side. In the grafted animal .beta.-Gal positive
cells were seen at the injection site in the middle of the intact
striatum. Cells were also seen in a diagonal band stretching caudally and
laterally through the striatum towards the parietal cortex. .beta.-Gal
positive cells were seen approaching and within the side opposite to
implantation. They formed in a dense band along the lower ventricular
margin of the corpus callosum, and encircled the area of the infarct. They
were particularly prominent in the residual strip of striatum, and some
had left the corpus callosum caudally to enter the cortex. These grafted
cells showed morphologies of several types, including bipolar cells, glial
cells and neuronal cells of both pyramidal and medium spiny neuron
appearance, suggesting a diverse pattern of differentiation.
For estimating lesion volume 50 .mu.m sections were cut from 3.7 to 6.3 mm
before bregma in all rats subjected to MCAo, with and without grafts, and
intact controls. Every tenth section was collected giving an inter-section
distance of 500 .mu.m and a total of 20 sections per brain. Sections were
strained with Cresyl Fast Violet. Images of each section were taken using
a stereo microscope and estimations of lesion volume were carried out. For
control rats there was no difference between the hemispheres. In rats
subjected to 60 min of MCAo there was a substantial infarct representing
approximately 26% of the total brain volume. In stroke rats with MHP36
grafts, lesion volume was decreased to approximately 16% of the total
brain volume, and was significantly smaller (p<0.05) than in rats with
MCAo and sham grafts.
A later study compared the effect of grafts into either the ipsi-lateral
or contra-lateral sides, or into the ventricles. Grafted cells were
labelled with the fluorescent marker PKH26 to assist their identification.
Behaviour was measured for 12 weeks after transplantation. Over this
period there was a significant improvement in the bilateral asymmetry test
in rats with grafts in both the ipsi-lateral and contra-lateral side, but
not in those with intraventricular grafts. However, rats with
intraventricular grafts, unlike those with intraparenchymal grafts, showed
improved spatial learning and memory in the water maze. These results
indicate that the site of grafting affects behavioural recovery and
support the claim that use of multiple sites may be advantageous. In
contrast to the finding in the earlier study, where contra-lateral grafts
restored spontaneous and amphetamine-induced rotation in animals tested 10
months after transplantation, there was no improvement in rotation bias in
any of the grafted groups tested 10-12 weeks after grafting. These
findings suggested that the time course of recovery may differ for
The brains of all the rats were processed for histology as described
above. Grafted cells were visualised by PKH26 fluorescence, and double
labelled with antibodies to neuronal and glial markers to identify cells
that differentiated into these phenotypes. Site of implantation influenced
cell survival and the pattern of migration. In general, more cells
survived with grafts implanted contra-lateral to the lesion than into the
ventricles, with ipsi-lateral grafts being intermediate. However, there
was a similar proportion of neuronal cells, seen primarily in the midline
regions, in all granted groups. Importantly, grafts from all three sites
migrated across the midline to the opposite side of the brain. Thus about
a third of grafted cells placed in the lesioned side were found in the
intact side of the brain, whilst a similar proportion of grafted cells
also migrated from the intact to the lesioned side, as in the earlier
study. These surprising findings indicated that grafted cells not only
responded to signals arising from injury, but were also attracted to the
intact side, possibly by signals arising from processes of reorganisation.
Lesion volume, measured as described above, showed that the area of damage
comprised approximately 18% of the total brain volume. However there was
no difference between sham grafted animals with MCAo, and those with
grafts. Thus grafts did not significantly reduce lesion volume, measured
14 weeks after transplantation, in contrast to the reduction seen at 11
months after transplantation in the earlier study. This may indicate that
grafts give some protection against secondary degeneration, and the effect
is only clearly evident at a late time point.
In a later study, the remaining brains were sectioned and the lesion
volumes determined for each group by measuring the volumes of the ipsi-lateral
and contra-lateral hemisphere. Volume measurements of the lesion size
revealed 18% loss of total brain volume in animals with 60 minutes MCAo.
No difference in lesion size was found between the MCAo groups regardless
of transplantation site. When the total number of transplanted cells was
analysed according to implantation site, it emerged that grafted cells
implanted contra-laterally were significantly greater than those grafted
in the ventricles, although there was no statistical difference when
compared to cells grafted ipsi-laterally. It was also apparent that there
had been extensive migration away from the implantation site into the
The above experiments aimed to see whether grafts of MHP36 cells, from a
conditionally immortalised clonal line, would promote functional recovery
from stroke damage when placed in the intact hemisphere contra-lateral to
the infarct cavity. The findings indicated that both sensorimotor and
motor asymmetries were normalised in rats with grafts initially sited in
the intact hemisphere.
The evidence for recovery of sensorimotor and motor functions is robust,
because improvements were seen over an extended time period. For example,
in MCA-occluded rats without grafts, tape-removal deficits were seen
consistently over an 18 week period of testing, with no hint of
spontaneous recovery. Improvement in grafted rats was also consistent over
this period. The late rotational data is interesting in that spontaneous
deficits were manifest in mild rotation to the right, possibly reflecting
a stronger push by the unaffected left paw, relative to the right.
However, dopamine agonist drugs induced marked rotation to the left,
consistent with activation of dopamine receptors on the right (intact)
side of the brain. This asymmetry was not evident in rats with grafts
sited in the intact side, even though one might expect the asymmetry to be
amplified, if grafted cells on the intact side differentiated into
tyrosine hydroxylase-positive (TH) neurons.
The control, grafted and non-grafted stroke groups showed a similar degree
of locomotor stimulation in response to amphetamine (i.e. the number of
rotations increased substantially above baseline) so that the drug
affected activity in all groups. If the normalisation of rotation bias in
the grafted group reflected lesion-induced insensitivity to dopamine
stimulation on both sides of the brain, one would not expect to see such a
marked increase in rotation in response to amphetamine. Therefore it is
reasonable to suppose that MHP36 grafts normalised rotational behaviour by
providing dopaminergic inervation on the side of the infarct. However,
since this is a late effect, some other compensatory mechanism, possibly
involving reduced retrograde degeneration, cannot be excluded.
There was some evidence of grafted cells on the side of implantation, not
only around the sites of injection, but also forming a ventral stream of
migration through the striatum. Thus it may be premature to conclude that
grafted cells exert functional effects only if they cross to the side of
damage. They may also be involved in reorganisation of the intact
hemisphere. This conclusion is supported by finding that cells implanted
on the lesion side migrated to the intact contra-lateral side.
Claim 1 of 11 Claims
1. A method for treating brain damage in
a mammal, comprising administering pluripotent neuroepithelial cells into
the mammal's damaged brain, wherein administration is into the brain
hemisphere contra-lateral to that containing the site of damage, wherein
said pluripotent neuroepithelial cells have been genetically modified to
be conditionally immortal, wherein said pluripotent neuroepithelial cells
are immortal prior to administration and differentiate after
administration, and wherein administration of said pluripotent
neuroepithelial cells improves said brain damage of the mammal.
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