Title: Neural transplantation
using pluripotent neuroepithelial cells
United States Patent: 7,048,921
Issued: May 23, 2006
Inventors: Sinden; John
(London, GB); Gray; Jeffrey A. (London, GB); Hodges; Helen (London, GB);
Kershaw; Timothy (London, GB); Rashid-Doubell; Fiza (Oxford, GB)
Assignee: ReNeuron Limited
Appl. No.: 760274
Filed: January 12, 2001
The subject invention pertains to a novel
method of correction of behavioral and/or psychological deficits made
possible by the intracerebral transplantation of pluripotent
neuroepithelial cells. Cells, cell lines, pharmaceutical preparations,
medicaments, methods for the production and maintenance of the cell lines
for use in the method of the invention are encompassed by the invention.
Description of the Invention
The present application relates to the
correction of behavioural and/or psychological deficits by the
intracerebral transplantation of neural cells, and to cells and
medicaments therefor. The invention also concerns methods for the
production and maintenance of the cell lines.
(a) obtaining neuroepithelial cells from
a human fetus, the cells being at a stage early enough in the
developmental pathway that they have the ability to differentiate into a
variety of different brain cell types,
(b) introducing into those cells DNA
which comprises a sequence capable of causing the cells to be
conditionally immortal under the control of appropriate control elements,
(c) maintaining the cells in vitro under
Behavioural and/or psychological deficits are caused by many diseases and
may also be caused when the brain undergoes trauma. For example, motor
dysfunction is one symptom of Parkinson's disease. As yet, in most cases,
there is no satisfactory treatment available.
The present invention provides for a method of treatment of a behavioural
and/or psychological deficit which comprises intracerebral transplantation
of a therapeutically effective amount of pluripotent neuroepithelial
The present invention is based in part on the observation that, when
transplanted into a damaged or diseased brain, pluripotent neuroepithelial
cells appear to respond to signals from the damaged or diseased brain by
taking up a phenotype that is able to replace or compensate for functional
deficits to which the damage or disease otherwise leads.
The term "pluripotent" is used herein to denote a an undifferentiated
neuroepithelial 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
For use in the present invention the neuroepithelial 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
differentiation 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 called or known as "stem cells"
and those called or known as "precursor cells".
The pluripotent neuroepithelial cells are advantageously, and will
generally be, conditionally immortal.
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.
The present invention provides a mammal which has undergone treatment
according to the present invention.
The present invention provides isolated human, pluripotent neuroepithelial
The present invention especially provides human, conditionally immortal
pluripotent neuroepithelial cells.
The present invention further provides a conditionally immortal,
pluripotent, neuroepithelial cell line, especially for therapeutic use,
more especially for the treatment of a behavioural and/or psychological
The cells of the present invention are capable of correcting a behavioural
or psychological deficit when implanted into a damaged part of the human
brain. The term "damage" used herein includes reduction or loss of
function. 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 behavioural and/or psychological 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, 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
The present invention further provides a process for the production of
human, conditionally immortal, pluripotent, neuroepithelial cells which
comprises the steps of:
The process may further include the step of cloning the cells to obtain
one or more cell lines.
A further aspect of the invention provides for pluripotent,
neuroepithelial cells, optionally in isolated form, for therapeutic use,
especially in humans. The therapeutic use may be treatment of a
behavioural and/or psychological deficit.
A further aspect of the invention provides for conditionally immortal,
pluripotent, neuroepithelial cells for therapeutic use, especially in
humans. The therapeutic use may be treatment of a behavioural and/or
The present invention further provides for the use of pluripotent,
neuroepithelial cells, optionally in isolated form, in the manufacture of
a medicament for the treatment of a behavioural and/or psychological
deficit. The medicament to be administered comprises pluripotent
The present invention especially provides for the use of conditionally
immortal, pluripotent, neuroepithelial cells in the manufacture of a
medicament for the treatment of a behavioural and/or psychological
deficit. The medicament to be administered comprises conditionally
immortal, pluripotent, neuroepithelial cells.
The conditionally immortal cells according to, and used in, the present
invention may be from clonal cell lines or may be of mixed population.
Cells from clonal cell lines may be preferred. Cells from a single cell
line may be used or a mixture of cells from two or more cell lines may be
The invention further provides a pharmaceutical preparation comprising
cells according to the invention and a pharmaceutically acceptable
Transplantation of conspecific fetal neural tissue into a damaged brain
has been studied in animal experiments and consequent repair has been
observed at the neuroanatomical, physiological and behavioural levels (Dunnett
& Bjorklund, 1994). There has been some application of this work in the
treatment of the motor dysfunction of Parkinson's disease (Lindvall, 1994)
but widespread use of this technique is handicapped by the need for tissue
derived from conspecific fetal brain. The fetal tissue required must be
specific to the type of damage one aims to repair and it must be taken at
a precise, time-limited stage during brain development that differs
according to both brain region and cell type. This leads to both practical
and ethical problems.
Work on fetal tissue transplant (Sinden, 1995) in certain types of damage
has shown that very specific matching of cell types is required to obtain
improvement in cognitive function.
We have found that when conditionally immortal pluripotent neuroepithelial
cells are implanted into a damaged brain the cells differentiate into the
correct form of cell required to repair the damage and the differentiated
cells are able to form the appropriate connections required to improve
function. The phenotype of the differentiated cells may be the same as the
phenotype of the damaged or lost cells, however, the differentiated cells
may be of a different phenotype, or of a number of phenotypes. In any
case, the cells take up a phenotype that is capable of functionally
integrating and compensating for the damaged or lost cells. That is
assisted by the propensity, that we have discovered, of the cells to
migrate to, and seek out, damaged tissue.
The use of pluripotent cells means that with one clonal cell line it is
possible to repair damage in number of different areas of the brain. It
also means that if more than one particular cell type is required to
repair damage in a given area then a single pluripotent cell line will be
capable of differentiating into the different types of cells required.
Conditionally immortal cells are cells which are immortal under certain
permissive conditions but are not immortal under nonpermissive conditions.
In the present case this means that by conditionally immortalising
pluripotent precursor cells extracted from fetal tissue and maintaining
them under permissive conditions the development of the precursor cells
may be arrested at a chosen stage and they may be propagated for long
periods. Use of conditionally immortalisation allows the development of
clonal lines which are readily expandable in vitro. If the conditions
under which the cells are maintained are switched to nonpermissive
conditions, the development of the cells is allowed to continue. If the
correct conditions are provided the cells will continue to develop and
Immortalised cells are usually prepared by the transduction of an oncogene
into cells. There is therefore a risk of tumour formation in the long
term, so such cells are not preferred for use in the present invention.
Conditionally immortal cells have the advantages of immortal cells in that
they are "frozen" in the desired stage of development, are easily
maintained and multiply well when under permissive conditions but they may
be used in transplants as long as the environment into which they are
transplanted has nonpermissive conditions. In the case of the cells of the
present invention the gene used to confer conditional immortality should
be chosen so that the conditions present in the brain will correspond to
The usual way to immortalise the cells is by transduction of an oncogene.
The use of conditionally immortal cells means that under nonpermissive
conditions the cells do not have oncogenic properties and so this excludes
any possibility of the implantation of cells leading to tumour growth.
If non-immortal cells are used then these may be maintained in vitro in
culture media with the addition of growth factors.
The gene which is used to confer conditional immortality may be
incorporated into cells after extraction from a fetal animal.
Alternatively, transgenic animals, other than humans, whose neural
epithelial cells comprise a gene for conferring conditional immortality
may be prepared and bred. If transgenic animals are bred then the cells
collected from the fetal animal tissue are already conditionally immortal
and do not require further treatment.
The cells used in the treatment of humans should preferably be derived
from human cells to reduce problems with immune rejection. This requires
the use of fetal tissue. The use of conditionally immortal cells means
that once a population of cells has been established it is not necessary
to use fetal tissue again. For example, cells are taken from a human fetus
at the appropriate stage of development and the DNA necessary to cause
conditional immortality in the cells is inserted. Those cells may then be
propagated or they may be cloned and individual cell lines selected.
Maintenance of the mixed populations and/or of selected cell lines
provides a constant source of material for implantation.
To treat a patient it is generally of assistance to know where damage has
occurred in the brain. 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 damaged area may be carried out. In many
cases, however, the location and/or type of damaged tissue may be unknown
or only poorly characterised. For example, neurodegenerative diseases may
lead to widespread damage to different types of cells. Treatment of such
damage is still possible and is assisted by the ability of pluripotent
neuroepithelial cells to migrate extensively once transplanted and to seek
out damaged tissue. The pluripotent cells may be transplanted at a single
site, or preferably at multiple sites, and are 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. Post mortem analysis of the brains of rats that had
received transplants of cells of a conditionally immortal pluripotent
neuroepithelial cell line showed that the cells aggregated in the area of
the damaged tissue, see Example 9 below, thus illustrating the propensity
of the cells to establish and integrate themselves in the area of damage
rather than in an area of undamaged tissue. The pluripotent nature of the
cells and their propensity for damaged tissue means that treatment with
cells from a single cell line of high potency is able to lead to
compensation for widespread damage of a number of different cell types.
After treatment the progress of the patient may be monitored using
behavioural and/or psychological tests and/or, if desired, tests which
monitor brain activity in selected areas of the brain. For example, tests
for cognitive function may be performed before and after transplantation.
Preferably, treatment will substantially correct a behavioural and/or
psychological 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 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.
The present invention is illustrated by work we have carried out on rats
which have brain damage. In the experiments described below conditionally
immortal cells used for transplantation are derived from the H-2Kb-tsA58
transgenic mouse developed by M. Noble and his associates at the Ludwig
Institute for Cancer Research (Jat et al., 1991). All cells from this
mouse possess a temperature-sensitive oncogene (tsA58, the temperature
sensitive mutant of the SV40 large T antigen under the control of the
interferon-inducible H-2Kb promotor) such that the cells divide
at the permissive temperature (lower than body temperature, 33° C.) but
differentiate only when restored to mouse body temperature (38° C.-39°
C.). It is this feature that provides them with conditional immortality.
This allowed us to clone and expand cell lines in vitro which then
differentiated upon transplantation into a host brain. A number of cell
lines were cloned from a population of cells taken originally from the
transgenic mouse, specifically, from embryonic day 14 (E 14) hippocampus.
We studied the rats, which received transplants of those cells, for at
least 8 months and in no case did the cells, after transplantation, form
tumours. Furthermore, in post-mortem histological preparations, the
transplanted cells (marked by prior transfection with a lac-z reporter
gene) have the appearance of differentiated cells appropriate to the
rodent nervous system.
The cloned cell lines show the potential, in vitro, to differentiate into
more than one phenotype, e.g., into both astroglial and neuronal
phenotypes, see Example 4 below.
The lesion-and-behaviour model in which we have demonstrated that cloned
cell lines are able to restore function in the damaged brain is one that
we have previously studied intensively using fetal conspecific transplants
(see Sinden et al., 1995). It utilises rats in which the technique for
four-vessel occlusion (4 VO), simulating human heart attack, causes
relatively circumscribed and specific damage to the CA 1 pyramidal cells
of the dorsal hippocampus, along with a cognitive deficit manifest as
difficulty in locating a submerged and invisible platform in a swimming
pool. This lesion and behaviour model provides a model of cognitive
dysfunction occurring as a consequence of a common form of brain damage,
i.e., transient loss of blood supply to the brain, for example, as may
occur during cardiac arrest.
We have previously demonstrated that, for fetal cell-suspension
transplants to restore performance in this task, they must be highly
specific to the damage caused by 4VO: transplants containing CA 1
pyramidal cells are effective; transplants containing cholinergic cells
from the basal forebrain, granule cells from the dentate gyrus, or even a
different class of pyramidal cells (CA3) from the hippocampus are
ineffective. Examples 5 to 8 below described experiments in which both the
clonal cell lines and a mixed population of E 14 hippocampal
neuroepithelial cells taken from the H-2Kb-tsA58 transgenic
mouse provide effective transplants for restoration of cognitive function
in this model.
We have found that two of the three clonal cell lines tested, the MHP36
cell line (previously known as the C36 cell line) and the MHP3 cell line
are as effective at restoring cognitive function as fetal rat transplants
containing CA 1 pyramidal cells. The third cell line tested, the MHP15
cell line (previously known as the C15 cell line) leads to an improvement
of function but does not show as great an improvement as MHP36 and MHP3.
The chances that we happened to pick upon cell lines that would
differentiate into CA 1 pyramidal cells, irrespective of the nature of the
host brain environment, are small. Thus it appears that the cell lines are
capable of responding to damage-associated signals so as to differentiate
into cells, of one or more types, that are able to re-establish the
necessary connections and restore the function(s) discharged by the
damaged tissue. It is this capacity that provides both a strategy and a
material basis for transplant therapies with which to target a wide range
of behavioural and psychological deficits consequent upon an equally wide
range of forms of damage to the human brain, while circumventing the
ethical and practical problems associated with the use of human fetal
The two cell lines which have shown the greatest ability, so far, to
restore function are both FGF2-responsive, i.e., they substantially
increase their proliferation in both permissive and non-permissive culture
in the presence of that growth factor, whereas the third cell line is only
slightly responsive. Cells and cell lines which show significantly
increased proliferation in response to the addition of a growth factor to
their culture environment are therefore generally preferred. The cells may
be tested under permissive conditions and/or nonpermissive conditions.
Cells showing the greatest increase in proliferation are generally most
preferred. The growth factor used to test the cells should preferably be
appropriate to the area of the brain in which the cells are intended for
use, i.e., a growth factor secreted in that area. For example, cells
intended for the repair of tissue in the hippocampus may be tested with
FGF2 (also known as bFGF). Cells responsive to FGF2 are generally
preferred. Other mitogenic growth factors may be used in testing,
including EGF and NGF.
The invention therefore provides a method of testing comprising
maintaining a population of cells of a conditionally immortal pluripotent
neuroepithelial cell line in vitro and culturing portions of the cells
under permissive conditions, in the presence and absence of a growth
factor, for example, FGF2, and determining the proliferation of the cells.
Preferably the cells are also tested under nonpermissive conditions. Those
cells which are responsive, i.e., show significantly increased
proliferation in the presence of the growth factor under both permissive
conditions, and preferably also under nonpermissive conditions, appear to
be cells which are especially suitable for use in the treatment of the
invention. The growth factor may, for example, be used at a concentration
of 10 ng/ml.
The temperature-sensitive oncogene which confers conditional immortality
upon cells derived from the H-2Kb-tsA58 transgenic mouse can be
introduced into human cells in vitro. Well known techniques for the
introduction of exogeneous DNA exists and these may be used, for example,
the gene may be introduced by transfection of the cells. Normal screening
techniques for checking that the gene has been incorporated may be used,
for example, Southern blotting may be used to screen for DNA insertion
sites. In some cases markers may be used or, if the ts SV40 large T
antigen gene is used then cells may be screened at the permissive
temperature for expression of SV40 as described in Example 4.
It should be understood that although the experiments described in the
Examples below have been carried out using the ts SV40 large T antigen
gene to confer conditional immortality on the cells, any other gene which
is capable of causing conditional immortality may be used. Such genes may
be constructed from known oncogenes. For example, a conditionally immortal
gene has been constructed from the c-myc oncogene and is described by
Hoshimaiuaru et al, 1996.
In the experiments on rats which are described in Examples 6, 7 and 8
below conditionally immortal pluripotent cells have been used to repair a
very specific type of damage. The uses of cells according to the invention
are not limited to repair of that particular type of damage.
Transplantation into any area of the brain is envisaged with consequent
improvement in function.
The part of the fetal brain from which the neuroepithelial cells are taken
and the precise time (stage and development) may vary. If pluripotent
cells are desired then the cells must, however, be taken at a point early
enough in the developmental pathway that they have the ability to
differentiate into the desired variety of different types and/or
phenotypes of brain cell types. For example, in the case of cells taken
from the embryonic mouse hippocampus the cells may be taken on embryonic
day 14 to 15. Human cells may be taken at the equivalent developmental
stage. For example, cells may be taken from human fetuses at about 8
Cells which have been removed may be screened in vitro to ensure that they
are still able to differentiate, in particular, to differentiate into the
appropriate type or phenotype of cell. Different areas of the brain when
damaged may produce different signals, for example, growth factors, and/or
different types of damage may cause different signals. The ability to
differentiate may be determined in vitro in the presence of the
appropriate signal, for example, the appropriate growth factor. Example 4
below describes a procedure in which the ability of cells to differentiate
into neuronal and glial phenotypes may be shown.
Some behavioural and/or psychological deficits are caused by the absence
of one or more chemicals in an otherwise healthy brain. It has previously
been proposed that transplants of transgenic cells could be used to supply
the missing chemicals. The present invention is not specifically concerned
with such problems. Although the cells of the present invention may be
genetically modified to include extra genes which express desired
products, this will not usually be necessary because the cells used
according to the present invention, once transplanted, differentiate and
then function fully as replacements for cells which have been lost or
damaged. The cells achieve functional integration and replace or
compensate for the missing or damaged cells. They become a functional part
of the brain rather than being merely a sophisticated method of drug
administration. Genetic modification of the cells will therefore usually
be restricted to the insertion of genes necessary for conditional
immortalisation and cloning. Genes required for cloning may be, for
example, a gene providing resistance to a selected antibiotic to enable
selection. Genetic modification to enable secretion of pharmacologic
agents is not preferred.
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. They may also be implanted into
ventricular spaces in the brain. If implanted into the neonate then they
may disperse throughout the entire brain.
Claim 1 of 17 Claims
1. A method for treating brain
damage in a mammal, said method comprising intracerebrally transplanting
pluripotent, nestin-positive, neuroepithelial cells into the damaged part of
the brain of said mammal, wherein said cells have been genetically modified
to be conditionally immortal, wherein said cells are immortal prior to said
transplanting and differentiate after said transplanting, and wherein said
transplanting improves brain function of said mammal.
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