Note: Descriptions are shown in the official language in which they were submitted.
_,_...._ ~ 02399589 2002-08-22
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~rovBL aRO~rs ~~cTOR-asepo~rs=v~ paocB~rzTOR cBLLs
w8IC8 cll~t 8B hROLIPBR71T8D IN VI'~RO
FIELD OF THE INVENTII~N
This invention relates to a method for the ~
vitro culture of neural progenitor cells, and to the
use of these cells as tissue grafts. In one aspect,
this invention relates to a method for the isolation
and in vitro perpetuation of large numbers of non-
tumorigenic neural progenitor cells which can be
induced to differentiate and which can be used for
neurotransplantation into an animal to alleviate the
symptoms of neurologic disease, neurodegeneration and
central nervous system (CNS) trauma. In another
aspect, this invention relates to a method of
generating nerve cells for the purposes of drug
screening of putative therapeutic agents targeted at
the nervous system. In another aspect, this invention
also relates to a method of generating cells for
autologous transplantation. In another aspect, the
invention relates to a method for the in situ
proliferation and differentiation of the progenitor
cells in the host.
The development of the nervous system begins at an
early stage of fetal development. Neurogenesis, the
generation of new neurons, is complete early in the
postnatal period. However, the synaptic connections
involved in neural circuits are continuously altered
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throughout the life of the individual, due to synaptic
plasticity and cell death.
The first step in neural development is cell
birth, which is the precise temporal and spatial
sequence in which precursor or progenitor cells
proliferate and differentiate. Proliferating cells will
give rise to neuroblasts, glioblasts and stem cells.
The second step is a period of cell type
differentiation and migration when progenitor cells
become neurons and glial cells and migrate to their
final positions. Cells which are derived from the
neural tube give rise to neurons and glia of the
central nervous system (CNS), while cells derived from
the neural crest give rise to the cells of the
15' peripheral nervous system (PNS). Certain factors
present during development, such as nerve growth factor
(NGF), promote the growth of neural cells. NGF is
secreted by cells of the neural crest and stimulates
the sprouting and growth of the neuronal axons.
The third step in development occurs when cells
acquire specific phenotypic qualities, such as the
expression of garticular neurotransmitters. At this
time, neurons also extend processes which synapse on
their targets. Neurons do not divide subsequent to
differentiation.
Finally, selective cell death occurs, wherein the
degeneration and death of specific cells, fibers and
synaptic connections "fine-tune" the complex circuitry
of the nervous system. This "fine-tuning~~ continues
throughout the life of the host. hater in life,
selective degeneration due to aging, infection and
CA 02399589 2002-08-22
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other unknown etiologies can lead to neurodegenerative
diseases.
The treatanent of neurodegenerative diseases has
become a major concern in recent years, due to the
expanding elderly population which is at greatest risk
for these diseases. These diseases, which include
Alzheimer's Disease, Huntington's Chorea and
Parkinson's Disease, have been linked to the
degeneration of neurons in specific locations in the
brain, which leads to the inability of the brain region
to synthesize and release neurotransmitters which are
vital to neuronal signalling. Another disease which is
thought to arise from the lack of neurotransmitters is
schizophrenia.
Neurodegeneration also encompasses those
conditions and diseases which result in neuronal loss.
These conditions can include those which arise as a
result of CNS trauma, including stroke and epilepsy.
Other diseases such as amyotrophic lateral sclerosis
and cerebral palsy also result in neuronal loss.
Because of a lack of understanding of the interactions
among specific areas of the brain, and of how these
interactions generate the outward manifestations of
their functions, treatment of CNS degeneration is
hampered. Studies of the intricate cellular
interactions and their effect on behavior and cognitive
function are made more difficult due to the large
number of cells and the complex neural circuits which
they form.
The objective of most CNS therapies is to regain
the particular chemical function or enzymatic activity
which is lost due to cellular degeneration.
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Degeneration in a brain region known as the basal
ganglia can lead to diseases with various cognitive and
motor symptoms, depending on the exact location. The
basal ganglia consists of aany separate regions,
including the striatum (which consists of the caudate
and putamen), the globus pallidus, the substantia
nigra, substantia innominata, ventral pallidum, nucleus
basalis of Meynert, ventral tegmental area and the
subthalamic nucleus.
In the case of Alzheimer's Disease, there is a
profound cellular degeneration of the forebrain and
cerebral cortex. In addition, upon closer inspection,
a localized degeneration in an area of the basal
ganglia, the nucleus basalis of Meynert, appears to be
selectively degenerated. This nucleus normally sends
cholinergic projections to the cerebral cortex which
are thought to participate in cognitive functions
including memory.
Many motor deficits are a result of degeneration
in the basal ganglia. Huntington's Chorea is
associated with the degeneration of neurons in the
striatum, which leads to involuntary jerking movements
in the host. Degeneration of a small region called the
subthalamic nucleus is associated with violent flinging
movements of the extremities in a condition called
ballismus, while degeneration in the putamen and globus
pallidus are associated with a condition of slow
writhing movements or athetosis. In the case of
Parkinson's Disease, degeneration is seen in another
area of the basal ganglia, the substantia nigra par
compacts. This area normally sends dopaminergic
connections to the dorsal striatum which are important
in regulating movement. Therapy for Parkinson's
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Disease has centered upon restoring dopaminergic
activity to this circuit.
Other forms of neurological impairment can occur
as a result of neural degeneration, such as amyotrophic
lateral sclerosis and cerebral palsy, or as a result of
CNS trauma, such as stroke and epilepsy.
To date, treatment for CNS dysfunction has been
mainly via the administration of pharmaceutical
compositions. Unfortunately, this type of treatment
has been fraught with many complications including the
limited ability to transport drugs across the blood-
brain barrier, and the drug-tolerance which is acquired
by patients to whom these drugs are administered long-
term. For instance, partial restoration of
dopaminergic activity in Parkinson's patients has been
achieved with levodopa, which is a dopamine precursor
able to cross the blood-brain barrier. However,
patients become tolerant to the effects of levodopa,
and therefore, steadily increasing dosages are needed
to maintain its effects. There are presently no
effective pharmaceutical treatments for Alzheimer's
Disease.
Recently, the concept of neurological tissue
grafting has been applied to the treatment of
neurological diseases such as Parkinson's Disease.
Neural grafts may avert the need not only for constant
. drug administration, but also for complicated drug
delivery systems which arise due to the blood-brain
barrier. However, there are limitations to this
technique as well. First, cells used for
transplantation which carry cell surface molecules of a
differentiated cell from another host can induce an
immune reaction in the host. In addition, the cells
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must be at a stage of development where they are able
to form normal neural connections with neighboring
cells. For these reasons, initial studies on
neurotransplantation centered on the use of fetal
cells. Perlow, et al. describe the transplantation of
fetal dopaminergic neurons into adult rats with
chemically induced nigrostriatal lesions in "Brain
grafts reduce motor abnormalities produced by
destruction of nigrostriatal dopamine system," Sc~~e ce
~: 643-647 (1979). These grafts showed good
survival, axonal outgrowth and significantly reduced
the motor abnormalities in the host animals. Lindvall,
et al., in "Grafts of fetal dopamine neurons survive
and improve motor function in Parkinson's Disease,~~
Science X47: 574-577 (1990), showed that neural
transplantation of human fetal mesencephalic dopamine
neurons can restore dopamine synthesis and storage, and
reduce rigidity and bradykinesia in patients suffering
from Parkinson~s disease. Freed, et al. in
"Transplantation of human fetal dopamine cells for
Parkinson~s Disease," ~chL,Ng~~r~.. 47: 505-512 (1990)
also show improvement in a patient who received a fetal
transplant.
The above references disclose that mammalian fetal
brain tissue has good survival characteristics upon
immediate transplantation. The increased survival
capability of fetal neurons is thought to be due to the
reduced susceptibility of fetal neurons to anoxia than
adult neurons, and also to the lack of cell surface
markers on fetal cells whose presence may lead to the
rejection of grafted tissue from adults. However,
although the brain is considered an immunologically
privileged site, some rejection of fetal tissue can
occur. Therefore, the ability to use fetal tissue is
limited, not only due to tissue rejection of the fetal
CA 02399589 2002-08-22
tissue isolated from another host, and because of the
resultant need for immunosuppressant drugs, but also
due to ethical problems in obtaining fetal tissue.
However, neonatal brain tissue possesses limited
capacity for survival and adult mammalian CNS neurons
generally do not survive transplantation into the
brain.
Although adult CNS neurons are not good candidates
to for neurotransplantation, neurons from the adult
peripheral nervous system (PNS) have been shown to
survive~transplantation, and to exert neurotrophic and
gliotrophic effects on developing host neural tissue.
One source of non-CNS neural tissue for transplantation
is the adrenal medulla. Adrenal chromaffin cells
originate from the neural crest like PNS neurons, and
receive synapses and produce carrier and enzyme
proteins similar to PNS neurons. Although these cells
function in an endocrine manner in the intact adrenal
medulla, in culture these cells lose their glandular
phenotype and develop neural features in culture in the
presence of certain growth factors and hormones
(Notter, et al., "Neuronal properties of monkey adrenal
medulla in vit,rg, r'ell ~'~~s"~ue Researr,~ 244 : 69-76
[1986 0 . When grafted into mammalian CNS, these cells
survive and synthesize significant quantities of
dopamine which can interact with dopamine receptors in
neighboring areas of the CNS.
In U.S. Patent No. 4,980,174, transplantation of
3o monoamine-containing cells isolated from adult rat
pineal gland and adrenal medulla into rat frontal
cortex led to the alleviation of learned helplessness,
a form of depression in the host. In U.S. Patent No.
4,753,635, chromaffin cells and adrenal medullary
tissue derived from steers were implanted into the
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brain stem or spinal cord of rats and produced
analgesia when the implanted tissue or cell was induced
to release nociceptor interacting substances (i.e.
catecholamines such as dopamine). Adrenal medullary
-cells have been autologously grafted into humans, and
have survived, leading to mild to moderate improvement
in symptoms (Watts, et al., "Adrenal-caudate
transplantation in patients with Parkinson's Disease
(PD) :1-year follow-up," ~l~;~gl~ 39 S~~pl ~~ 12?
[1989], l;urtig, et al., "Post-mortem analysis of
adrenal-medulla-to-caudate autograft in a patient with
Parkinson's Disease," ~,~~~,1~~~ Neurolgav 25i fi07-614
[1989]). However, adrenal cells do not obtain a normal
neural phenotype, and are therefore probably of limited
use for transplants Where synaptic connections must be
formed .
Another source of tissue for neurotransplantation
is from cell lines. Cell lines are immortalized cells
which are derived either by transformation of normal
cells with an oncogene (Cepko, "Immortalization of
neural cells via retrovirus-mediated oncogene
transduction," Ann. Rev. Neurosci. 12: 47-65 [1989]) or
by the culturing of cells with altered growth
characteristics ~~, v~,.tro (Ronnett, et al., "Human
cortical neuronal cell line: Establishment from a
patient with unilateral megalenceghaly," Scienge 248:
603-605 [1990]). Such cells can be grown in culture in
large quantities to be used for multiple
transplantations. Some cell lines have been shown to
differentiate upon chemical treatment to express a
variety of neuronal properties such as neurite
formation, excitable membranes and synthesis of
neurotransmitters and their receptors. Furthermore,
upon differentiation, these cells appear to be
amitotic, and therefore non-cancerous. However, the
CA 02399589 2002-08-22
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potential for these cells to induce adverse immune
responses, the use of retroviruses to immortalize
- cells, the potential for the reversion of these cells
to an amitotic state, and the lack of response of these
cells to normal growth-inhibiting signals make cell
lines less than optimal for widespread use.
Another approach to naurotransplantation involves
the use of genetically engineered cell types or gene
therapy. Using this method, a foreign gene or
transgene can be introduced into a cell which is
deficient in a particular enzymatic activity, thereby
restoring activity. Cells which now contain the
transferred gene can be transplanted to the site of
neurodegeneration, and provide products such as
neurotransmitters and growth factors (Rosenberg, et
al., "Grafting genetically modified cells to the
damaged brain: Restorative effects of NGF Expression,"
Science 242: 1575-1578, [1988]) which may function to
alleviate some of the symptoms of degeneration.
However, there still exists a risk of inducing an
immune reaction with these cells and the retrovirus-
mediated transfer may result in other cellular
abnormalities. Also, cell lines produced by -
retrovirus-mediated gene transfer have been shown to '
gradually inactivate their transferred genes following
transplantation (Palmer, et al. "Genetically modified
skin fibroblasts persist long after transplantation but
gradually inactivate introduced genes," Pros. Nato".
Acad. Sci. 88: pp. 1330-1334 [1991]). In addition,
these cells may also not achieve normal neuronal
connections with the host tissue.
The inability in the prior art of the transplant
to fully integrate into the host tissue, and the lack
of availability of cells in unlimited amounts from a
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reliable source for grafting are, perhaps, the greatest
limitations of neurotransplantation.
Therefore, in view of the aforementioned
deficiencies attendant with prior art methods of neural
cell culturing and transplantation, it should be
apparent that there still exists a need in the art for
a reliable source of unlimited numbers of cells for
neurotransplantation, which are capable of
differentiating into neurons. Furthermore, given that
heterologous donor cells may be immunologically
rejected, there exists a need for the isolation,
perpetuation and transplantation of autologous
progenitors from the juvenile or adult brain that are
capable of differentiating into neurons. In addition,
there exists a need for the repair of damaged neuronal
tissue in a relatively non-invasive fashion, that is by
inducing progenitor cells to proliferate and
differentiate into neurons in situ.
Accordingly, a major object of the present
2o invention is to provide a reliable source of an
unlimited number of cells for neurotransplantation,
which are capable of differentiating into neurons.
It is another object of the present invention to
provide a method for the in vitro proliferation of
neural progenitor calls from embryonic, juvenile and
adult brain, for differentiation of these progenitors
into neurons, and for neurotransplantation of the
progenitor cells into a heterologous or autologous
host. Progenitor cells may also be induced to
proliferate and differentiate ~ln situ thereby averting
the need for transplantation.
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A still further object of the invention is to
provide a method of generating nerve cells for the
purpose of screening putative therapeutic agents
targeted at the nervous system.
SUMMARY OF THE INVENTION
This invention provides in one aspect a
composition for inducing the proliferation of a
progenitor cell comprising a culture medium
supplemented with at least one growth factor,
preferably epidermal growth factor or transforming
growth factor alpha.
The invention also provides a method for the 'fin
vitro proliferation and differentiation of progenitor
cells, comprising the steps of (a) isolating the cell
from a mammal, (b) exposing the cell to a culture
medium containing a growth factor, (c) inducing the
cell to proliferate, and (d) inducing the cell to
differentiate. Proliferation and perpetuation of the
progenitor cells can be carried out either in
suspension cultures, or by allowing cells to adhere to
a fixed substrate. Proliferation and differentiation
can be done before or after transplantation, and in
various combinations of in vitro or in vivo conditions,
including (1) proliferation and differentiation ~
vitro, then transplantation, (2) proliferation ~
vitro, transplantation, then further proliferation and
differentiation i~ivo, and (3) proliferation ~
yitro, transplantation and differentiation in vivo.
The invention also provides for the proliferation and
differentiation of the progenitor cells in situ, which
can be done directly in the host without the need for
transplantation.
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In another aspect, the invention provides for
preservation of progenitor cells by cryogenesis.
The invention also provides a method for the ~
vivo transplantation of a progenitor cell, treated as
in any of (1) through (3) above, which comprises
implanting, into a mammal, these cells which have been
treated with at least one growth factor.
Furthermore, the invention provides a method for
treating neurodegenerative diseases comprising
administering to a mammal progenitor cells which have
been treated as in any of (1) through (3), have been
treated with a growth factor and induced to
differentiate into neurons. The invention also
provides a method for treating neurodegenerative
disease comprising stimulating ~n situ mammalian CNS
progenitor cells to proliferate and differentiate into
neurons.
The invention also provides a method for the
transfection of these pragenitor cells with vectors
which can express the gene products for growth factors,
growth factor receptors, and peptide neurotransmitters,
or express enzymes which are involved in the synthesis
of neurotransmitters, including those for amino acids,
biogenic amines and neuropeptides, and for the
transplantation of these transfected cells into regions
of neurodegeneration.
In a still further aspect, the invention provides
a method for the screening of potential neurologically
therapeutic pharmaceuticals.
With the foregoing and other objects, advantages
and features of the invention that will become
CA 02399589 2002-08-22
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hereinafter apparent, the nature of the invention may
be more clearly understood by reference to the
following detailed description of the invention and to
the appended claims.
BRIEF DESC$I_~,j'ION OF ;~J~E D,$$W,~'NGS
Figure 1. EGF-indu~~, ~~~glia~nt off" a c~,uster of
undifferentjatea cs,~,ls from a si.n"al,_e ra,~ll. With
limiting dilution of the cell suspension, single cells
were plated and identified at the base of 96-well
plates. (A-D). Phase contrast micrographs of a single
cell (A) after 3 days in vitro (DIV). The same cell
began to divide after 5 DIV (B) and generated a cluster
of cells observed after 7 (C) and 10 (D) DIV.
Scratches in the plastic (arrowhead) serve to identify
the field. Bar, lO~Cm. (E-F). Phase contrast
micrograph (E) of 10 DIV cluster of cells, generated in
response to EGF. The majority of cells in this cluster
contained nestin-IR (F). Bar, 10 Vim.
Figure 2. NSE-ZR cel~in EGF-~~GFa-inducp~
~roliferati~a cell s~ly~st~rs. The presence of NSE-IR
cells in EGF and TGFa-induced proliferating clusters
was examined at 20 and 25 DIV. Prior to 15 DIV no NSE-
IR cells were detected. NSE-IR cells appeared after
16-18 DIV and were seen initially in the proliferating
core. (A, H). After 20 DIV, numerous NSE-IR cells were
identified in both EGF(A)-and TGFa(B)-treated cells.
Few NSE-IR cells were found outside the proliferating
core and no differences were apparent between the NSE-
IR cells in the EGF- and TGFa-treated cultures. (C, D).
After 25 DIV hundreds of NSE-TR cells were observed.
In addition to large numbers of NSE-IR cells in the
proliferating core, many IR cells had migrated away.
At this stage, some differences in neuronal morphology
... ~.~ a..~ vvl.VJ
CA 02399589 2002-08-22
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between (C) EGF- and (D) TGFa-treated cells became
apparent. While EGF-treated cells had short neurites,
some of the NSE-IR cells in the TGFa-treated cultures
had developed fairly long processes (arrows). Bar,
20~tm.
Figure 3. lrpyZ~,r~;~~ce of GFAP-IR cells ~ EGF- and
~GFa-treated cult~~res. GFAP-IR first appeared at 16-18
DIV. 8y 20 DIV a few GFAP-IR cells were observed
migrating away from the proliferating core. There was
no apparent difference between EGF- and TGFa-treated
cultures. At 25 DIV, a large majority of the cells
which had migrated away from the central core were
GFAP-IR. At this stage all of the GFAP-IR cells in
EGF-treated cultures had a stellate morphology, while
many of the GFAP-IR in the TGFa-treated cultures
' assumed the morphology of immature protoplasmic
astrocytes (some stellate cells were still apparent).
By 30 DIV, all of the GFAP-IR cells in the EGF-treated
cultures exhibited stellate morphology. In contrast,
nearly all of the GFAP-IR cells in the TGFa-treated
cultures were protoplasmic. Bar, 20~cm.
Figure 4. phenotype of neurons 4enerated by EGF-
induced proliferation of adult striatal ~rooenitor
Fluorescence micrographs of cells with neuronal
morphology, fn 21 DIV cultures of a single EGF-
generated sphere plated on a fixed substrate, after
staining for GAGA (a) and substance P (b).
Figure 5. Transglanted mouse neurosoheres
differentiate infi~o_ neu:~g~~,s in the rat CNS. A
monoclonal antibody, Ms, which recognizes a surface
protein found only on mouse neurons was used to label
transplanted mouse progenitors which had differentiated
into neurons in the rat host. Positive labelled cell
CA 02399589 2002-08-22 _ _ .
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bodies (large arrows) and neurites (small arrows) are
identified .
The present invention provides for the isolation
of neural progenitor cells from an animal, for
perpetuation of these cells in vitro or inin viyg in the
presence of growth factors, for the differentiation of
these cells into neurons and glia, for the cryogenesis
l0 of progenitor cells, and for the use of these cells for
neurotransplantation into a host in order to treat
neurodegenerative disease and neurological trauma, and
for drug-screening applications. The induction of
proliferation and differentiation in these progenitor
cells can be done either by growing adherent or
suspension cells in tissue culture, or, under
appropriate conditions, in the host in the following
combinations: (1) proliferation and differentiation ~
~titro, then transplantation, (2) proliferation j~
vitro, transplantation, then further proliferation and
differentiation in vivo, (3) proliferation in vitro,
transplantation and differentiation in vivo, and (4)
proliferation and differentiation ,fin ,situ. .
Proliferation and differentiation i~,~ _situ, involves a
non-surgical approach which coaxes progenitor cells to
proliferate in situ with pharmaceutical manipulation.
Progenitor cells are thought to be under a tonic
inhibitory influence which maintains~the progenitors in
a suppressed state until their differentiation is
required. This tonic inhibitory influence may be a
soluble factor, as fractions of neural lysates added
back to progenitor cells can inhibit their
proliferation in y~,tro. In culture, these progenitor
cells may not be subject to this same inhibitory
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-is-
influence. These cells are now able to be
proliferated, and unlike neurons which are terminally
differentiated and therefore non-dividing, they can be
produced ink unlimited number and are therefore highly
suitable for transplantation into heterologous and
autologous hosts with neurodegenerative diseases.
By progenitor is meant a~n oligopotent or
multipotent stem cell which is able to divide without
limit and under specific conditions can produce
daughter cells which terminally differentiate into
neurons and glia. These cells can be used for
transplantation into a heterologous or autologous host.
By heterologous is meant a host other than the animal
from which the progenitor cells were originally
~ derived. By autologous is meant the identical host
from which the cells were originally derived.
Cells can be obtained from embryonic, post-natal,
juvenile or adult neural tissue from any animal. By
any animal is meant any multicellular animal which
contains nervous tissue. More particularly, is meant
any insect, fish, reptile, bird, amphibian or mammal
and the like. The most preferable donors are mammals,
especially mice and humans.
In the case of a heterologous donor animal, the
animal may be euthanized, and the brain and specific
area of interest removed using a sterile procedure.
Brain areas of particular interest include any area
from which progenitor cells can be obtained which will
serve to restore function to a degenerated area of the
host's brain. These regions include areas of the
. central nervous system (CNS) including the cerebral
cortex, cerebellum, midbrain, brainstem, spinal cord
and ventricular tissue, and areas of the peripheral
CA 02399589 2002-08-22
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nervous system (PNS) including the carotid body and the
adrenal medulla. More particularly, these areas
include regions in the basal ganglia, preferably the
striatum which consists of the caudate and putamen, or
various cell groups such as the globus pallidus, the
subthalamic nucleus, the nucleus basalis which is found
to be degenerated in Alzheimer~s Disease patients, or
the substantia nigra gars compacta which is found to be
degenerated in Parkinson~s Disease patients. This
latter cell group is named for and identified by its
dark color due to the presence of high levels of the
pigment, melanin. Melanin is derived from tyrosine or
dopa through the action of tyrosinase.
Human heterologous neural progenitor cells may be
derived from fetal tissue obtained from elective
abortion, or from a post-natal, juvenile or adult organ
donor. Autologous neural tissue can be obtained by
biopsy, or from patients undergoing neurosurgery in
which neural tissue is removed, in particular during
epilepsy surgery, and more particularly during temporal
lobectomies and hippocampalectomies. -
Cells can be obtained from donor tissue by
dissociation of individual cells from the connecting
extracellular matrix of the tissue. Dissociation can
be obtained using any known procedure, including
treatment with enzymes such as trypsin, collagenase and
the like, or by using physical methods of dissociation
such as with a blunt instrument. Dissociation of fetal
cells can be carried out in tissue culture medium,
while a preferable medium for dissociation of juvenile
and adult cells is artificial cerebral spinal fluid
_,. ~ (aCSF). Regular aCSF contains 124 mM NaCl, 5 mM KCl,
1.3 mM MgCl=, 2 mM CaCl2, 26 mM NaIiCO" and 10 mM D-
glucose. Low Ca++ aCSF contains the same ingredients
WO 93/OIZi5 ~ 02399589 2002-08-22 p[~'/CA92/00283
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except for MgClz at a concentration of 3.2 mM and CaCh
at a concentration of o.1 mM.
Dissociated cells can be placed into any known
culture medium cagable of supporting cell growth,
including MEM, DMEM, RPMI, F-12, and the like,
containing supplements which are required for cellular
metabolism such as glutamine and other amino acids,
vitamins, minerals and useful proteins such as
transferrin and the like. Medium may also contain
antibiotics to prevent contamination with yeast,
bacteria and fungi such as penicillin, streptomycin,
gentamicin and the like. In some cases, the medium may
contain serum derived from bovine, equine, chicken and
the like. A particularly preferable medium for cells
is a mixture of DMEM and F-12.
Conditions for culturing should be close to
physiological conditions. The pH of the culture media
should be close to physiological pH, preferably between
pH 6-8, more preferably close to pH 7, even more
particularly about pH 7.4. Cells should be cultured at
a temperature close to physiological temperature,
preferably between 30°C-40°C, more preferably between
32°C-38°C, and most preferably between 35°C-37°C.
Cells can be grown in suspension or on a fixed
substrate, but proliferation of the progenitors is
preferably done in suspension to generate large numbers
of cells. In the case of suspension cells, flasks are
shaken well and the neurospheres allowed to settle on
the bottom corner of the flask. The spheres are then
transferred to a 50 ml centrifuge tube and centrifuged
.. ~.t.low speed. The medium is aspirated, the cells
resuspended in a small amount of medium with growth
CA 02399589 2002-08-22
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factor, and the cells mechanically dissociated. The
cells are then counted and replated.
Culturing of cells on a fixed substrate can lead
to differentiation of the progenitor into a terminally
differentiated cell which is no longer capable of
dividing, but can be used for transplantation to form
connections with other neural targets. Differentiation
can also be induced in suspension if the cells are
cultured for a period of time without dissociation and
re-initiation of proliferation.
Growth factors which may be used for inducing
proliferation include any trophic factor which allows
progenitor cells to proliferate, including any molecule
which binds to a receptor on the surface of the cell to
exert a trophic, or growth-inducing effect on the cell.
Such factors include nerve growth factor (NGF), acidic
and basic fibroblast growth factor (aFGF, bFGF),
platelet-derived growth factor (PDGF), thyrotropin-
releasing hormone (TRH), epidermal growth factor (EGF),
an EGF-like ligand, amphiregulin, transforming growth
factor alpha and beta (TGFa, TGF~), insulin-like growth
factor (IGF.,) and the like.
The most preferable factors are EGF and TGFa.
These growth factors are single chain polypeptides of
53 and 50 amino acids, respectively, which compete for
binding of the EGF receptor (EGFR). EGF and TGFa have
been demonstrated to influence cellular
differentiation. TGFa immunoreactivity (IR), EGF-IR
and EGFR-IR, as well as TGFa and EGF mRNAs have been
observed in the rodent and human CNS. The results of
ligand binding studies have demonstrated that the
number of binding sites for [~~I]EGF in the rodent brain
increase during. gestation and are higher in the first
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post-natal week than in the adult, consistent with a
role for this factor in CNS development. EGF induces
the proliferation of a multipotent progenitor cell from
the striatum that gives rise to stellate astrocytes and
neurons with short processes, and TGFa induces the
proliferation of a multipotent progenitor cell that
gives rise to protoplasmic astrocytes and neurons with
long, well developed neurites. The concentration of
growth factors used is 1 fg/ml - 1 mg/ml, preferably 20
ng/ml.
Neuronal and glial cells can be derived following
the differentiation of the multigotent progenitor
cells. Differentiation of the cells can be induced by
any method known in the art which activates the cascade
of biological events which lead to growth, which
include the liberation of inositol triphosphate and
intracellular Ca;+, liberation of diacyl glycerol and
the activation of protein kinase C and other cellular
kinases, and the like. Treatment with phorbol esters,
growth factors and other chemical signals, as well as
growth on a fixed substrate such as an sonically
charged surface such as poly-L-lysine and poly-L-
ornithine and the like can induce differentiation.
Differentiation can also be induced by leaving the
cells in suspension in the continued presence of growth
factor, in the absence of reinitiation of
proliferation.
Cells from the particular neural region are
removed from the animal using a sterile procedure, and
are mechanically dissociated using any method known in
the art. Embryonic or early post-natal brain tissue
are dissociated directly into culture medium, while
juvenile or adult brain tissue are dissociated into
artificial cerebral spinal fluid (CSF). Dissociated
_. . ... _. _ _ . _ ~ 02399589 2002-08-22
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cells are centrifuged at low speed, between 200 and
2000 rpm, and more particularly between 400 and 800
rpm, and then resuspended in culture medium. Cells are
resuspended at a approximately 5 x 10' to 2 x 105
cells/ml, preferably 1 x lOs cells/ml. Cells plated on
a fixed substrate are plated at approximately 2-3 x lo'
cells/cmi, preferably 2.5 x 103 cells/cmz.
Cell suspensions in culture medium are
supplemented with any growth factor which allows for
the proliferation of progenitor cells and seeded in any
receptacle capable of sustaining cells, particularly
culture flasks, culture plates or roller bottles, and
more particularly in small culture flasks such as 25 cmz
culture flasks. Cells proliferate within 3-4 days in a
37'C incubator, and proliferation can be reinitiated at
any time after that by dissociation of the cells and
resuspension in fresh medium containing growth factors.
In the absence of substrate, cells lift off the
floor of the flask and continue to proliferate in
suspension forming a hollow sphere of undifferentiated
cells. After approximately 3-10 days in ~'tro, and
more particularly approximately 6-7 days ~ vitro, the
proliferating clusters (neurospheres) are fed every 2-7
days, and more particularly every 2-4 days by gentle
centrifugation and resuspension in medium containing
growth factor.
After 6-7 days in vitro, individual cells in the
neurospheres can be separated by physical dissociation
of the neurospheres with a blunt instrument, more
particularly by triturating the neurospheres with a
pipette, especially a fire polished pasteur pipette.
Single cells from the dissociated neurospheres are
suspended in culture medium containing growth factor,
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_22_
and a percentage of these cells proliferate and form
new neurospheres largely composed of undifferentiated
cells. Differentiation of cells can be induced by
plating the cells on a fixed substrate, including poly-
L-lysine or poly-L-ornithine coated flasks, plates or
coverslips in the continued presence of EGF, TGFa or
any factor capable of sustaining differentiation such
as basic fibroblast growth factor (bFGF) and the like.
Differentiation can also be induced by leaving the
cells in suspension in the presence of a growth factor,
including EGF or TGFa, without reinitiation of
proliferation.
In order to identify cellular molecules on the two
types of cells which arise upon growth-factor-induced
~ differentiation, namely neurons and glia,
immunocytochemistry is performed using antibodies
specific for known cellular markers. Antibodies
_ specific for any neuronal or glial proteins can be used
to distinguish the cellular characteristics or
phenotypic properties of neurons from glia. In
particular these cellular markers include, for neurons,
neuron specific enolase (NSE) and neurofilament, and
for glia, glial fibrillary acidic protein (GFAP),
galactocerebroside and the like.
Also useful for identifying neuronal cells are
antibodies against neurotransmitters. These include
antibodies to acetylcholine (ACh), dopamine (DA),
epinephrine (E), norepinephrine (NE), histamine (H),
serotonin or 5-hydroxytryptamine (5-HT), neuropeptides
such as substance P (SP), adrenocorticotrophic hormone
(ACTH), vasopressin or anti-diuretic hormone (ADH),
oxytocin, somatostatin, angiotensin II, neurotensin,
and~bombesin, hypothalamic releasing hormones such as
thyrotropic-releasing hormone (TRH) and luteinizing
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hormone releasing hormone (LHRH), gastrointestinal
peptides such as vasoactive intestinal peptide (VIP)
and cholecystokinin (CCK) and CCK-like peptide, opioid
peptides such as endorphins like ~-endorphin and
enkephalins such as met- and leu-enkephalin,
prostaglandins, amino acids such as gamma-aminobutyric
acid (GABA), glycine, glutamate, cysteine, taurine and
aspartate and dipeptides such as carnosine.
Antibodies to neurotransmitter-synthesizing
enzymes are also useful such as glutamic acid
decarboxylase (GAD) which is involved in the synthesis
of GABA, choline acetyltransferase (CAT) for ACh
synthesis, dopa decarboxylase (DDC) for dopamine,
dopamine-~-hydroxylase (DBH) for NE, and amino acid
decarboxylase (AADC) for 5-HT.
Antibodies to enzymes which are involved in the
deactivation of neurotransmitters may also be useful
such as acetyl cholinesterase (AChE) which deactivates
ACh. Antibodies to enzymes involved in the reuptake of
neurotransmitters into neuronal terminals such as
monoamine oxidase (MAO) and catechol-o-methyl
transferase (COMT) for DA, MAO for 5-HT, and GAGA
transferase (GAGA-T) for GAGA may also identify
neurons.
Other markers for neurons include antibodies to
neurotransmitter receptors such as the AChE nicotinic
and muscarinic receptors, adrenergic receptors al, a=, ~,
and ~_, the dopamine receptor and the like.
Cells which contain a high level of melanin, such
as those which would be found in the substantia nigra,
could be identified using an antibody to melanin.
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Progenitor cells can be identified with antibodies
which specifically bind to proteins found only in
progenitor cells, and not cells which have
differentiated. In particular are antibodies to an
intermediate filament protein, and more particularly an
antibody called Rat401 which is directed to nestin, an
intermediate filament protein found specifically in
neuroepithelial stem cells.
Any secondary antibody or heterologous anti-
immunoglobulin, can be used which binds to the primary
antibody, and which allows for its detection by being
conjugated to a particular enzyme, isotope, particle or
dye. Such antibodies can be raised in any animal, such
as goats, sheep, rabbits, swine, horses, mice, rats and
the like, and be directed to a primary antibody raised
in any heterologous animal of the same such varieties.
Secondary antibodies can be conjugated to dyes such as
fluorescein, rhodamine or other dye, can be
radiolabelled with any isotope such as 1~T or conjugated
to a radiolabelled protein such as digoxin, or can be
conjugated to particles which can be seen in the
microscope such as immuno-gold particles and the like.
Secondary antibodies can also be conjugated to any
enzymes which can react with a substrate to form a
visible or detectable reaction product, such as
horseradish-peroxidase and alkaline phosphatase.
Particularly useful antibodies are secondary antibodies
consisting of fluorescein conjugated affinipure goat
anti-mouse IgG and rhodamine conjugated affinipure goat
anti-rabbit IgG.
Cells are cultured on any substrate on which cells
__ can.grow and differentiate, and which can be analyzed
using microscopy, such as culture plates, glass slides,
cover slips and the like. Cells are cultured up to 6
CA 02399589 2002-08-22
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weeks, and more preferably for 14-30 days in v,~tro, and
can be fixed in any dehydrating solution such as an
alcohol or acetone, or in a solution of cross-linking
aldehydes. Cells are then stained using primary
antibodies directed to any particular cellular feature
which is to be identified, followed by reaction with
secondary conjugated antibodies for detection.
Transplanted cells can be administered to any
animal with abnormal neurological or neurodegenerative
to symptoms obtained in any manner, including those
obtained as a result of chemical or electrolytic
lesions, as a result of experimental aspiration of
neural areas, or as a result of aging processes.
Particularly preferable lesions in non-human animals
are obtained with 6-hydroxy-dopamine (6-OIiDA), 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),
ibotenic acid and the like.
Because immunosuppressants carry some risk of
morbidity and mortality as a result of infections which
may occur, only autologous transplantation or
heterologous transplantation using tissue from parents
or siblings, can,be done in the absence of these drugs.
Transplantation can be done bilaterally, or, in the
case of a patient suffering from Parkinson's Disease,
contralateral to the most affected side. Surgery is
performed in a manner in which particular brain regions
may be located, such as in relation to skull sutures,
particularly with a stereotaxic guide. Cells are
delivered throughout any affected neural area, in
particular to the basal ganglia, and preferably to the
caudate and putamen, the nucleus basalis or the
. substantia nigra. Cells are administered to the
particular rmgion using any method which maintains the
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-26-
integrity of surrounding areas of the brain, preferably
by injection cannula.
Cells administered to the particular neural region
preferably form a neural graft, wherein the cells form
normal neuronal or synaptic connections with
neighboring neurons, and maintain contact with glial
cells which may form myelin sheaths around the neuron's
axon, and provide a trophic influence for the neurons.
As these transplanted cells form connections, they re-
establish~the neuronal networks which have been damaged
due to disease and aging.
Progenitor cells can be induced to proliferate and
differentiate ~,n situ by administering to the host, any
growth factor or pharmaceutical composition which will
15~ induce proliferation and differentiation of the cells.
These growth factors include any growth factor known in
the art, including the growth factors described above
for in vitro proliferation and differentiation, and in
particular EGF and TGFa. Pharmaceutical compositions
include any substance which blocks the inhibitory
influence and/or stimulate the progenitor to
proliferate and ultimately differentiate.
Administration of growth hormones can be done by any
method, including injection cannula, transfection of
cells with growth-hormone-expressing vectors,
injection, timed-release apparati which can administer
substances at the desired site, and the like.
Pharmaceutical compositions can be administered by any
method, including injection cannula, injection, oral
administration, timed-release apparati and the like.
Survival of the graft in the living host can be
examined using various non-invasive scans such as
computerized axial tomography (CAT or CT scan), nuclear
CA 02399589 2002-08-22
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magnetic resonance or magnetic resonance imaging (NI~t
or lrBtl ) or more preferably positron emission tomography
- (PET) scans. Post-mortem examination of graft survival
can be done by removing the neural tissue, and
examining the affected region macroscopically, or more
preferably using microscopy. Cells can be stained with
any stains visible under light or electron microscopic
conditions, more particularly with stains which are
specific for neurons and glia. Particularly useful are
monoclonal antibodies which identify neuronal cell
surface markers such as the M6 antibody which
identifies mouse neurons. Most preferable are
antibodies which identify any neurotransmitters,
particularly those directed to gamma amino butyric acid
(GAGA) and substance P, and to enzymes involved in the
synthesis of neurotransmitters, in particular, glutamic
acid decarboxylase (GAD). Transplanted cells can also
be identified by prior incorporation of tracer dyes
such as rhodamine- or fluorescein-labelled
microspheres, fast blue, bis-benzamide or retrovirally
introduced histochemical markers such as the lac Z gene
which produces beta galactosidase.
Functional integration of the graft into the -
host's neural tissue can be assessed by examining the
effectiveness of grafts on restoring various functions,
including but not limited to tests for endocrine,
motor, cognitive and sensory functions. Motor tests
which can be used include those which quantitate
rotational movement away from the degenerated side of
the brain, and those which quantitate slowness of
movement, balance, coordination, akinesia or lack of
movement, rigidity and tremors, Cognitive tests
include various tests of ability to perform everyday
tasks, as well.as various memory tests, including maze
performance.
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In another embodiment of the invention, progenitor
cells are transplanted into a host, and induced to
proliferate and/or differentiate in that host by either
(1) proliferation and differentiation ~.n vitro, then
transplantation, (2) proliferation ~n vitro,
transplantation, then further proliferation and
differentiation i~~yQ,. or (3) proliferation in vitro,
transplantation and differentiation ~Zg'~vo.
Alternatively, the cells can be induced to proliferate
and differentiate in situ by induction with particular
growth factors or pharmaceutical compositions which
will induce their proliferation and differentiation.
Therefore, this latter method is non-surgical, and thus
circumvents the problems associated with physical
intervention and immune reactions to foreign cells.
Any growth factor can be used, particularly EGF, TGFa,
aFGF, bFGF and NGF. These growth factors can be
administered in any manner known in the art in which
the factors may either pass through or by-pass the
blood-brain barrier (BBB).
Methods for allowing factors to pass through the
blood-brain barrier include minimizing the size of the
factor, or providing hydrophobic factors which may pass
through more easily.
Methods for by-passing the blood-brain barrier
include transfection of the progenitor cells with
expression vectors containing genes which code for
growth factors, so that the cells themselves produce
the factor. Cells can be transfected by any means
3D known in the art, including CaP04 transfection, DEAE-
dextran transfection, polybrene transfection, by
protoplast fusion, electroporation, lipofection, and
the like.
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Any expression vector known in the art can be used
to express the growth factor, as long as it has a
promoter which is active in the cell, and appropriate
termination and polyadenylation signals. These
expression vectors include recombinant vaccinia virus
vectors including pSCll, or vectors derived various
viruses such as from Simian Virus 40 (SV40, i.e. pSV2-
dhfr, pSV2neo,~ pko-neo, pSV2gpt, pSVT7 and pBABY), from
Rous Sarcoma Virus (RSV, i.e. pRSVneo), from mouse
mammary tumor virus (MMTV, i.e. pMSG), from adenovirus
(pMT2), from herpes simplex virus (HSV, i.e. pTK2 and
pHyg), from bovine papillomavirus (BPV, i.e. pdBPV and
pBV-1MTHA), from Epstein-Barr Virus (EBV, i.e. p205 and
pHEBo) or any other eukaryotic expression vector known
in the art.
Any growth factor known to induce proliferation,
growth or differentiation of the progenitor cells can
be used for expression in the progenitor cells,
including NGF, FGF, EGF TGFa and the like. Cells may
also be transformed with receptors for any growth
factor such as for bFGF, NGF, EGF and the like. In
addition, cells may be transfected with any gene coding
for a neurotransmitter or neurotransmitter-synthesizing
enzyme which was listed above or any other for which
expression in the host is desired.
Other methods for providing growth factors to the
area of transplantation include the implantation into
the brain in proximity to the graft of any device which
can provide an infusion of the factor to the
surrounding cells.
._, Progenitor cells can be cryopreserved by any
method known in the art, by suspending the cells in an
isotonic solution, preferably a cell culture medium,
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containing a particular cryopreservant. Such
cryopreservants include dimethyl sulfoxide (DMSO),
glycerol and the like. These cryopreservants are used
at a concentration of 5-15~, preferably 8-10~. Cells
are frozen gradually to a temperature of -10°C to
-150°C, preferably -20°C to -100°C, and more preferably
-70°C to -80°C.
Progenitor cells cultured in vitro can be used for
the screening of potential neurologically therapeutic
compositions. These compositions can be applied to
cells in culture at varying dosages, and the response
of the cells monitored for various time periods.
Physical characteristics of the cells can be analyzed
by observing cell and neurite growth with microscopy.
The induction of expression flf new or increased levels
of proteins such as enzymes, receptors and other cell
surface moleculss, or of neurotransmitters, amino
acids, neuropeptides and biogenic amines can be
analyzed with any technique known in the art which can
identify the alteration of the level of such molecules.
These techniques include immunohistochemistry using
antibodies against such molecules, or biochemical
analysis. Such biochemical analysis includes protein
assays, enzymatic assays, receptor binding assays,
enzyme-linked immunosorbant assays (ELISA),
electrophoretic analysis, analysis with high
performance liquid chromatography (i~iPLC), Western
blots, and radioimmune assays (RIA). Nucleic acid
analysis such as Northern blots can be used to examine
the levels of mRNA coding for these molecules, or for
enzymes which synthesize these molecules.
Alternatively, cells treated with these pharmaceutical
.. cs~mpositions can be transplanted into an animal, and
their survival, ability to form neuronal connections,
CA 02399589 2002-08-22
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and biochemical and immunological characteristics
examined as described above.
The following examples are presented in order to
more fully illustrate the preferred embodiments of the
invention. They should in no way be construed,
however, as limiting the scope of the invention, as
defined by the appended claims.
14-day-old CD, albino mouse embryos (Charles River)
were decapitated and the brain and striata were removed
using sterile procedure. Tissue was mechanically
dissociated with a fire-polished Pasteur pipette into
serum-free medium composed of a 1:1 mixture of
Dulbecco~s modified Eagle s medium (DMEM) and F-12
nutrient (Gibco). Dissociated cells were centrifuged
at 800,r.p.m. for 5 minutes, the supernatant aspirated,
and the cells resuspended in DMEM/F-12 medium for
counting.
Brain tissue from juvenile and adult mouse brain
tissue was removed and dissected into 500 ~m sections
and immediately transferred into low calcium oxygenated
artificial cerebrospinal fluid (low Ca** aCSF)
containing 1.33 mg/ml trypsin, 0.67 mg/ml
hyaluronidase, and 0.2 mg/ml kynurenic acid. Tissue
was stirred in this solution for 90 minutes at 32~C-
35'C. aCSF was poured off and replaced with fresh
oxygenated aCSF for 5 minutes. Tissue was transferred
.~ to,D~IEM/F-12/10% hormone solution containing 0.7 mg/ml
ovomucoid and triturated with a fire polished Pasteur
' pipette. Cells were centrifuged at 40o r.p.m, for 5
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minutes, the supernatant aspirated and the pelleted
cells resuspended in DMEM/F-12/10% hormone mix.
Dissociated cells prepared as in Examples 1 and 2
were diluted to approximately 1 cell/well in a 96 well
(100 y,l/well) tissue culture plate to examine the
clonal nature of EGF-induced proliferation. The
presence of a single cell in a well was confirmed with
phase contrast microscopy.
~~~,E 4
2500 cells/cm2 prepared as in Example 1 were plated
on poly-L-ornithine-coated (15 ~cg/ml;Sigma) glass
coverslips in 24 well Nunclon (0.5 ml/well) culture
dishes. The culture medium was a serum-free medium
composed of DMEM/F-12 (1:1) including glucose (0.6%),
glutamine (2 ~cM), sodium bicarbonate (3 mM), and HEPES
(4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid)
buffer (5 mM) (all from Sigma except glutamine
[Gibco]). A defined hormone mix and salt mixture
(Sigma) that included insulin (25 ~g/ml), transferrin
(100 ~g/ml), progesterone (20 nM), putrescine (50 ~cM),
and selenium chloride (30 nM) was used in place of
serum. Cultures contained the above medium together
with 16-20 ng/ml EGF (purified from mouse sub-
maxillary, Collaborative Research) or TGFa (human
recombinant, Gibco). After 10-14 days in vitro, media
(DMEM only plus hormone mixture) and growth factors
were replaced. This medium change was repeated every
two to four days. The number of surviving cells at 5
days _i~~itro was determined by incubating the
coverslips in 0.4% trypan blue (Gibco) for two minutes,
washing with phosphate buffered saline (PBS, pH 7.3)
~ . v ~~.. ........
CA 02399589 2002-08-22
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and counting the number of cells that excluded dye with
a Nikon Diaphot inverted microscope.
Dissociated mouse brain cells prepared as in
Examples 1 and 2 (at 1 x 10f cell/ml) were suspended in
the culture medium described in Example 4 together with
20 ng/ml of EGF or TGFa. Cells were seeded in a TZ5
culture flask and housed in an incubator at 37'C, 100%
humidity, 95% air/5% COz. Cells began to proliferate
l0 within 3-4 days and due to a lack of substrate lifted
off the floor of the flask and continued to proliferate
in suspension forming a hollow sphere of
undifferentiated cells. After 5-7 days ~.n vitro the
proliferating clusters (neurospheres) were fed every 2-
4 days by gentle centrifugation and resuspension in
DMEM with the additives described above.
After 6-7 days in vitro, individual cells in the
neurospheres from Example 5 were separated by
triturating the neurospheres with a fire polished -
pasteur pipette. Single cells from the dissociated
neurospheres were suspended in tissue culture flasks in
DMEM/F-12/10% honaone mix together with 20 ng/ml of
EGF. A percentage of dissociated cells began to
proliferate and formed new neurospheres largely
composed of undifferentiated cells. The flasks Were
shaken well,and neurospheres were allowed to settle in
the bottom corner of the flask. The neurospheres were
then transferred to 50 ml centrifuge tubes and
_" 30 cent~rifuged.at 300 rpm for S minutes. The medium was
aspirated off, and the neurospheres Were resuspended in
1 ml of medium containing EGF. The cells were
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dissociated with a fire-narrowed Pasteur pipette and
triturated forty times. 20 microliters of cells were
removed for counting and added to 20 microliters of
Trypan Blue diluted 1:2. The cells were counted and
re-plated at 50,000 cells/ml. Differentiation of cells
was induced by maintaining the cells in the culture
flasks in the presence of EGF or TGFa at 20 nq/ml
without reinitiating proliferation by dissociation of
the neurospheres or by plating on poly-ornithine in the
continued presence of EGF or TGFa.
EXAMPL'~ 7
Indirect immunocytochemistry was carried out with
cells pregared as in Examples 1 and 2 which had been
cultured for 14-30 days in vitro on glass coverslips.
For anti-NSE (or anti-nestin) and anti-GFAP
immunocytochemistry, cells were fixed with 4%
paraformaldehyde in PHS and 95% ethanol/5% acetic acid,
respectively. Following a 30 minute fixation period,
coverslips were washed three times (10 minutes each) in
PBS (pH ~ 7.3) and then incubated in the primary
antiserum (NSE 1:300, nestin 1:1500 or GFAP 1:100) in
PHS/10% normal goat serum/0.3% Triton-X-100) for two
hours at 37'C. Coverslips were washed three times (10
minutes each) in PHS and incubated with secondary
antibodies (goat-anti-rabbit-rhodamine for anti-NSE or
anti-nestin and goat-anti-mouse-fluorescein for anti-
GFAP, both at 1:50) for 30 minutes at 37'C. Coverslips
were then washed three times (10 minutes each) in PBS,
rinsed with water, placed on glass slides and
3o coverslipped using Fluorsave, a mounting medium
preferable for use with fluorescein-conjugated
... antibodies. Fluorescence was detected and photographed
With a Nikon Optiphot photomicroscope.
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Rats were anesthetized with nembutal (25 mg/kg
1.p) and injected with atropine sulfate (2 mg/kg 1.p.).
Animals sustained an ibotenate lesion of the striatum.
7 days after the lesion, the animals received an
injection of cells prepared as in Examples 1 or 2 under
stereotaxic control. Injections were made to the
lesioned area via a 21-gauge cannula fitted with a
teflon catheter to a microinjector. Injected cells
l0 were labelled with fluorescein-labelled microspheres.
Animals were given behavioral tests before the lesion,
after the lesion, and at various intervals after the
transplant to determine the functionality of the
grafted cells at various post-operative time points,
then killed and perfused transcardially with 4%
buffered paraformaldehyde, 0.1% glutaraldehyde and 5%
sucrose solution at 4'C. The brains were frozen in
liquid nitrogen and stored at -20'C until use. Brains
sections were sliced to 26 ~Cm on a cryostat, fixed in
4% paraformaldehyde and stained using the M6 monoclonal
antibody to stain for mouse neurons, and then reacted
with a secondary anti-rat fluorescein-conjugated
antibody. Neuronal and glial phenotype was identified
by dual labelling of the cells with antibody to NSE and
GFAP.
Cells prepared according to Example 2 were
cultured for 14-30 days ~in vitro on glass coverslips
and then were fixed with 4% paraformaldehyde in PBS.
Following a 30 minute fixation period, coverslips were
.. washed three times (10 minutes each) in PBS (pH 7.3)
and then incubated in the primary antiserum (anti-GAGA
1:3000, anti-glutamine decarboxylase 1:500, and anti-
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-36-
substance P 1:100, in PBS containing 10% normal goat
serum/0.3% Triton-X-100) for two hours at 37'C.
Coverslips were washed three times (l0 minutes each) in
PBS and incubated with secondary antibodies (goat anti-
rabbit or goat anti-sheep conjugated to fluorescein or
rhodamine at 1:50) for 30 minutes at 37'C. Coverslips
- were then washed three times (10 minutes each) in PBS,
rinsed with water, placed on glass slides and
coverslipped using Fluorsave~'as the mounting medium.
l0 Fluorescence Was detected and photographed with a Nikon
Optiphot photomicroscope.
EXAMPLE 10
Cells prepared as in Example 2 were centrifuged at
300 r.p.m. for 3 minutes, the medium aspirated and then
the cells resuspended in warm DMEM/F-12 medium
containing 10% glycerol at a concentration of
approximately 1 x 10' cells, ml, and slowly frozen to
-20°C for 2 hours in a pre-cooled Styrofoam box with
the lid open. Once frozen, the cells Were transferred
to -80°C.. The cells were thawed by placing the
cryovial in a 37°C water bath, mixed by gentle
agitation, an equal volume of warmed medium added to
cells every 5 minutes for l5 minutes, and then the
volume brought up to 10 ml. The cells were then
centrifuged for 3 minutes at 300 rpm and the
supernatant discarded. 10 microliters of cells were
removed to determine viability and 10 microliters of
cells were removed and stained with Trypan Blue and
counted on a haemocytometer. Cells were resuspended in
3D DMEM/F-12/10% hormone solution at 100,000 cells/flask
and cultured 3-4 days at 37°C in T25 culture flasks.
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Cells are obtained from ventral mesencephalic
tissue from a human fetus aged 8 weeks following
routine suction abortion which is collected into a
sterile collection apparatus. A 2 x 4 x 1 mm piece of
tissue is dissected and dissociated as in Example 1.
The cells are then cultured as in Example 5.
Cells prepared as in Example 11 are used for
l0 neurotransplantation into a blood-group matched host
with a neurodegenerative disease. Surgery is performed
using a HRW computed tomographic (CT) stereotaxic
guide. The patient is given local anesthesia
supplemented with intravenously administered midazolam.
The patient undergoes CT scanning to establish the
coordinates of the region to receive the transplant.
The injection cannula consists of a 17-gauge stainless
steel outer cannula with a 19-gauge inner stylet. This
is inserted into the brain to the correct coordinates,
then removed and replaced with a 19-gauge infusion
cannula that has been preloaded with 30 ~cl of tissue
suspension. The c~lls are slowly infused at a rate of
3 ~cl/min as the cannula is withdrawn. Multiple
stereotactic needle passes are made throughout the area
of interest, approximately 4 mm apart. The patient is
examined by CT scan post-operatively for hemorrhage or
edema. Neurological evaluations are performed at
various post-operative intervals, as well as PET scans
to determine metabolic activity of the implanted cells.
CA 02399589 2002-08-22
' WO 93/01275 PCT/CA92/00283
-38-
ALE 13
Neural tissue is obtained from the ventricular
area by biopsy of a human subject and cultured as in
Example 5.
EXAMPLE 14
Cells proliferated as in Examples 1 or 2 are
transfected with expression vectors containing the
genes for the bFGF receptor or the NGF receptor.-
Vector DNA containing the genes are diluted in O.1X TE
(1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of
40~tg/ml. 220~e1 of the DNA is added to 2501 of 2X HBS
(280 mM N$Cl, 10 mM KCl, 1.5 mM Na~FIP04~2HZ0, 12 mM
dextrose, 50 mM HEPES) in a disposable, sterile 5 ml
plastic tube. 31 u1 of 2 M CaClz is added slowly and
the mixture is incubated for 30 minutes at room
temperature. During this 30 minute incubation, the
cells are centrifuged at 800g for 5 minutes at 4°C.
The cells are resuspended in 2o volumes of ice-cold PBS
and divided into aliquots of 1 x 10' cells, which are
again centrifuged. Each aliquot of cells is
resuspended in 1 ml of the DNA-CaClz suspension, and
incubated for 20 minutes at room temperature. The
cells are then diluted in growth medium and incubated
for 6-24 hours at 37°C in 5%-7% CO=. The cells are
again centrifuged, washed in PHS and returned to 10 ml
of growth medium for 48 hours.
Cells transfected as in Example 14 are
- transplanted into a human patient as in Example 12.
WO 93/01275 ~ 02399589 2002-08-22 p[7 /CA92/UU2~3:3
-39-
While the invention has been described and
illustrated herein by references to various specific
materials, procedures and examples it is understood
that the invention is not restricted to the particular
material combinations of material, and procedures
selected for that purpose. Numerous variations of such
details can be implied as will be appreciated by those
skilled in the art.
