Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Integration Of Transplanted Neural Progenitor Cells
Into Neural Tissue Of Immature And Mature
Dystrophic Recipients
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S.
Provisional Application No. 60/119,642, filed on
February 11, 1999, the whole of which is hereby
incorporated by reference herein.
BACKGROUND OF THE INVENTION
Proliferative cells present in the hippocampus of
adult rodent have been isolated, cultured, and
transplanted to various sites within the central nervous
system (CNS). These cells are capable of
differentiating into neurons when grafted to sites where
neurogenesis is known to occur (Suhonen 1996,
Shihabuddin 1997, Gage 1995). However, prior attempts
to use transplanted neurons to repopulate areas of
pathological cell loss within the CNS of adult mammals
have largely failed, because donor neural cells tend not
to integrate with host cells. For instance, attempts to
transplant neurons into the eye have not demonstrated
morphological integration with the host retina (del
Cerro 1992, Silverman 1992, Aramant 1994, Berson &
Jacobiec 1999).
As part of the central nervous system, both
developmentally and phenotypically, the retina shares
the recalcitrance of brain and spinal cord with respect
to functional repair. This is unfortunate because,
among heritable conditions alone, over 100 examples of
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diseases exist that involve the loss of retinal neurons
(Bird, 1995; Simunovic and Moore, 1998).
One attempted strategy for replacing diseased
retinal neurons has been to transplant retinal tissue
from healthy donors to the retina of the diseased host
(Gouras et al., 1994; Silverman and Hughes, 1989).
While the results of such studies have been encouraging
in terms of graft survival, the problem of morphological
and functional integration between graft and host has
remained daunting. The graft-host interface is often
well demarcated histologically, with ultrastructural
studies revealing the presence of a dense glial scar
across which few neurites are seen to cross (Inert et
al., 1998).
Thus, prior art findings have not provided a viable
solution to neural degenerative disorders, particularly
in adult animals.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to methods of
treating dystrophic neural tissue, particularly damaged
or diseased, differentiated neural tissue, in humans and
other animals. It is shown that neural progenitor cells
can functionally and morphologically migrate and
integrate into mature and immature neural tissue. In
particular, disclosed is the first successful, stable
morphological integration of neural progenitor cells,
e.g., adult hippocampal progenitor cells (AHPCs), into
the neural tissue of animals of various ages, including
immature, nondystrophic retina of syngeneic recipients
(e.g., Fischer rat-derived AHPCs into immature retina of
Fischer rats), and notably, diseased adult retina in
allogeneic recipients (e. g., Fischer rat-derived AHPCs
into dystrophic Royal College Surgeon (RCS) rats).
Surprisingly, AHPCs have also been found to integrate
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succesfully into a xenogeneic recipient: e.g., rat AHPCs
into the retina of dystrophic rd-1 mice.
Thus, the invention encompasses methods of
repairing, replacing, augmenting, or rescuing damaged or
diseased, differentiated neural tissue, by introducing
adult-derived neural progenitor cells into a human or
other animal recipient, whether the recipient is
syngeneic (of the same species and genetic strain),
allogeneic (of the same species but a different strain),
or xenogeneic (of a different species) to the donor. In
particular, the method comprises introducing neural
progenitor cells derived from a healthy donor into
dystrophic neural tissue of an animal recipient,
including an adult or a young animal. One embodiment of
the invention encompasses repopulating or rescuing a
dystrophic retina or optic nerve with neurons, by
introducing neural progenitor cells, e.g., AHPCs,
derived from an adult donor animal, into the dystrophic
eyes of an animal recipient.
The neural progenitor cells may be introduced into
dystrophic neural tissue by placement within a
recipient's central nervous system, an eye, an optic
nerve, or vitreous. The recipient can be either an
immature (young) or immature (adult) animal.
Advantageously, the neural progenitor cells are
derived from adult brain tissue, such as the hippocampus
or the ventricular zone. Neural progenitor cells are
preferably clonally derived. The neural progenitor
cells may, prior to introduction into a dystrophic
neural tissue site, have been cultured in vitro in a
culture medium comprising at least one trophic factor
selected from the group consisting of: a neural growth
factor; a neurotrophin; a mitogen; a cytokine; a growth
factor; a hormone; and a combination thereof.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1a-f depict the localization of grafted
AHPCs to specific retinal layers in recipient rats of
different ages (4 weeks (a-d); 10 weeks (e), and 18
weeks (f));
Figures 2a-i depict confocal images of expression
of neuronal markers by grafted AHPCs in animal grafted
at: 4 weeks, examined 4 weeks after grafting (a-c); at
weeks, examined 4 weeks after grafting (d-f): at 16
10 weeks, examined 1 week after grafting (g-I);
Figures 3a-h depict confocal images of grafted
cells treated with anti-synaptophysin/Cy3 (red) antibody
in animals grafted at 4 weeks, and examined 4 weeks
after grafting; and
Figures 4a-c present confocal images of GFP+
neurites projecting, via the host optic fiber layer,
into the optic nerve head 4 weeks after grafting (a+b).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to successful
transplantation of neural progenitor cells into
dystrophic neural tissue. In application, the invention
encompasses a method of treating dystrophic neural
tissue, comprising introducing neural progenitor cells
derived from an adult animal donor into dystrophic
neural tissue in an animal recipient, e.g., by grafting
or applying adult progenitor cells into tissue affected
by the disorder.
The recipient may be an young ( immature ) animal or
an adult (mature) animal. The neural progenitor cell
donor and recipient may be of different species
(xenogeneic). Exemplary donor-recipient pairs include,
but are not limited, to: a donor rat and a recipient
mouse; a donor mouse and a recipient rat; a donor pig
and a recipient human. The donor and recipient may be
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of the same species (e. g., human-to-human, rat-to-rat,
mouse-to-mouse), and be allogeneic (of different
strains, i.e., have different histocompatibility genes)
or syngeneic (of the same strain, i.e., having identical
histocompatibility genes).
Examples of dystrophic neural tissue that can be
treated by the invention include the central nervous
system (CNS) and neural tissue of the eye, particularly
the retina or optic nerve. Thus, in one embodiment, the
invention encompasses a method of repopulating or
rescuing a dystrophic retina with neural cells,
comprising introducing neural progenitor cells derived
from an adult donor (e. g., AHPCs) into dystrophic neural
tissue of an animal recipient. The method is
particularly useful for treating dystrophic retinal
tissue caused by an optic neuropathy, e.g., glaucoma.
As used herein, the term "dystrophic neural tissue"
encompasses damaged, injured, or diseased neural tissue,
which neutral tissue includes differentiated neural
tissue. Thus the present invention provides methods for
treating a neuronal or neural disorder or neural injury.
A "neuronal disorder" or "neural disorder" is any
disorder or disease that involves the nervous system.
One type of neuronal disorder is a neurodegenerative
disorder. Neurodegenerative disorders include but are
not limited to: (1) diseases of central motor systems
including degenerative conditions affecting the basal
ganglia (e. g., Huntington's disease, Wilson's disease,
Striatonigral degeneration, corticobasal ganglionic
degeneration, Tourettes syndrome, Parkinson's disease,
progressive supranuclear palsy, progressive bulbar
palsy, familial spastic paraplegia, spinomuscular
atrophy, ALS and variants thereof, dentatorubral
atrophy, olivo-pontocerebellar atrophy, paraneoplastic
cerebellar degeneration, cerebral angiopathy (both
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hereditary and sporadic)); (2) diseases affecting
sensory neurons (e. g., Friedreich's ataxia, diabetes,
peripheral neuropathy, retinal neuronal degeneration);
(3) diseases of limbic and cortical systems (e.g., s
cerebral amyloidosis, Pick's atrophy, Retts syndrome;
(4) neurodegenerative pathologies involving multiple
neuronal systems and/or brainstem (e. g., Alzheimer's
disease, AIDS-related dementia, Zeigh's disease, diffuse
Lewy body disease, epilepsy, Multiple system atrophy,
Guillain-Barre syndrome, lysosomal storage disorders
such as lipofuscinosis, late-degenerative stages of
Down's syndrome, Alper's disease, vertigo as result of
CNS degeneration; (5) pathologies arising with aging and
chronic alcohol or drug abuse (e.g., with alcoholism the
degeneration of neurons in locus oeruleus, cerebellum,
cholinergic basal forebrain; with aging degeneration of
cerebellar neurons and conical neurons leading to
cognitive and motor impairments; and with chronic
amphetamine abuse degeneration of basal ganglia neurons
leading to motor impairments; and (6) pathological
changes resulting from focal trauma such as stroke,
focal ischemia, vascular insufficiency, hypoxic-ischemic
encephalopathy, hyperglycemia, hypoglycemia or direct
trauma.
The presence of a neuronal or neurodegenerative
disorder or injury may be indicated by subjective
symptoms, such as pain, change in sensation including
decreased sensation, muscle weakness, coordination
problems, imbalance, neurasthenia, malaise, decreased
reaction times, tremors, confusion, poor memory,
uncontrollable movement, lack of affect,
obsessive/compulsive behavior, aphasia, agnosia, visual
neglect, etc. Frequently, objective indicia, or signs
observable by a physician or a health care provider,
overlap with subjective indicia. Examples of objective
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indicia include the physician's observation of signs
such as decreased reaction time, muscle fasciculations,
tremors, rigidity, spasticity, muscle weakness, poor
coordination, disorientation, dysphasia, dysarthria, and
imbalance. Additionally, objective signs can include
laboratory parameters, such as the assessment of neural
tissue loss and function by Positron Emission Tomography
(PET) or functional Magnetic Resonance Imaging MRI),
blood tests, biopsies and electrical studies such as
electromyographic data.
"Treating" dystrophic neural tissue is intended to
encompass repairing, replacing, augmenting, rescuing, or
repopulating the diseased or damaged neural tissue, or
otherwise compensating for the dystrophic condition of
the neural tissue.
"Introduction" of neural progenitor cells into
dystrophic neural tissue (e. g., a damaged or diseased
retina or optic nerve), may be accomplished by any means
known in the medical arts, including but not limited to
grafting and injection. It should be understood that
such means of introducing the neural progenitor cells
also encompass placing, injecting or grafting them into
a site separate and/or apart from the diseased or
damaged neural tissue site, since the neural progenitor
cells are capable of migrating to and integrating into
that dystrophic site. For example, dystrophic retinal
or optic nerve tissue can be treated by placing neural
progenitor cells into the vitreous of the eye.
The neural progenitor cells used in the invention
are derived from a healthy adult animal donor, and may
come from brain tissue, such as the hippocampus or
ventricular zone. Advantageously, adult -hippocampal
progenitor cells (AHPC) may be used, particularly
clonally derived AHPCs cultured in vitro under
proliferative conditions.
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_ g _
As used herein, the term "progenitor cell" refers
to cells which have the ability to differentiate,
including stem cells and progenitor cells. In contrast
to undifferentiated cells, differentiated cells have a
clearly defined morphology that identifies it as a
member of a defined histological type. The cell can be a
mammalian cell. In one embodiment, the mammalian cell is
a rodent cell. In another embodiment, the cell is a
primate cell, such as a human cell. Progenitor cells
employed herein refer to both undifferentiated cells
whose lineal descendants differentiate along the
appropriate pathway to produce a fully differentiated
phenotype, as well as founder cells of embryonic or
other cell lineage, which are undifferentiated cells
displaying high proliferative potential, generating a
wide variety of differentiated progeny including the
principal phenotypes of the tissue, possessing the
capacity for self-renewal and retaining their multi-
lineage potential over time (Gage et al. (1995) Annu.
Rev. Neurosci. 18:159-192, each herein incorporated by
reference). All differentiated cells have, by
definition, a progenitor cell type. For example,
"neural progenitor cells" such as neuroblasts are
progenitors for neurons and germ cells for gamete cells.
Additionally, it is readily appreciated that progenitor
cells do not differentiate into only one type of cell.
For example, neural progenitor cells give rise primarily
to neurons, however, such cells can also rise to
astrocytes, glial cells and oligodendrocytes. Those of
skill in the art will readily recognize the associated
progenitor cells for differentiated cells. Stem cells
are capable of dividing to produce two daughter cell
types with different fates: one is another stem cell
identical to the mother cell, and the other is a lineage
progenitor cell which will divide to produce more
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differentiated cells. In adult mammals, stem cells occur
in most tissue systems. For example, the bone marrow
gives rise to all blood cells and muscle.
The therapeutic benefit of the invention can be
evaluated or assessed by any of a number of subjective
or objective factors indicating a response of the
condition being treated. Such indices include measures
of increased neural or neuronal proliferation or more
normal function of surviving brain areas. In addition,
macroscopic methods of evaluating the effects of the
invention can be used which may be invasive or
noninvasive. Further examples of evidence of a
therapeutic benefit include clinical evaluations of
cognitive functions including object identification,
increased performance speed of defined tasks as compared
to pretreatment performance speeds, and nerve conduction
velocity studies.
In another aspect of the invention, the neural
progenitor cells have preferably been cultured in vitro
in a culture medium comprising at least one trophic
factor, or even combinations of such factors. As used
herein, the term "trophic factor" refers to compounds
with trophic actions that promote and/or control
proliferation, differentiation, migration, survival
and/or death (e. g., apoptosis) of their target cells.
Such factors include cytokines, neurotrophins, growth
factors, mitogens, co-factors, and the like, including
epidermal growth factor, fibroblast growth factor,
platelet-derived growth factor, insulin-like growth
factors, ciliary neurotrophic factor and related
molecules, glial-derived growth factor and related
molecules, schwanoma-derived growth factor, glial growth
factor, stiatal-derived neuronotrophic factor,
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platelet-derived growth factor, hepatocyte growth
factor, scatter factor (HGF-SF), transforming growth
factor-beta and related molecules, neurotransmitters,
and hormones. Those of ordinary skill in the art will
recognize additional trophic factors that can be
employed in the present invention (see, e.g., Aebischer
et al. Neurotrophic Factors (Handbook of Experimental
Pharmacology, Vol 134) (Springer Verlag, 1998); Meyers,
R.A. Encyclopedia of Molecular Biology and Molecular
Medicine: Denaturation of DNA - Growth Factors (VCH Pub,
1996); Meager & Robinson, Growth Factors . Essential
Data (John Wiley and Sons, 1999); McKay & Brown, Growth
Factors and Receptors: A Practical Approach (Oxford
University Press, 1998); Leroith & Bondy, Growth Factors
and Cytokines in Health and Disease, Vol lA and 1B . A
Multi-Volume Treatise (JAI Pr, 1996); Lenfant et al.,
Growth Factors of the Vascular and Nervous Systems:
Functional Characterization and Biotechnology:
International Symposium on Biotechnology of Grow (S.
Karger Publishing, 1992).
"Trophic factors" have a broad range of biological
activities and their activity and specificity may be
achieved by cooperation with other factors. Although
trophic factors are generally active at extremely low
concentrations, high concentrations of mitogen together
with high cell density are often required to induce
proliferation of multipotent neural progenitor cell
populations. For example, growth factors for early
progenitors may be useful for enhancing the viability of
progenitor cells as well as treating disorders by
renewal of mature cells from the progenitor cell pool.
Preferred trophic factors contemplated for use in
the present invention are mitogenic growth factors, like
fibroblast growth factor-2 (FGF-2) (Gage, F.H., et al.,
1995, Proc. Natl Acad. Sci. USA 92:11879-11883) and
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epidermal growth factor (EGF) (Lois, C., and Alvarez-
Buylla, A., 1993, Proc. Natl. Acad. Sci. USA 90(5):2074-
2077), which induce proliferation and/or propogation of
progenitor cells, e.g., neural progenitor cells isolated
from the brain. Studies from single cells in culture
demonstrate that FGF-2 (Gritti, A., et al., 1996, J.
Neurosci. 16:1091-1100) and EGF (Reynolds, B.A., and
Weiss, S., 1996, Develop. Biol. 175:1-13) are mitogens
for multipotent neural stem cells and likely cooperate
with other trophic factors (Cattaneo, E., and McKay, R.,
1990, Nature 347:762-765; Stemple, D.L., and Anderson,
D.J., 1992, Cell 71:973-985), some of which are yet
unknown (Davis, A.A., and Temple, S., 1994, Nature
372:263-266 Temple, S., 1989, Nature 340:471-473;
Kilpatrick, T.J., and Bartlett, P.F., 1993, Neuron
10:255-265 Palmer, T.D., et al., 1997, Mol. Cell.
Neurosci. 8:389-404) to achieve specificity.
As used herein, the neural progenitor cells can be
cultivated in the presence of a trophic factor, or
combinations of trophic factors. For example, these
cells can be cultivated in medium having "neurotrophins"
(or "neurotrophic factor") that promote the survival and
functional activity of nerve or glial cells, including a
factor that enhances neural differentiation, induces
neural proliferation, influences synaptic functions,
and/or promotes the survival of neurons that are
normally destined to die, during different phases of the
development of the central and peripheral nervous
system. Exemplary neurotrophins include, for example,
ciliary neurotrophic factor (CNF), nerve growth factor
(NGF), fibroblast growth factor (FGF), brain-derived
neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), glia
derived neurotrophic factor (GDNF), and the like. Such
factors are characterized by their trophic actions,
their expression patterns in the brain, and molecular
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aspects of their receptors and intracellular signaling
pathways. Neurotropic factors that have been identified
include NT-4 , NT-5 , NT-6 , NT-7 , ciliary
neuronotrophic factor (CNTF), Glial cell line-derived
neurotrophic factor (GDNF), and Purpurin. Neuron-
specific enolase (NSE) has been found to be a neuronal
survival factor. Other factors possessing a broader
spectrum of functions, which have neurotrophic
activities but are not normally classified as
neurotrophins, also are contemplated for use in the
invention. These factors include epithelial growth
factor (EGF), heparin-binding neurite-promoting factor
(HBNF), IGF-2, a-FGF and b-FGF , PDGF , neuron-specific
enolase (NSE), and Activin A. Other factors have been
identified which specifically influence neuronal
differentiation and influence transmitter phenotypes
without affecting neuronal survival. Although the
intracerebral administration of FGF-2 has been shown to
stimulate neurogenesis in the adult rat SVZ, FGF-2 alone
in the adult rat hippocampus has a limited effect on the
proliferation of neural stem/progenitor cells (Kuhn et.
al. (1997); Wagner et al. (1999) each herein
incorporated by reference).
In a preferred embodiment of the present, the
present invention employs FGF and FGF-like factors,
including a-FGF, b-FGF such as FGF-2, FGF-4, FGF-6, and
the like. A particularly advantageous medium for
culturing neural progenitor cells comprises one of the
following: fibroblast growth factor (FGF) alone
(particularly basic FGF or FGF-2), FGF plus epidermal
growth factor (EGF), or FGF plus EGF plus heparin, which
is mitogenic.
For example, the neural progenitor cells may be
derived by the following steps:
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(a) isolating fresh neural progenitor cells from
an adult donor animal;
(b) culturing said freshly isolated neural
progenitor cells on a polyornithene/laminin-coated
substrate, in a culture medium containing at least one
trophic factor, selected from the group consisting of
FGF-2 alone, FGF-2 plus EGF, and FGF-2 plus EGF plus
heparin;
(c) incorporating an identifying, genetic marker
into said cultured progenitor cells; and
(d) cloning individual hippocampal progenitor cell
lines from the cultured cells resulting from step (c).
Additionally, the methods of the invention can
further comprise, prior to introducing the neural
progenitor cells into a recipient, confirming the
lineage potential of each clone of clonally derived
adult hippocampal progenitor cells by inducing a sample
of said clonally derived hippocampal progenitor cells to
differentiate in "conditioned medium", a term of art
referring to medium or supernatant removed from cultures
of living cells and then filtered.
The invention also encompasses a kit for generating
neural progenitor cell lines derived from an adult donor
animal, comprising the following:
(a) a polyornithine/laminin-coated substrate
(e. g., a coated tissue culture vessel);
(b) a culture medium containing at least one
trophic factor selected from the group consisting
of or a combination thereof;
(c) a vector comprising an identifying
genetic marker, for incorporation into hippocampal
progenitor cells (HPC) isolated from an adult
animal, upon culture of those cells (e. g., green
fluorescent protein (GFP));
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(d) an article of manufacture comprising
instructions for cloning at least one hippocampal
progenitor cell line from hippocampal progenitor
cells isolated from an adult donor animal.
'
In yet another embodiment of the present invention,
there are provided methods for treating neuronal
disorders, the method comprising increasing the level of
adult progenitor cells in dystrophic tissue. Progenitor
cells can be grafted into the tissue ex vivo (by
cultivating the cells in vitro) or can be cultivated in
vivo. As contemplated herein, the progenitor cells can
be native to the dystrophic tissue but propagated and/or
proliferated by the administration of trophic factors in
vivo or in vitro. In a preferred embodiment of the
present invention, the progenitor cells can be
propagated or proliferated in vitro, and incorporated or
re-incorporated into the dystrophic tissue.
Alternatively, trophic factors can be administered to
the dystrophic tissue to increase the level of native or
transplanted progenitor cells.
The invention is further described by way of the
following, non-limiting examples.
Example I: Intra-Species, Allogeneic Retinal Transplant
Clonally derived, adult rat hippocampal progenitor
cells (AHPCs), genetically modified to express green
fluorescent protein (GFP), were injected into the eyes
of dystrophic RCS rats of various ages. When
subsequently examined, the retinas of these animals
exhibited widespread migration of green fluorescent
protein-expressing (GFP+) donor cells into all layers of
the host retina. The transplanted cells survived for at
least 2 months post-grafting, without provoking a
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prominent immune response. Furthermore, GFP+ cells
aligned themselves with the existing cytoarchitecture
and exhibited extensive arborization in configurations
appropriate for retinal neurons. Similar results were
obtained with both immature and visually mature,
recipient animals. These results indicate that the
dystrophic retina can be substantially repopulated by
using a line of adult-derived, neural progenitor cells
from an allogeneic donor, and that these cells can be
functionally integrated, since they arborize extensively
within the host neuropil.
It has recently been shown that proliferative cells
present in the adult rodent hippocampus (Altman and Das,
1965) can be isolated (Palmer et al., 1997), cultured
(Gage et al., 1995 and 1998), and transplanted into
various sites within the CNS, where they can
differentiate into neurons (Gage et al., 1995;
Shihabuddin et al., 1997; Suhonen et al., 1996). The
present data indicate that transplanted, adult
hippocampal progenitor cells (AHPCs) provide a more
effective source of donor material for retinal
transplantation. Specifically, the data show that these
cells can migrate into the dystrophic retina of adult
Royal College of Surgeons (RCS) rats, an extensively
studied model of retinal degeneration (LaVail et al.,
1975; Matthes and LaVail, 1989; Villegas et al., 1998).
That is, transplanted AHPC cells can migrate into, and
differentiate within, the mature retina during the
active phase of neuronal degeneration.
Methodology
Donor cell line: Hippocampal progenitor cells were
clonally derived from adult Fischer 344 rats,
genetically modified to express the modified jellyfish
(Aequorea victoria) enhanced green fluorescent protein
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GFP (eGFP). In some cases, the cells were pulsed prior
to transplantation with BrdU (e.g., 5 um, 2 days, or
more preferably, 50 ng/ml, 3 pulses over 3 days).
Specifically, AHPCs were cultured and differentiated as
follows. Primary adult hippocampal progenitor cultures
were prepared from hippocampal tissues of 3-month-old
female Fisher 344 rats as previously described (Gage et
al. 1995a). Dissociated cells were cultured on
polyornithine/laminin coated dishes using a mixture of
DMEM/Ham' F-12 (1:1) supplemented with N2 (Gibco) and 20
ng / ml FGF-2 (human recombinant, prepared in E. coli,
kindly provided by A. Baird). Individual cells were
genetically marked using replication-defective
retroviral vectors expressing GFP from a tetracycline-
regulatable, minimal human cytomegalovirus immediate
early promoter fused to a tet-operator (NIT-GFP).
Cloned cultures were derived from bulk-injected
cultures. Each AHPC clone carried a neomycin
phosphotransferase gene (neo) and the enhanced green
fluorescence protein (GFP) gene. To confirm the lineage.
potential of each clone prior to grafting, AHPCs were
induced to differentiate in 4-well chamber slides at a
cell density of 2,500 cells per cm2 by withdrawal of
FGF-2 and treatment for 14 days in DMEM/F12 + N2,
supplemented with 0.5 uM all-traps retinoic acid and
0.5o fetal bovine serum. These conditions were
previously shown to favor the differentiation of
neurons, astrocytes, and oligodendrocytes in a single
well (Palmer et al. 1997). AHPCs were prepared for
grafting in the following manner. Cultured AHPCs were
harvested with trypsin, washed with high glucose
Dulbecco's PBS (D-PBS, Gibco), and suspended at a
density of 100,000 cells per ul in D-PBS containing 20
ng of FGF-2 per ml.
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Recipient animals and transplantation: At the age of
3-28 days, pigmented dystrophic RCS rats (graft duration
before sacrifice: 1 week, n=22; 4 weeks n=41; 10 weeks,
n=6; 18 weeks, n=4; 36 weeks, n=9), and albino
dystrophic rats (graft duration: 1 week, n=8; 10 weeks,
n=6) received injections of AHPCs into the vitreous or
subretinal space under general (Ketamine/xylazine) and
topical (proparacaine) anesthesia. Injections were
performed under direct observation using coaxial
illumination via binocular surgical microscope (holler)
through a dilated pupil (topical tropicamide lo). The
injections were made via a beveled glass micropipette
(outer diameter of 1 mm) connected to a 50-ul Hamilton
microsyringe via PE tubing. The sharp tip of the
micropipette allowed direct entry to the vitreous cavity
through a self-sealing wound, the entry point being just
vitread to the corneo-scleral junction. This approach
to the vitreous avoided trauma to the ciliary body and
lens, but necessarily resulted in focal perforation of
the intervening uvea and peripheral retina. A total of
50,000-100,000 cells in 1-2 ul of DMEM/F12 media were
injected. As a control, cells that were freeze-thawed 3
times (from -70 °C) were also injected (n=6).
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Tissue preparation and histology: Recipient animals
were killed with an overdose of sodium pentobarbitol at
1, 2, 4, 8, and 16 weeks post-transplantation. The eyes
were removed and immersion-fixed with 40
paraformaldehyde for 4 hours at 4 °C. The anterior
segment and lens were then removed, and the posterior
segment cryoprotected in 30% sucrose/PBS overnight at 4
°C, followed by embedding in OCT and subsequent
sectioning at 7-14 um on a cryostat. Sections were
processed for haematoxilin and eosin, anti-BrdU (1:400),
anti-synaptophysin (1:200) and anti-GFP (1:500), anti-
calbindin (1:1000), anti-rhodopsin (1:200), or anti-NF-
200 (1:40), anti-MAP-5 (1:500), anti-GFAP (1:200),
followed by reaction with Cy3-conjugated secondary
antibodies (1:150), thus allowing co-localization of
these markers with the endogenous GFP expressed in
transplanted AHPCs. Confocal microscopy was carried out
on a subset of material that was of particular interest.
Morphology: Retinae containing high numbers of grafted
cells were analyzed to determine their laminar
localization at 4 and 8 weeks post-transplantation. Age
at time of transplantation (1, 4, and 10 weeks) was also
compared. A total of nine 50 um wide regions of
sectioned retina were analyzed for each animal, chosen
so that both central and peripheral regions were
included.
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Results
Clonally derived AHPCs from adult Fischer 344 rats,
which were genetically modified to express green
fluorescent protein (GFP) and also labeled with BrdU in
some cases, were transplanted into both immature (3 days
postnatal, P3) and mature (4-36 weeks postnatal),
dystrophic eyes of RCS rats. Following transplantation,
donor-derived cells were found to maintain high levels
of GFP expression. GFP+ cells were clearly evident
under FITC illumination and were verified to be of graft
origin based upon anti-GFP immunoreactivity, anti-BrdU
immunoreactivity, as well as constitutive GFP expression
(data not shown). The GFP+ cells were quite striking in
appearance and were easily distinguished from
autofluorescence of host photoreceptor outer segments in
the recipient, based on intensity, morphology, location,
and spectral specificity. Subsequent identification of
donor-derived cells was therefore based on GFP
fluorescence alone, obviating the need for prelabeling
with BrdU or the use of anti-GFP antibodies.
At 4 weeks following the injection of AHPCs into
the vitreous of immature and mature dystrophic RCS rats,
at least 500 of the injected cells survived and
maintained high levels of GFP expression in
approximately 800 of the 1, 4, 10, and 18 week old
recipients, while no surviving cells were found in the
36 week old recipients.
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Already at one week post-grafting, grafted AHPCs
could be seen adhering to the vitreal surface of the
graft recipient or host's eye, migrating into the host
retina, and taking up residence within the cellular
retinal laminae of the host, including the outer nuclear
layer. In some cases, grafted cells were seen in the
host photoreceptor layer, and when examined with anti-
BrdU, were found to be double labeled with GFP and BrdU,
confirming the cells' derivation from the transplanted
AHPCs. No evidence of viable donor cells, or host GFP
expression, was seen following injection of freeze-
thawed GFP+ AHPCs (negative control), confirming
observations reported in Takahashi 1998, incorporated
herein by reference.
At subsequent times post-grafting, widespread
migration and morphological integration of grafted AHPCs
into the host retina was seen. GFP+ cells were found
within the retina of 600, 350, 480, and 600 of animals
grafted at the ages of 1, 4, 10, and 18 weeks,
respectively. At 8 weeks post-grafting, intra-retinal
GFP+ cells were found in 800 of the recipients who were
1 week old at the time of grafting, and 500 of those
initially 4 weeks old.
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TABLE I
Percentage of GFP-expressing cells found
in each region of the retina
1 week 4 week 10 week
old old old
recipient recipient recipient
Region 4 weeks 8 weeks 4 weeks 8 weeks 4 weeks
post- post- post- post- post-
transplanttransplanttransplanttransplanttransplant
Vitreous74,4+18.715.3+4.1 28.7+7.9 2.2+2.0 98.86.7
GCL/IPL 5.81.5 7.2+4.2 4.9+1.2 32.36.3 7.91.7
INL OPL 4.9+2.4 17.5+3.4 17.05.7 5.41.6 32.53.3*
ONL SRS 14.9+4.7 60.0+10.6 49.39.7 60.17.6 10.8+3.2*
1 0 GCL/ILP = ganglion cell layer and inner plexiform layer
INL/OPL = inner nuclear layer and outer plexiform layer
ONL/SRS = outer nuclear layer and subretinal space
*N.B. At this time point clearly defined outer plexiform and outer nuclear
1 5 layers are not present
Table 1 shows the laminar distribution of migrating
20 AHPCs in representative 1, 4, and 10 week old
recipients. The majority of grafted cells left the
vitreous and entered the retina, where they migrated
into the various laminae. Although grafted cells were
also found in the ganglion cell and inner nuclear
25 layers, they showed a predilection for the outer retina,
particularly the outer nuclear layer, subretinal debris
zone and intervening layer of photoreceptor elements
(collectively designated "ONL/SRS"). At a later time
point (8 weeks post-injection), the number of cells in
30 the ONL/SRS was greater yet. GFP+ cells appeared to
gain access to the retina either by direct radial
migration through the undamaged vitreal surface or, in
greater numbers, by way of the peripheral injection
tract with subsequent lateral migration. In the latter
35 case, cells could be found migrating into as much as 600
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of the longitudinal extent of the neuroretina.
Regardless of the course taken by the migrating AHPCs,
GFP+ cells were found in all layers of the host
neuroretina, but not in the retinal pigment epithelium,
choroid, or sclera.
Figures la-f depict the localization of grafted
AHPCs to specific retinal layers in rats of different
ages (4 weeks (a-d); 10 weeks (e), and 18 weeks (f)).
Cells (green) were grafted into the vitreous of 4 (a-d),
10 (e), and 18 (f) week old rats, and examined 4 weeks
later. Retina sections were labeled with anti-
synaptophysin/Cy3 antibody (red) to demarcate the
synaptic and cellular layers of the host retina, and
viewed under FITC and Cy3 fluorescent illumination. The
arrow in Fig. la indicates cell seen in Fig. 1b at
higher power; the arrow in Fig. 1c indicates cell seen
in Fig. 1d at higher power (vit, vitreous; gcl, ganglion
cell layer; ipl, inner plexiform layer; inl, inner
nuclear layer; opl, outer plexiform layer; onl, outer
nuclear layer; srs, subretinal debris and degenerating
photoreceptor elements).
In Figure 1, numerous GFP+ cells exhibiting
neuronal morphologies can be seen. These cells were
found in all cellular layers of the host retina, yet
tended to respect the plexiform layers (particularly the
inner plexiform layer) where they elaborated arbors.
Moreover, the configuration of the neuritic processes
extended by grafted cells often resembled those of
normal retinal neurons: neurites preferentially
projected either laterally (i.e. resembling those of
horizontal or amacrine cells) or radially (i.e.
resembling bipolar cell processes; see Fig. lb).
Whether this reflects intrinsic or extrinsic
developmental factors, or is simply a consequence of
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restrictions imposed by the local retinal
cytoarchitecture remains to be determined.
Additionally, further study of recipient animals
treated according to the method of the invention (aged 1
week at time of transplant), has shown that the grafted
cells exhibit axonal growth into the optic nerve at or
about 8 weeks post-graft.
A number of markers were evaluated to determine
whether grafted cells had adopted mature neuronal
phenotypes. The results are shown in Figure 2a-i.
Figure 2a-i depict confocal images of expression of
neuronal markers by grafted AHPCs. Figs. 2a-c are from
animals grafted at 4 weeks of age, examined 4 weeks
after grafting: constituitive GFP expression (a), anti-
calbindin/Cy3 immunoreactivity (b), and merged image
(c). The arrows indicate 2 cells co-expressing these
labels. Figs. 2d-fare from animals grafted at 10 weeks
of age, examined 4 weeks after grafting: constitutive
GFP expression (d), MAP-5/Cy3 immunoreactivity (e),
merged image (f). The arrows indicate 2 cells co-
expressing these labels. Fig. 2g-i are from animals
grafted at 16 weeks of age, examined 1 week after
grafting: constituitive GFP expression (g), anti-NF-200
/Cy3 immunoreactivity (g), and merged image (i). Arrows
indicate 2 cells co-expressing these labels.
A subpopulation of GFP+ cells were found to co-
express calbindin, a marker found on some retinal
interneurons (Fig. 2a-c), while others co-expressed the
neuronal marker MAP-5 (Fig. 2 d-f) or NF-200 (Fig 2 g-
I). These results contrast to an earlier report, in
which AHPCs grafted into the developing eye of normal
animals failed to express neuronal markers (Takahashi
1998). While these markers are not retina-specific,
they do show that hippocampal-derived progenitor cells
are capable of developing mature neuronal phenotypes
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when grafted to a novel site such as the retina.
Furthermore, the expression of these markers was
regionally appropriate, with calbindin expression
confined to transplanted AHPCs in the inner nuclear
layer, and NF-200 expression seen predominantly in the
ganglion cell layer. Significantly, grafted AHPCs did
not show any evidence of GFAP expression or astrocytic
morphological development, suggesting a preference for
neuronal differentiation in the microenvironment of the
degenerating retina.
As GFP+ cells frequently developed elaborate
neuronal arbors, the relationship between donor neurites
and synaptophysin expression was investigated. Although
widely dispersed throughout the retina, the vast
majority of synaptophysin seen was localized to the
plexiform layers, consistent with host origin. From
their positions in the cellular layers, grafted cells
frequently extended processes into these layers,
apparently in a directed manner.
Figure 3a-h depicts confocal images of grafted
cells treated with anti-synaptophysin/Cy3 (redj
antibody, which show grafted AHPCs (green) sending
processes into the inner plexiform layer (a-d), or the
outer plexiform layer (e-h) (grafted at 4 weeks,
examined 4 weeks after grafting). In Fig. 3a-b, a cell
is shown merged (a), and reconstructed to show entire
neuritic arbor (b). In Fig. 3c-h, AHPCs send neurites
into the inner plexiform layer (c, higher power in d),
and outer plexiform layers (e + g, higher power in f +
h, respectively). These processes intermingle with, and
appear to contact synaptophysin-positive profiles of the
host.
In Figs. 3 a-d, large GFP+ cells are observed to
send neuritic processes into the host inner plexiform
layer, while Figs. 3 e-h show cells with elaborate
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arbors intermingling with the host outer plexiform
layer. Figs. 3 a-b provide one example of the way in
which the configuration of GFP+ arbors frequently
reflects the orientation of the host plexiform layers.
One process tracks along the INL/IPL interface while
another, originating from an position offset within the
ILP, assumes a parallel course in the opposite direction
despite the lack of a laminar interface to guide it.
Confocal analysis confirmed that large numbers of GFP+
processes come into direct apposition to host
synaptophysin + profiles (Fig. 3e-h; 4 weeks post-
grafting into 4 week old hosts).
Grafted AHPCs are also capable of extending
processes into the host optic nerve. Grafted cells
residing in the ganglion cell layer extend neurites with
large growth cones that approach, but do not cross, the
level of the scleral outlet at 4 weeks post-grafting
into 1 week old hosts (Figs. 4a-b). Figures 4a-c show
confocal images of GFP+ neurites projecting, via the
host optic fiber layer, into the optic nerve head 4
weeks after grafting. These fibers have large growth
cones (arrows in Fig. 4a, and in higher power in
Fig. 4b), which approach, but do not cross, the scleral
outlet (labeled "sc") at 4 weeks post grafting into
initially 1 week-old hosts. When animals were examined
8 weeks after grafting, numerous growth cone-tipped
processes were found to have entered the optic nerve,
extending over 300 um beyond the scleral outlet
(Fig. 4c).
When examined at 8 weeks post-grafting, large
numbers of growing neurites were found to cross the
scleral outlet, and extend long processes at least 300
um into the optic nerve (Fig. 4c). The apparent
increased density of GFP+ processes at successive time
points indicate that AHPCs continue to develop along a
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neuronal-like pathway for at least 8 weeks post-
grafting.
No evidence of immunological rejection, decreased
cell survival, or decreased gene expression was observed
over the course of this study. The range of graft
survival and incorporation obtained in different aged
hosts (high level of incorporation in animals up to 10
weeks of age at time of transplant, lower level of
incorporation seen in 18 week-old recipients, no
survival in 36 week-old recipients) suggests that the
progressive degeneration occurring in the RCS retina
(which begins at 3 weeks of age) contributes to this
variability. As the rat retina is fully developed
before the end of the 3rd postnatal week, the widespread
incorporation seen at 4 and 10 weeks indicates that
developmental maturity is not a barrier to the
acceptance of AHPCs by the diseased mammalian retina.
Discussion
This study shows that neuronal progenitor cells
derived from adult, differentiated neural tissue (e. g.,
hippocampus), can migrate in large numbers into all
layers of the dystrophic neuroretina of mature animals,
including, in some cases, the photoreceptor layer.
Following migration, transplanted AHPCs respect the
local laminar organization and exhibit a surprising
ability to differentiate into neurons with morphological
characteristics suggestive of native retinal cell types.
The cell processes extended by AHPCs within the retina
tend to resemble the neuritic profiles of specific
retinal neurons, including sublamina-specific
ramifications within the inner plexiform layer
suggestive of bipolar and horizontal cells (bowling,
1970). Furthermore, the presence of distinct bands of
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diffuse GFP-derived fluorescence along these sublaminar
zones suggests a network of fine terminals within the
host neuropil.
These data indicate that neural progenitor cells
such as AHPCs are capable of functional integration into
the retina of animals up to 10 weeks of age, as well as
limited incorporation into 18-week-old recipients, an
age when the RCS retina has degenerated severely and
other interventions are ineffective. At 36 weeks of
age, however, AHPCs not only fail to enter the retina
but show very little survival, suggesting the loss of an
important trophic influence late in the course of the
dystrophy.
Having migrated into the retina from the vitreous,
grafted AHPCs disperse within the host tissue rather
than remaining adherent to each other, as is typically
seen with embryonic neural grafts. After taking up
residence, these cells differentiate along neuronal (as
opposed to filial) lines and extend processes within the
host plexiform layers. Furthermore, the orientation of
many of these processes is reminiscent of the
arborization pattern of retinal amacrine cells. AHPCs
in the ganglion cell layer frequently extend neurites
into the optic fiber layer and optic nerve.
More recently, neural progenitor cells have
reportedly been found to differentiate into cells of the
hematopoietic lineage (Bjornson 1999), suggesting that a
hippocampal to retinal fate shift should not be
dismissed. Morphologically, AHPC arborizations appear
to respond to extrinsic retinal cues in preference to
any intrinsic hippocampal developmental programs.
Finally, while the finding reported here that graft-
derived neurites are intimately associated with host
synaptophysin profiles does not conclusively demonstrate
synapse formation, another laboratory using this same
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AHPC cell line has recently provided electron
microscopic evidence of synapse formation in vitro, as
well as excitatory post-synaptic potentials (Toda 1999).
These results reinforce the conclusion that the
neuronal repopulation method achieved here represents
morphological and functional integration, rather than
simply cellular infiltration or random migration and
neurite extension.
Neural progenitor cells such as AHPCs migrate and
integrate into neonatal, nondystrophic, syngeneic
Fischer rat hosts (see Takahasi 1998, incorporated
herein by reference). AHPCs also readily migrate into
mechanically injured retina of adult, syngeneic hosts as
well as diseased retina of mature, allogeneic RCS rat
hosts.
The preceding results are consistent with more
recent studies in which stem or progenitor cells seem to
respond to the presence of pathology. For instance,
neural stem cells grafted to the bloodstream of
irradiated mice repopulate the bone marrow (Bjornson
1999), while similar cells grafted to the cerebral
ventricles of neonatal shiverer mice replace lost
oligodendrocytes (Yandava 1999). Neural progenitor
cells clearly possess a high degree of plasticity
(Johansson 1999, Flax 1998, Brustle 1998, Morrison 1999)
and provide a new tool for studying mechanisms of neural
development and degeneration.
The data presented here provide the first
definitive evidence for the survival, migration, and
neuronal differentiation of a transplanted cell in
diseased, mature retina. This study shows that neural
progenitor cells can overcome many of the obstacles to
neuronal integration present in the mature mammalian
central nervous system.
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The observations here of widespread morphological
integration in an allogeneic situation, also argues for
the importance of the specific microenvironment of the
host retina in promoting migration and differentiation
of grafted precursor cells. The same is expected for
other specialized or differentiated neural tissue into
which AHPCs are integrated.
The present invention will enable the attainment of
the ultimate goal of restorative neuronal
transplantation into the eye: introduction of new
photoreceptor cells. The data demonstrate that
neuroprogenitor cells such as AHPCs are capable of
repopulating the outer nuclear layer of the dystrophic
retina with cells resembling neurons. The surprising
degree of plasticity exhibited by AHPCs transplanted to
the diseased eye indicates that using neural progenitor
cells to repopulate the eye with photoreceptor cells,
seemingly impossible only a few years ago, is now a
realistic objective.
One of ordinary skill in the art of neuronal
transplantation will appreciate how to practice the
present invention and to manipulate AHPCs and other
neural progenitor cells to account for such factors as
functional capability, host immunological tolerance, and
the long-term consequences of grafting (e. g., promoting
graft survival and controlling undesired proliferation).
The demonstration here of survival in a dystrophic,
allogeneic environment for at least 2 months, indicates
the ultimate immunological success of progenitor cell
transplantation to the diseased central nervous system.
Neuroprogenitor cells like AHPCs are capable of
reaching all layers of the retina, and differentiating
into cells with local phenotypic characteristics. These
cells represent an exciting new tool for studying and
manipulating retinal development in mammalian species.
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Since neural progenitor cells can be propagated in vitro
and, following transplantation, can extensively
repopulate an actively degenerating retina in visually
mature animals, they will also be useful in treating
retinal diseases involving neuronal cell loss. In view
of the results discussed herein, it is reasonable to
expect that AHPCs and other neural progenitor cells
would similarly be able to differentiate into the
appropriate neuronal cell lineage of other neural sites
into which these progenitors are transplanted in vivo.
Therefore, AHPC. transplantation can be useful also to
treat other neurological diseases and injuries involving
neuronal loss or damage.
Example II: Xenogeneic Retinal Transplants
The survival of adult rat-derived, hippocampal
neural progenitor cells transplanted into the dystrophic
mouse retina was investigated. These transplanted cells
were capable of integrating into the murine host retina
and of maintaining expression of the green fluorescence
protein (GFP) gene inserted into the progenitor cells.
Methodology
Neural progenitor cells, cultured from the
hippocampus of adult Fischer 344 rats, were genetically
modified to express GFP and a clonal cell line was
isolated, as previously described. These cells were
then transplanted into the vitreous of 7-day-old
"rd-1" mice (50,000 cells in 1 ul), without
immunosuppression. After 2-4 weeks post-transplant, the
eyes were removed and sectioned.
D~~."l tc. or,rl Tli cn"cci nn
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At both survival times (2 weeks and 4 weeks post-
transplant), large numbers of GFP+ cells were found in
the vitreous of host mice. Many cells were adherent to
the inner surface of the retina, where they extended
long, axon-like processes. In some cases, cells were
found to have migrated into the host retina, where they
developed neuron-like phenotypes, and extended numerous
processes into the host neuropil.
Rat, adult neural progenitor cells transplanted to
a xenogeneic environment without immunosuppression are
capable of surviving for at least 4 weeks and
maintaining expression of a GFP marker. These cells can
also migrate into the host retina, where they developed
neuron-like phenotypes. The use of xenogeneic,
pluripotent progenitor cells as a source of donor tissue
in transplantation protocols offers a viable new
technology for studying and manipulating neural
development and neural tissue plasticity, and repairing
damaged central nervous system (CNS) tissue. In the
case of human disease, the present technology will
enable the use of xenogenic, neural tissue, such as pig-
derived neural progenitor cells, to treat retinal and
other neurological diseases and injuries involving
neuronal loss.
Example III: Physiological improvement in rats receiving
neural progenitor cell transplants
Neuroprogenitor cells such as AHPCs have the
capacity to restore vision in blind rodents. Recent
experiments have demonstrated that grafts of AHPCs into
the eye of RCS rats leads to behavioral recovery, as
measured by a optokinetic nystagmas (OKN) reflex test.
OKN is an involuntary reflex, which depends upon visual
acuity level to generate a response to rotating contrast
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gratings. These gratings can be varied by intensity,
contrast, and frequency to precisely determine visual
acuity. Animals grafted with AHPCs can possess an OKN
response, whereas control animals do not. Two possible
mechanisms can explain this visual behavior:
1) Grafted AHPCs are actively integrated in
the retinal cytoarcitecture, and are contributing
in some manner to the visual pathway, either as
photoreceptors, interneurons, and/or retinal
ganglion cells; and/or
2) Grafted AHPCs lead to rescue of host
photoreceptors. If the latter is true, AHPC
grafting has great promise in the growing field of
growth factor delivery, as grafted cells can
integrate into the host retina in a stable manner,
and can be modified in vitro to secrete specific
molecules into the host retina.
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Other uses of multipotent neural stem/progenitor cells
for cell replacement in different disorders.
AHPCs have been grafted in adult rat hippocampus
where they migrate and differentiate into neurons in the
dentate gyrus (Gage et al., 1995). The site specific
migration and integration of these cells have been
tested by grafting them in the rostral migratory pathway
leading to the olfactory bulb (Suhonen et al., 1997).
Cells migrated along the RMP and then laterally to
granule cell and gromular cell layers. Cells migrating
to the granule cell layer became calbindin/NeuN+ cells.
and those migrating to gromeruli became tyrosine
hydroxylase+ neurons (typical phenotypes of cells
residing in these regions). Similarly, clonal
populations of AHPCs grafted in neonate eyes migrated to
different layers assuming the morphological
characteristics of cells present at those layers.
However, they did not express any of the markers
specific for eye cells (Takahashi et al., 1998). In
addition, adult spinal cord-derived progenitor cells,
when grafted in the spinal cord, have been found to
generate only glial cells. In contrast, in the
hippocampus, they migrate in a similar fashion to AHPCs
and differentiate into neurons only in the dentate gyres
(Shiabuddin, Horner, Ray and Gage, unpublished results).
These results indicate that AHPCs are plastic and that
their ultimate fate has been guided by the external
stimuli present in a specific region of the organ and
not by their internal programming. These observations
suggest that these cells can be used for grafting in
organs very different from their site of origin. This
hypothesis has recently been confirmed by a report
showing that stem/progenitor cells derived from adult
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mouse brain when transplanted into irradiated mice
produce a variety of blood cell types (Bjornson et al. ,
1999). Two recent reports have shown that fetal human
brain-derived neural stem cells grafted into embryonic
rats or new born mice participate in aspects of normal
development. Grafted cells migrate, incorporate into
all major compartments of the brain, and differentiate
into multiple developmentally and regionally appropriate
cell types (Flax et al., 1998, Brustle et al., 1998).
These data indicate that xenografts of multipotent
neural stem/progenitor cells not only survive, they
behave like endogenous cells of the recipient species.
The ultimate fate of the grafted cells is determined by
the endogenous stimuli present in specific brain
regions.
Grafting of multipotent neural stem/progenitor cells in
various organs for reconstitution in various diseases
and disorders.
The plastic and pluripotent nature of multipotent
neural stem/progenitor cells derived from rat, mouse and
human have made them ideal candidates for their use as a
source of cells which can be used to replace or correct
for cells lost in disease or injury. The utility of
these cells for transplantation can be tested in the
following disease models.
Liver diseases: The liver plays a central role in
the pathophysiology of many inherited metabolic
diseases. Despite the unusual ability of the adult liver
to regenerate after injury, the liver is an important
target for cell therapy. Two separate transgenic mouse
models have been established wherein the animals produce
a toxic by-product that damages or kills hepatocytes. In
albumin-urokinase (Alb-uPA) transgenic mice,
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hepatocyte-targeted expression of a hepatotoxic
transgene creates a functional liver deficit leading to
chronic stimulus of liver growth (Rhim et al., 1994). In
a second murine model for human hereditary liver
disease, tyrosinaema type I (HTI), a recessive liver
disease, is caused by deficiency of fumarylacetoacetate
hydroxylase (FAH). Transplantation of hepatocytes from
normal animals to spleens of adult transgenic animals
showed that the transplanted cells can repopulate 80-90%
of the diseased livers. We propose to transplant adult
rat hippocampal-derived progenitor cells (AHPCs) or
adult mouse-brain derived progenitor cells (AMPCs), or
fetal or adult human brain-derived progenitor cells, in
to these animal models to determine whether
brain-derived progenitor cells can respond to local
environment of the spleen, become hepatocytes, and
replace the dying cells to correct the disease
phenotypes.
Diabetes: Recent experimental data from immune and
endocrine studies using spontaneous or transgenic models
of the disease have emphasized the role of the islet of
Langerhans, and particularly beta cells, in autoimmune
insulin-dependent (Type 1) diabetes mellitus (IDDM)
pathogenesis. IDDM is a chronic disorder that results
from the destruction of the insulin-producing beta cells
of the pancreatic islets. In its initial phase, T
lymphocytes and other inflammatory cells invade the
islets, eventually destroying them. The pathological
consequence is the inability of the animals to maintain
glucose homeostasis. Most work has focused on the
spontaneous model of the disease, the non-obese diabetic
(NOD) mouse, which in addition to providing genetic
data, appears to be useful for sequential study of the
early developmental, immune and endocrine events that
occur in IDDM pathophysiology. A transgenic line
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overexpressing a T-cell receptor (TCR) that recognizes a
natural autoantigen recognized in IDDM has been
developed. Viable islet cells isolated from pancreas
have been transplanted, resulting in complete reversal
of hypoglycaemia in diabetic animals (Thomas et al.,
1990). AHPCs, AMPCs, or fetal or adult human
brain-derived progenitor cells can be grafted in the
pancreas of the NOD or transgenic mice to determine
whether hypoglycemia can be corrected by the replacement
of the damaged cells with the grafted cells.
Muscle disorders: Duchenne muscular dystrophy (DMD)
is characterized by slow and progressive muscle weakness
affecting limb and respiratory muscles, which degenerate
until fatal cardiorespiratory failure. Myodystrophy of
the Duchenne type results from mutations affecting the
gene for dystrophin, a cytoskeletal protein. Several
types of mutations have been described, which encompass
the complete absence of dystrophin to its presence in
reduced levels or the presence of partially functional
truncated forms, and which lead to severe to very mild
forms of the disease (Gills, 1996). The mdx mice that
showed complete absence of dystrophin have been used as
a model for DMD and have been tested for cell therapy.
Normal myoblasts have also been transplanted into the
muscle of patients with DMD. A form of congenital
dystrophy caused by a deficiency of the a2 subunit of
the basement membrane protein laminin/merosin is termed
merosin-deficient congenital muscular dystrophy (MCMD).
Most patients with MCMD are never able to walk. Null
mutant dyw mice have been generated (Kung et al.,
1998). Expression of human LAMA2 gene in the skeletal
muscle of dyw mice dramatically improves the muscle
disease in these animals. Both mdx mice and dyw mice can
be used for transplantation of AHPCs, AMPCs or fetal or
adult human brain-derived progenitor cells into muscles,
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to determine whether these cells can become myoblasts
and replace degenerating muscle cells.
Cardiovascular disease: The regulation of
cardiovascular function is complex and depends on many
factors interacting in a defined and temporal fashion.
Knock-out mice lacking desmin, needed to maintain the
integrity of the myocardium to develop cariomyopathy
(Lie et al., 1996; Milner et al., 1996, Thornell et al.,
1997). AHPCs, AMPCs, or fetal or adult human
brain-derived progenitor cells can be grafted in the
myocardium of these mice to determine whether the
transplanted cells can replace the diseased cells and
improve heart function.
Pulmonary disease (Cystic fibrosis): Cystic
fibrosis, the most common autosomally inherited disease,
is caused by the defective gene Cftr, which encodes an
ion channel at the cell membrane. By homologeous
recombination, several groups have disrupted the Cftr
gene. All null mutation mice developed symptoms of
cystic fibrosis (Dorm et al., 1992). AHPCs, AMPCs, or
fetal or adult human brain-derived progenitor cells can
be grafted into the lung of these mutant mice to
determine whether these cells can replace the diseased
cells having defective ion channels, and restore normal
lung function.
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