Note: Descriptions are shown in the official language in which they were submitted.
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SERTOLI CELLS AS NELTRORECOVERY INDUCING CELLS
FOR NEURODEGENERATIVE DISORDERS
TEC~TICAL FIELD
The present invention generally
relates to cell transplantation and specifically to
a method of transplanting cells which, following
transplantation into the central nervous system
(CNS), ameliorates the behavioral and functional
deficits associated with neurological and
neurodegenerative disorders.
BACKGROUND OF THE INVENTION
In -treating disease it is often useful to
treat tissue locally, rather than systemically,
with trophic factors, particularly areas of tissue
damage as for,example-in wound healing.
As a further example, transplantation of
neural tissue into the mammalian central nervous
system (CNS) is becoming an alternative treatment
for neurological and neurodegenerative disorders
including epilepsy, stroke, Huntington~s diseases,
head injury, spinal injury, pain, Parkinson~s
disease, myelin deficiencies, neuromuscular
disorders, neurological,pain, amyotrophic,lateral
sclerosis, Alzl2eimer~s disease, and affective
disorders,.of the, brain. Preclinical and. clinical
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data indicate. that transplanted cells (thegraft)
used in cell transplantation protocols for these
types of neurodegenerative diseases survive and
integrate with the host tissue, and provides
functional recovery. (Sanberg et al., 1994).
The primary source for these grafts has
been the fetus. For example, fetal ventral
mesencephalic tissue has been demonstrated to be a
viable graft source in Parkinson~s disease.
(Lindvall et al., 1990; Bjorklund, 1992).
Likewise, fetal striatal tissue has been utilized
successfully as graft material in Huntington~s
disease. (Isacson et al., 1986; Sanberg et al.,
1994) .
Neurologically dysfunctional animals have
been transplantedwith non-fetal cells and non-
neuronal cells/tissue. For-example, chromaffin
cells from adult donors have been used in the
treatment of=Parkinson~s disease. The major-
advantage of this type of transplantation protocol
is that the graft source is not a fetal source and,
thereby,, circumvents the ethical and logistical
problems associated with acquiring fetal tissue.
Utilizing the chromaffin cell protocol;
normalization of behavior is observed. However,
the functional recovery of this behavior is
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temporary and the animals revert to their pre-
transplantation status (Bjorklund and Stenevi,
1985; Lindvall et -al., 1987). The inability of -
this type of treatment protocol to maintain normal
behavioral activity in animals in the Parkinson's
disease model renders clinical application of this
protocol as well as other treatment therapies
premature. -
Administration of growth facto-rs as a
means of treating neurological and-
neurodegenerative diseases has been contemplated in
the art. However, delivering these agents to the
brain is fraught with great difficulties that have
yet to be successfully overcome. Generally, these
agents cannot be administered systemically and
infusion into-the brain is an impractical and
imperfect solution. Engineering cells to delive-r
specific, single trophic factors when implanted in
the brain has been suggested, but stable
transfection and survival of the cells when
implanted in the brain continues to be problematic.-
Additionally, it is becoming increasingly
recognized that multiple trophic factors acting in
concert are likely to be necessary for-the
successful treatment of neurological and
neurodegenerative conditions.
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Long term maintenance of functional
recovery has been observed in a diabetic animal
model utilizing a novel transplantation treatment
protocol utilizing isolated islet cells and Sertoli
cells. It-is clear that the efficacy of the
treatment is due to the presence of the Sertoli
cells, in part, due to their known
immunosuppressive secretory factor. (Selawry and
Cameron, 1993; Cameron et al., 1990). Sertoli
cells are also known to secrete a number of
important trophic growth factors.
Accordingly, it would be desirable to
utilize Sertoli cells alone as a source for
diseases where growth and trophic factor support of
damaged tissue is useful.- Examples include, wound
healing and neurological disorders-including
neurodegenerative disorders. The Sertoli cells can
be used to function as an in situ factory~for
trophic factors to thereby hasten wound healing and
to ameliorate functional and behavioral deficits
associated with neurological and neurodegenerative
disorders.
25.
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SLT1~ARY OF THE INVENTION
In accordance with the present invention, there is
provided a method of generating in situ trophic factor
production by transplanting Sertoli cells into a mammal, the
cells secreting trophic factors in situ.
In a broad aspect, the present invention relates to
the use of Sertoli cells to generate in situ trophic factors
by transplanting Sertoli cells into a tissue in need of
trophic factors of a mammal, the cells creating trophic
factors in situ.
In another broad aspect, the present invention
relates to Sertoli cells which are useful in generating
situ trophic factor production by transplanting porcine
Sertoli cells into the central nervous system of a subject,
the cells secreting trophic factors in situ, for treating
neurological disorders including epilepsy, stroke,
Huntington's diseases, head injury, spinal injury, pain,
Parkinson's disease, myelin deficiencies, neuromuscular
disorders, neurological pain, amyotrophic lateral sclerosis,
Alzheimer's disease, and affective disorders of the brain.
In yet another broad aspect, the present invention
relates to Sertoli cells which are useful in generating ~
situ trophic factor production by transplanting Sertoli cells
into an area of tissue damage of a subject, the Sertoli cells
secreting trophic factors in situ.
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BRIEF DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be
readily appreciated as the same becomes better understood by
reference to the following detailed description when
considered in connection with the accompanying drawings
wherein:
Figure 1 is a graph showing the results of
apomorphine-induced rotational behaviour, animals from both
groups exhibited >7 rotations per minute or, at least, a total
of 210 rotations for 30 minutes (contralateral to the lesion)
when challenged with apomorhine pre-transplant, at post-
transplant periods, animals receiving media alone continued to
display significant rotations, in contrast, animals receiving
the Sertoli cells had a marked reduction (more than 600) in
their rotational behaviour across the post-transplant periods;
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Figure 2 is a graph showing biased swing
behavior, animals from both groups displayed >800
biased swing activity (contralateral to the lesion)
as revealed by the elevated body swing test, at
post-transplant periods, animals receiving the
media alone continued to display significant biased
swing activity, in contrast-, animals receiving the
Sertoli cells did not exhibit any biased swing
behavior across the post-transplant periods;
Figure 3A-C are light micrographs
illustrating cells from the ventral mesencephalon
of fetal rats (VM) isolated and cultured for seven
days in control medium (CM) or Sertoli cell pre-
conditioned medium (SCM) and photographed with
darkfield, interference contrast optics, wherein
(A) depicts VM cells incubated in CM showing no
evidenceof stimulation or differentiation,
(B) depicts VM cells incubated in SCM appearing
highly stimulated, and (C) at higher magnification,
depicts VM cells incubated in SCM exhibiting
neurite outgrowth as a result of Sertoli secreted
trophic factors;
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Figure 4A-B are electron micrographs
illusCrating (A) the striatum of the brain showing
the penetration-tract (arrows) and the site of
Sertoli cell transplantation, and (B) shows the
boxed area in (A) at higher magnification, with
higher resolution, Sertoli cells (arrows) are
easily identified because of the 1~ latex bead
inclusions which were loaded into the cells prior
to transplantation; and
Figure 5A-B are two light micrographs
illustrating grafted Sertoli cells in situ labeled
with a florescent tag (DiI) prior to their
transplantation into the striatum of the brain
wherein-(A) depicts viable, florescent Sertoli
cells in a rat host that had not received
immunosuppression therapy with cyclosporine A
(CsA), and (B) shows viable, florescent Sertoli
cells in the rat host that had received
cyclosporine A immunosuppression therapy.
DETAILED DESCRIPTION OF, THE INVENTION
Generally, the present inventionprovides
a method for p>~omoting the repair, protection, and
support of dysfunctional tissue by mechanisms
2~ including. in situ. production of Sertoli cell-
derived growth and~regulatory factors referred to
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_g_
generally as trophic f actors. Additionally, the
present method provides a method ofgenerating in
situ trophic factor production. This is achieved
by transplanting isolated Sertoli cells into a
mammal, the cells secreting trophic factors in
situ.
One significant benefit of utilizing
Sertoli cells as an in situ factory for producing
trophic factors is that Sertoli cells have been _
shown to have an effective immunosuppressant
effect. Accordingly, concomitant adjunctive
therapy to produce immunosuppression is not
required. In other words, the Sertoli cells can be
used as a trophic factor source while also
providing a self-induced local immunosuppressive
effect .
Trophic factors secreted by Sertoli cells
include Sertoli cell-derived growth and regulatory
factors such as insulin-like growth factors I and
II, epidermal growth factor, transforming growth
factors a and.j3, and interleukin la (Griswold,
1992). For a more extensive list of Sertoli cell
secretory factors refer to Table 1. Such factors
have been shown to have an ameliorative effect on
25, behavioral and, functional deficits associated with
neurodegenerative diseases. These factors are well-
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known tropic factors which support normal cell and
tissue metabolism and function. lGriswold. 'i9~21
The present invention utilized the phenomenon that
Sertoli cells can produce a trophic-rich, growth-
s -supportive fluid microenvironment at the site of
cellular dysfunction or cellular/tissue damage.
Cellular/tissue damage can include, but is not
limited to, radiation damage, burns and wounds. In
contrast to the Sertoli cell/islet cell
transphantation protocol usedin the diabetic
model, the method of the present invention utilizes
only one type of cell, i.e. Sertoli cells, thereby
significantly reducing the logistic and procedural
problems inherent in attempting to transplant two
different cell types at one host site.
Although rat Sertoli cells are utilized
in the following examples, Sertoli cells from any
suitable source can be used. For example, human
Sertoli cells may be used for transplantation in
humans. Additionally, in a preferred embodiment of
the present invention, porcine Sertoli cells may be
transplanted into a mammal, such as a human.
Furthermore, veterinary uses of the present
invention are contemplated and allogenic Sertoli
cells.would~be selected for transplantation into
the desired mammalian host.
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As demonstrated in the experimental
section below, the present invention can be
utilized as a treatment for ameliorating the
behavioral and functional deficits associated with
neurodegenerative diseases, such as Huntington's
disease and Parkinson's disease. This can be
accomplished without the concomitant side effects
of previously utilized immunosuppressive adjuvant
therapy, such as the chronic use of cyclosporine A.
The Sertoli cells, to provide both the secretion of
the trophic factors and the immunosuppressive
effect .
As shown in the examples below, the
transplantation of Sertoli cells prior to inducing
or formation of a brain lesion can provide a
neuroprotective effect. For example, as
demonstrated below, implantation of Sertoli cells
prior to inducement o-f a Huntington's type disease
provided both neuroprotective and prophylactic
effects on a subsequent-brain lesion. Therefore,-
the implantation of =Sertoli cells early on
following diagnosis of a neurodegenerative disease
may provide useful treatment, prevention or
reduction of the disease. Additionally, Sertoli
cells may be transplanted in other types of CNS
- .trauma. such as head injury to treat, prevent,
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and/or prophylactically reduce the effects of CNS
inj uzy .
The following example demonstrates the
ability of the present invention to ameliorate
behavioral deficits associated with
neurodegenerative disorders.
EXAMPLE 1: SERTOLI CELL TRANSPLANTATION
Specific Protocol:
The protocol generally involves two basic
steps, (1) Sertoli cell isolation and (2) cell
transplantation both of which are briefly described
below (for greater details regarding the cell
isolation see Selawry and Cameron (1993) and for
details regarding cell transplantation, see
Pakzaban et al.(1993) both incorporated by
reference. - --
(lA) Sertoli Cell Isolation
The isolation procedure follows a well
defined method Selawry and Cameron, (1993) and is
routinely utilized. The cell culture medium used
in all isolation steps and in which the cells were
incubated was DMEM:Hams Fl2 supplemented with
retinol, ITS, and gentamicin sulfate (Cameron and
Muffly, 1991.). Testes.were,surgically.collected _ .
from sixteen day old male~Sprague-Dawley rats. The
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testes were decapsulated and prepared for--enzymatic
digestion to separate other testicularcell types
from the Sertoli cells. The enzymatic procedure
utilized collagenase (O.lo), hyaluronidase (O.lo),
5- and trypsin (0.25x) which is a typical procedure
used in many cell isolation protocols. After
sequential enzymatic digestion, the Sertoli cell
isolate was washed with culture medium, transferred
to sterile culture vessels and placed in a
humidified, 5o C02 - 95o air tissue culture
incubator. Following forty-eight hours of pre-
incubation in a 39°C incubator, the Sertoli cells
were washed to remove any contaminating debris.
The resultant Sertoli cell-enriched fraction was
resuspended into 0.25m1 of DMEM/F12 medium and
incubated at 37°C for at least 24 hours.
The Sertoli cells are then liberated from
the vessel floor with trypsin, transferred to a
sterile conical test tube, and repeatedly-washed by
centrifugation and treated with trypsin inhibitor
to cease the enzymatic action of the trypsin.
During the day of transplantation, the Sertoli
cell-enriched fraction is resuspended and suctioned
using aHamilton syringe with a 20 gauge spinal
needle.
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(1B) Isolation and Pretreatment of
Sertoli Cells
Alternatively, as previously described
(Cameron et al. 1987a; Cameron et al. 1987b)
decapsulated rat testes were subjected to-
sequential enzymatic treatment at 37°C using 0.250
trypsin (Sigma) and O.lo collagenase (Sigma, type
V) (Cameron et al. 1987a; Cameron et al. 1987b).
The resulting Sertoli cell aggregates were equally
distributed in a volume of 20m1 incubation medium
into 75cm2 tissue culture flasks (Costar). Plated
Sertoli aggregates were incubated at 39°C in 5a
C02-95o air for 48 hours after which cells were
subjected to hypotonic treatment with sterile 0_5mM
Tris-Hcl buffer for one minute (Galdieri et al.
1981) to expedite the removal of contaminating germ
cells. Following two'washes with incubation
medium, flasks were replenished with 20m1
incubation medium and returned to the COZ-injected
incubator-at 37°C in 5o C02-95o air.- The resulting
pre=treated Sertoli-enriched monocultures contained
greater than 95o Sertoli cells. Plating density
(< 2.0 X 106 Sertoli cells/cm~) did not result in a
confluent monolayer of-cells.-
2 5.
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(2) Cell Transplantation -
The transplantation protocol follows the
procedure as previously described (Pakzaban et al.,
1993). Animal surgery was carried out under
sterile conditions. All animals were initially -
anesthetized with 0.60 ml/kg sodium pentobarbital
and then were placed in a Koph stereotaxic
instrument. Unilateral striatal transplants were
performed using coordinates set at: anterop-osterior
_ +1.2, mediolateral = +/- 2.8, dorsoventral = 6.0,
5.9, and 5.8 (based on the atlas ofPaxinos and
Watson, 1984). The striatum ipsilateral to the
lesioned substantia nigra was transplanted with
Sertoli cells. Each striatum receives a total
volume of 3-~C1 of Sertoli cell suspension. One
microliter-of the Sertoli cell suspension was
infused overone minute per dorsoventral site.
Controls only received media. Another-five minutes
was allowed upon reaching the last dorsoventral
site before-retracting the needl e. After surgery,
the animals were placed on heating pads to recover.
Animals receive a short course of immunosuppression
using Cyclosporine-A (20 mg/kg/d, i.p.) immediately
after surgery and on the day following transplant.
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However, subsequent studies demonstrated that this
short course of Cyclosporine-A is not needed
(Figure 5A-B)
Sertoli cells are transplanted into
animal models of various neurodegenerative
disorders by stereotaxic coordinates-defined for
the specific disorder, as illustrated in the
Parkinson's disease example, and then are
systemically assayed for functional recovery by
techniques specific to that animal model.
The present study used Sprague-Dawley
male, eight week old rats with 6-OHDA-induced
hemiparkinsonism (n=12). At three weeks post-
lesion, the animals were subjected to~behavioral
tests that included the apomorphine-induced
rotational behavior and the swing behavior.
Baseline data showed significant apomorphine-
induced rotational behavior (contralateral to the
lesioned side of the CNS) in all these animals (at
least 200 turns for 30 minutes). Using the
elevated body swing test (EBST), significant right-
biased swing activity (more than 700) was also
noted.
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At three weeks post-lesion, one group of
animals (n=6) received Sertoli cells and one group
(n=6) was subjected to the same surgical procedure
but only received media (DMEM without serum) as
controls. All animals received cyclosporine
(20mg/kg) for the first two days following the
transplant. At one, one and a half, and two months
post-transplant, animals were again introduced in
the same behavioral tests.
The animals receiving Sertoli cells
exhibited significant reductions in rotations (mean
of 50 turns for 30 minutes) while the animals
receiving the media alone were at pre-transplant
rotational level (Figure 1). The normalization of
turning behavior persisted across the two month
test period. The right-biased swing activity
previously displayed by the Sertoli cells
transplanted animals was also significantly reduced
at post-transplant test sessions (Figure 2). The
animals receiving the media did not show any
significant reductions in their right-biased swing
responses. -
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~.lt autopsy, brains were removed from the
animals and fixed for vibratome sectioning at 40-
80~,m. Following staining, there was a marked
reduction of..activated glial cells at the
penetrationsite (i.e., lesion site) in Sertoli
cell transplanted rats when compared to the
penetration site in the lesioned animals not
transplanted with Sertoli cells.
EXAMPLE 2: GROWTH OF NEURAL CELLS
Incubation medium and Sertoli cell pre-
conditioned medium
The incubation medium used for Sertoli
cell culture and co-culture was Dulbecco's Minimum
Essential Medium: Hams F12Nutrient Medium
(Whittaker Bioproducts) mixed 1:1and supplemented
with 3mg/ml L-glutamine (Sigma, grade III),
O.Olcc/ml insulin-transferrin-selenium (ITS,
Collaborative Research, Inc.), 50 ng/ml retinol
(Sigma) , 19~..~.1/ml lactic acid (Sigma) and 0.01cc/ml
gentamicin sulfate (Gibco).
Following the first 48 hour incubation
peripd of isolated Sertoli cells, media was
collected and centrifuged at 1500rpm for5 minutes.
The..supernatent was collected and immediately
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frozen-in sterile test tubes. This medium was
identified as Sertoli pre-conditioned medium (SCM).
Isolation and incubation of fetal brain
cells
Fetal brain cells (FBC) were collected
from the ventral mesencephalonof fetal rats (15-17
days gestation). The fetal brain tissue was
suspended in medium and initially dispersed by
passing it through a series of sequentially
decreasing sized hypodermic needles (18-26 gauge).
The resulting suspension was treated with O.lo
trypsin for five minutes and followed by O.lo
trypsin inhibitor for two minutes. The suspended
FBC were washed (3X), resuspended in incubation
medium and plated in poly-L-lysine-coated culture
vessels.
Cells from the ventral mesencephalon of
fetal rats (ZTM) were isolated and cultured for
seven days in control medium (CM) or Sertoli cell
pre-conditioned medium (SCM) as shown in Figure 3A.
VM cells incubated in CM showed no evidence of
cellular stimulation ,or differentiation. Referring
to Figure 3B, vM cells. incubated in SCM were highly
stimulated. Figure 3C illustrates that at higher
magnification, VM cells incubated in SCM show
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neurite outgrowth as a response to Sertoli cell
secreted trophic factors.
EXAMPLE 3: IDENTIFICATION OF SERTOLI CELLS
- Incorporation of latex beads:
Sertoli cells were isolated and prepared
for incubation as described. Prior to
transplantation (approximately 12 hours), sterile
lam latex beads (10~1/ml medium; Pelco, 'I~stin, CA)
were added to the incubation medium. Sertoli cells
rapidly phagocytosed the beads. Immediately prior
to transplantation, the beaded Sertoli cells were
washed (three times) and resuspended in 1ml of
incubation medium.
Referring to Figure 4A, Sertoli cells
were transplanted into the striatum of the brain
wherein the penetration tract (arrows) and the site
of Sertoli cell transplantation are shown. At
higher magnification as shown in Figure4B, Sertoli
cells (arrows) were easily identified because of -
the inclusion of 1~ latex-beads which were loaded
into the.Sertoli cells prior to transplantation.
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EXAMPLE 4: EFFECTS OF CYCLOSPORINE A (CSA) ON THE
SURVIVAL OF TRANSPLANTED SERTOLI CELLS
Fluorescent cell .labelinct:
Immediately priorto transplantation
(approximately twohours), Sertoli -cell-
monocultures were treated with CM-DiI fluorescent
dye for cell tracking (100.1 stock/ml medium;-
Molecular Probes, Inc., Eugene, OR) for seven
minutes at 37°C and then placed at 4°C for an
additional 15 minutes.-- Fluorescent "tagged" Sertoli
cells were washed (3X) and resuspended in lml of
incubati-on medium.
The effect of cyclosporirie A on the
survival of grafted Sertoli cells in situ was
15_ examined. Grafted Sertoli cells were labeled.with
a fluorescent tag (DiI) prior-to transplantation
into the striatum of the brain. The tissue was
collected one month post-transplantation.
Referring to-.Figure 5A~ viable fluorescent Sertoli
cells were seeriin a rat host that had not received
immunosuppression therapy with cyclosporine A.
Referring to Figure 5B, viable fluorescent Sertoli
cells are shown in a rat host that had received
cyclosporine A.immunosuppression therapy. This
example demonstrates that cyclosporine A is not
ANtENDED SHEET
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necessary for the survival of Sertoli ce-lls
transplanted into the brain.
EXAMPLE 5: PROPHYLACTIC EFFECTS OF SERTOLI CELLS
The transplantation of Sertoli cells is
neuroprotective when implanted prior to inducing
brain lesions. This prophylactic effect of Sertoli
cells was demonstrated in an animal model for
Huntington's Disease (HD). This model is produced
1o by the systemic administration of the mitochondrial
inhibitor, 3-nitropropronic acid (3NP). It has
been. demonstrated by Sanberg and colleagues
(Koutouzis et al. 1994; Borlongan et al. 1995) and
others that the injection of 3NP _causes specific
lesions within the striatum which mimic the
pathology seen in Huntington's disease.
In the present experiment 8 rats were
transplanted with rat Sertoli cells (as described
previously) unilaterally into one striatum of
normal rats. Therefore, one side of the brain had
Sertoli cells and the other side was without. One
month later, the animals were injected with 3NP as
described elsewhere (Koutouzis et al. 1994;
Borlongan et al. 1995) to induceI~. Normal rats
when injected with 3NP demonstrate bilateral damage
of the striatum of the brain and have behavioral
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deficits which are equal on both sides o-f the body
(Koutouzis et al. 1994; Borlongan et la. 1995).
One month following 3NP administration
the animals demonstrated unilateral behavioral
deficits. This was seen by the demonstration of
apomorphine-induced rotations post-lesion in
Sertoli transplanted animals, but not in controls
(Number of Rotations; Controls=0.25~.6; Sertoli
transplanted=197~31.9, p<.0001). This asymmetric
rotational behavior was indicative of a lesion on
the side of the brain which was not transplanted
with Sertoli cells. Therefore, Sertoli cell
implants, vis-a-vis trophic mechanisms, have
neuroprotective and prophylactic effects on
subsequent brain lesions. This provides evidence
that Sertoli transplantation may also-be useful in
treating neurodegenerative diseases early, before
significant damage is present.
These results, taken together, show that
the Sertoli cells ameliorate the behavioral and
functional deficits of animal models of Parkinson's
disease and Huntington's disease. The mechanism
involved is most likely the secretion of Sertoli
cell-derived growth factors, as demonstrated by the
sprouting of neuronal tissue as shown in Example 2,
and regulatory factors which promote the repair and
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the prolonged support of the relevant nervous tissue.
Additionally, Sertoli cells may protect and promote nervous
tissue repair in the brain by inhibiting glial cell activation
at the lesion site. These results also demonstrate the
viability in situ of transplanted Sertoli cells.
Throughout this application various publications are
referenced by citation or number. Full citations for the
publication are listed below. These publications more fully
describe the state of the art to which this invention
pertains.
The invention has been described in an illustrative
manner, and it is to be understood the terminology used is
intended to be in the nature of description rather than of
limitation.
Obviously, many modifications and variations of the
present invention are possible in light of the above
teachings. Therefore, it is to be understood that within the
scope of the appended claims, the invention may be practiced
otherwise than as specifically described.
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TABLE 1
I. Sertoli Cell-Derived Growth and Regulatory Factors (Partial
List)
Cntegory and Protein Function
Hormones/GroWth Factors
Mullerian Inhibiting Substance inhibits Mullerian duct
Inhibin inhibits FSH release
Insulin-like Growth Factor
(Sommatomedins A and C, IGF) growth factor
Prodynorphin
Interleukin-la mitogen
Transforming Growth Factor a& f3 growth factors
Basic Fibroblast Growth Factor growth factor
LHRH-like Factor Leydig cell steroidogenesis
(unpurified or incompletely characterized)
Sertoli Secreted Growth Factor growth factor
Seminiferous Growth Factor
Leydig Cell Stimulatory Activity
Testins
CMB proteins
Vitamin Binding Proteins vitamin transport
Transport and Bioprotection
Transferrin iron transport
Ceruloplasm copper transport
Saposin binds glycosphingolipids
SGP-2 (Clusterin) lipid transport?
Androgen Binding Protein transports T and DHT
SPARC calcium binding protein?
IGF Binding Proteins IGF transport
Riboflavin Binding Protein riboflavin transport
Professes and Protease Inhibitors
Plasminogen Activator protease
Cyclic Protein-2 protease inhibitor
Cystatin. protease inhibitor
a2-Macroglobulin protease inhibitor
Type IV Collagenase protease
Metalloproteinases protease
Baseraent membrane
Collagen IV
Laminin
Proteoglycans
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REFERENCES CITED
Bjorklund and Stenevi, "Intracerebral neural
grafting: a historical perspective" in Bjorklund, A
and U. Stenevi, eds. Neural Grafting in the
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