CA2215343C - Sertoli cells as neurorecovery inducing cells for neurodegenerative disorders - Google Patents

Abstract

A method of generating in situ trophic factor production by transplanting Sertoli cells into a tissue in need of trophic factors of a mammal, the cell s creating trophic factors in situ.

Description

_1_ 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 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 WO 96/28030 . -- PCT/LTS96/03335 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.

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.

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.

- 5 (a) -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;

WO 9G128~30 PCTlUS96/03335 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;

WO 96128030 PCTlClS96l03335 _7_ 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 WO 96!28030 PCT/US96/03335 _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-WO 9G/28030 PCTlUS9Gl03335 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.

WO 96/28030 PCTlUS96/03335 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, WO 96!28030 PCT'/US96/03335 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 WO 96/28030 PC~'1US96/0333i 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.

WO 96128030 PCTlLlS96/03335 (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.

WO 96/28030 - - PCT/US96l03335 _14_ (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.

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.

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. -WO 96128030 PCT/US96/~3335 ~.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 WO 96!28030 PCT/US96/03335 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 WO 96/28030 PCTlUS96/03335 _lg_ 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.

\ CA 02215343 1997-09-12 PCTIUS q ~ / 0 3 3 3 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

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 WO 96/28030 PCTlLTS96/03335 _22_ ._ 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 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.

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 REFERENCES CITED
Bjorklund and Stenevi, "Intracerebral neural grafting: a historical perspective" in Bjorklund, A
and U. Stenevi, eds. Neural Grafting in the mammalian CNS, Amsterdam: Elsevier, 3-11 (1985).
Bjorklund, "Dopaminergic transplants in experimental Parkinsonism: Cellular mechanisms of graft-induced functional recovery" Current Biology, 2:683-689 (1992) .
Borlongan et al., "PR: Systemic 3-nitropropionic acid: Behavior deficits and striatal damage in rats"
Brain Research Bulletin, 36:549-556 (1995).
Cameron et al., "Successful islet/abdominal testis transplantation does not require Leydig cells"
Transplantation, 50:549-556 (1995).
Cameron and Muffly, "Hormonal regulation of spermatid binding to Sertoli cells in vitro." J. Cell Sci., 100:523-533 (1991).
Griswold, "Protein Secretion by Sertoli cells:
general considerations" in Russell, L.d. and M.D.
Griswold eds. The Sertoli Cell, Cache River Press, Clearwater, FL, 195-200 (1992).
Isacson et al., "Graft-induced behavioral recovery in an animal model of-Huntington's disease" Proc. Natl.
Acad. Sci., 83:2728-2732 (1986).
Koutouzis et al., "PR:Systemic 3-nitropropionic acid:
Long term effects on locomotor behavior" Brain Research, 646:242-246 (1994).
Lindvall et al., "Transplantation in Parkinson's disease: two cases of adrenal medullary grafts to-the putamen" Ann. Neurol. 22:457-468 (1987).
Lindvall et al., "Grafts of fetal dopamine neurons survive and improve motor function in Parkinson''s disease" Science, 247:574-577 (1990).
Pakzaban et al., "Increased proportion of-Ache-rich zones and improved morphological integration in host striatum of fetal grafts derived from the lateral but not the medial ganglionic eminence" Exp. Brain Res., 97:13-22 (1993).

Sanberg et al., "Cell transplantation for Huntington's disease" R.G. Landes Co., Boca Raton, FL, pp. l9-21 (1994).
Selawry and Cameron, "Sertoli cell-enriched fractions in successful islet cell transplantation" Cell Transplan., 2:123-129 (1993).
Wictorin et al., "Reformation of long axon pathways in adult rat CNS byhuman forebrain neuroblasts"
Nature, 347:556-558 (1990).

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. 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.

2. A use as set forth in Claim 1, wherein the tissue in need of trophic factors is the central nervous system of a mammal.

3. A use as set forth in claim 2 wherein the mammal suffers from a neurological disorder including a neural degeneration disorder, said use ameliorating behavioural and functional deficits caused by the disorder by the action of the secreted trophic factors.

4. A use as set forth in Claim 1, wherein the Sertoli cells are porcine Sertoli cells.

5. A use as set forth in claim 2, wherein said transplanting is further defined as protecting the central nervous system from degenerative disorders.

6. A use as set forth in claim 2 wherein said transplanting is further defined as repairing damaged central nervous system tissue.

7. A use as set forth in claim 3, wherein the neurological disorder or neural degeneration disorder includes epilepsy, stroke, Huntington's diseases, head injury, spinal injury, pain, Parkinson's disease, myelin deficiencies, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's diseases, and affective disorders of the brain.

8. Sertoli cells which are useful in generating in 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.

9. Sertoli cells which are useful in generating in situ trophic factor production as set forth in claim 8, wherein the subject is human.

10. Sertoli cells which are useful in generating in 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.