JPH11501818A - Sertoli cells as neural recovery-inducing cells for neurodegenerative diseases - Google Patents

Abstract

  (57) [Summary] A method of producing trophic factor production in situ by transplanting Sertoli cells, which produce trophic factors in situ, into mammalian tissues that require trophic factors.

Description

DETAILED DESCRIPTION OF THE INVENTION Sertoli cells TECHNICAL FIELD The present invention as neurorestorative induced cell for neurodegenerative diseases in general cell transplantation, particularly, after transplantation into the central nervous system (CNS), and neurological and neurodegenerative disorders The present invention relates to a method for transplanting cells to restore related behavioral and functional defects. BACKGROUND OF THE INVENTION In treating disease, it is often beneficial to treat tissue with trophic factors locally rather than systemically, for example, in the area of tissue damage as in wound healing. As yet another example, transplantation of neural tissue into the mammalian central nervous system (CNS) can include epilepsy, stroke, Huntington's disease, head trauma, spinal cord trauma, pain, Parkinson's disease, myelin deficiency, neuromuscular disease, neuralgia, muscle pain It is becoming another treatment for neurological and neurodegenerative diseases, including atrophic lateral sclerosis, Alzheimer's disease, and emotional disorders of the brain. Preclinical and clinical data indicate that transplanted cells (grafts) used in cell transplantation protocols for these types of neurodegenerative diseases survive, coalesce with host tissues, and confer functional recovery (Sanberg et al., 1994). The primary source of these grafts was the fetus. For example, fetal ventral midbrain tissue has been demonstrated to be a viable transplant source in Parkinson's disease (Lindvall et al., 1990; Bjorklund, 1992). Similarly, fetal striatal tissue has been successfully used as a graft material in Huntington's disease (Isacson et al., 1986; Sanberg et al., 1994). Animals with neurological dysfunction had been implanted with non-fetal cells and non-neuronal cells / tissue. For example, chromaffin cells from adult donors have been used to treat Parkinson's disease. A major advantage of this type of transplantation protocol is that the graft source is not a fetal source, thereby avoiding ethical and logical issues associated with obtaining fetal tissue. Using the chromaffin cell protocol, normalization of behavior is observed. However, the functional recovery of this behavior is temporary, and the animals return to their pre-transplant state (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 prematurely applies this protocol, as well as the clinical application of other treatments. The administration of growth factors as a means of treating neurological and neurodegenerative diseases has been contemplated in the art. However, delivering these drugs to the brain is still associated with many difficulties that need to be successfully resolved. In general, these drugs cannot be administered systemically, and infusion into the brain is an impractical and incomplete solution. While it has been suggested to engineer cells to deliver specific monotrophic factors when transplanted into the brain, stable transfection and survival of the cells when transplanted into the brain is an ongoing problem. In addition, there is an increasing recognition that co-acting multitrophic factors are probably necessary for the successful treatment of neurological and neurodegenerative conditions. Long-term maintenance of functional recovery has been observed in diabetic animal models using a novel transplantation treatment protocol using isolated islet cells and Sertoli cells. It appears that the efficacy of the treatment is due in part to the presence of Sertoli cells, and in part to their known immunosuppressive secretory factors (Selawry and Cameron, 1993; Cameron et al., 1990 ) . Sertoli cells are also known to secrete some important trophic growth factors. Therefore, it would be desirable to use Sertoli cells alone as a source for diseases where growth factor and trophic factor support of damaged tissue would be beneficial. Examples include neurological disorders, including wound healing and neurodegenerative disorders. Sertoli cells function as trophic factor in situ factories, thereby speeding wound healing and may be used to restore functional and behavioral deficits associated with neurological and neurodegenerative diseases. SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a method of producing in situ trophic factor production by transplanting Sertoli cells, which secrete trophic factors in situ, into a mammal. BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily understood and will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings. FIG. 1 is a graph showing the results of apomorphine-induced rotation behavior, showing> 7 rotations per minute when animals from both groups were challenged with apomorphine preimplantation, or at least a total of 210 rotations over 30 minutes (lesion On the other hand, during the post-transplantation period, animals receiving medium alone continue to show significant rotation, whereas, in contrast, animals receiving Sertoli cells show a significant decrease in their rotational behavior over the post-transplantation period ( (Greater than 60%). FIG. 2 is a graph showing biased rocking behavior, with animals from both groups having> 80% biased rocking activity (opposite to lesion) as evidenced by an elevated body rock test. In the post-implantation period, animals receiving medium alone continued to show significant biased rocking activity, whereas animals receiving Sertoli cells exhibited a biased rocking behavior over the post-implantation period. Did not show. Figures 3A-C show fetal rat ventral midbrain (isolated and cultured in control medium (CM) or Sertoli cell conditioned medium (SCM) for 7 days and photographed with darkfield, interference contrast optics). Light micrographs showing cells from (VM), (A) showing VM cells incubated in CM showing no evidence of stimulation or differentiation, (B) apparently highly stimulated VM cells incubated in SCM, and (C) shows, at high magnification, VM cells incubated in SCM showing neurite outgrowth as a result of Sertoli secretory trophic factor. FIGS. 4A-B are electron micrographs showing the striatum (A) of the brain showing the site of invasion (arrows) and Sertoli cell transplantation, and (B) is a high-resolution, high-resolution box-shaped image in (A). Areas are shown, Sertoli cells (arrows) are easily identified for 1μ latex bead inclusions, which were placed in cells prior to implantation. 5A-B are two light micrographs showing transplanted Sertoli cells labeled in situ with fluorescent labeling (Dil) prior to transplantation into the striatum of the brain, where (A) is cyclosporin A (CsA) (B) shows surviving fluorescent Sertoli cells in a rat host that did not receive immunosuppressive treatment with, and (B) shows live fluorescent Sertoli cells in a rat host that received cyclosporin A immunosuppressive treatment. DETAILED DESCRIPTION OF THE INVENTION In general, the present invention provides methods for promoting the recovery, protection, and support of dysfunctional tissues by mechanisms involving in situ production of Sertoli cell-derived growth factors and regulatory factors, commonly referred to as trophic factors. . Further, the present invention provides a method for producing in situ trophic factor production. This is done by transplanting isolated Sertoli cells, which secrete trophic factors in situ. One significant advantage of utilizing Sertoli cells as an in situ factory for producing trophic factors is that Sertoli cells have been shown to have effective immunosuppressive effects. Therefore, no concurrent additional therapy to produce immunosuppression is required. In other words, Sertoli cells can be used as a source of trophic factors and also provide a self-induced local immunosuppressive effect. The trophic factors secreted by Sertoli cells include Sertoli cell-derived growth factors and regulatory factors such as insulin-like growth factors I and II, epidermal growth factor, transforming growth factors α and β, and interleukin 1α (Griswold, 1992). Is mentioned. See Table 1 for a more extensive list of Sertoli cell secretion factors. Such factors have been shown to have a reversible effect on behavioral and functional deficits associated with neurodegenerative diseases. These factors are known trophic factors that support normal cell and tissue metabolism and function (Griswold, 1992). The present invention took advantage of the phenomenon that Sertoli cells can produce a nutrient-rich, growth-supporting liquid microenvironment at the site of cell dysfunction or cell / tissue damage. Cell / tissue damage includes, but is not limited to, radiation damage, burns and wounds. In contrast to the Sertoli cell / islet cell transplantation protocol used in the diabetes model, the method of the present invention uses only one type of cell, namely Sertoli cells, thereby combining two different cell types with one host site. Significantly reduce the inherent logical and operational problems when attempting to port to. Rat Sertoli cells are used in the examples below, but Sertoli cells from any suitable source may be used. For example, human Sertoli cells can be used for transplantation in humans. Further, in a preferred embodiment of the present invention, porcine Sertoli cells can be transplanted into a mammal, such as a human. Further, veterinary uses of the invention are contemplated, and syngeneic Sertoli cells will be selected for transplantation into a desired mammalian host. As demonstrated in the experimental part below, the present invention may be used as a treatment to reverse the behavioral and functional deficits associated with neurodegenerative diseases such as Huntington's disease and Parkinson's disease. This can be done without the attendant side effects of conventionally utilized immunosuppressive adjuvant therapies, such as chronic use of cyclosporin A. Sertoli cells provide both secretory and immunosuppressive effects of trophic factors. As shown in the examples below, transplantation of Sertoli cells prior to induction or formation of brain lesions can provide a neuroprotective effect. For example, as demonstrated below, transplantation of Sertoli cells prior to induction of Huntington's disease provided both neuroprotective and prophylactic effects on subsequent brain lesions. Therefore, transplantation of Sertoli cells early after diagnosis of a neurodegenerative disease may provide beneficial treatment, prevention or reduction of the disease. In addition, Sertoli cells can be transplanted in cases of other types of CNS trauma, such as head lesions, to treat, prevent, and prevent the effects of CNS damage. The following examples demonstrate the ability of the present invention to reverse the behavioral deficits associated with neurodegenerative diseases. Example 1 Sertoli Cell Transplantation Special Protocol: The protocol generally involves two basic steps, (1) Sertoli cell isolation and (2) cell transplantation, both of which are briefly described below (Cell See Selawry and Cameron (1993) for details on detachment and Pak-zaban et al. (1993) for details on cell transplantation; both are incorporated by reference). (1A) Sertoli Methods Cell Isolation Isolation operations described may, in accordance Selawry and Cameron (1993), is used in the routine. The cell culture medium used for all isolation steps and in which the cells were incubated was DMEM: Hams F12 (Cameron and Muffly, 1991) supplemented with retinol, ITS, and gentamicin sulfate. Testes were surgically collected from 16 day old male SD rats. The testes were decapsulated and prepared for enzymatic digestion to separate other testicular cell types from Sertoli cells. The enzymatic procedure is a typical procedure used in many cell separation protocols, using collagenase (0.1%), hyaluronidase (0.1%), and trypsin (0.25%). After serial enzymatic digestion, Sertoli cell isolates were washed with medium, transferred to sterile culture vessels, and placed in a humidified tissue culture incubator with 5% CO ₂ -95% air. After 48 hours of pre-incubation in a 39 ° C. incubator, Sertoli cells were washed to remove contaminating debris. The resulting Sertoli cell-enriched fraction was resuspended in 0.25 ml of DMEM / F12 medium and incubated at 37 ° C. for at least 24 hours. Sertoli cells were then released from the vessel bed with trypsin, transferred to sterile conical tubes, repeatedly washed by centrifugation, and treated with trypsin inhibitor to stop the enzymatic action of trypsin. During the day of implantation, the Sertoli cell-enriched fraction was resuspended and aspirated using a Hamilton syringe with a 20 gauge spiral needle. (1B) Isolation and pretreatment of Sertoli cells Also as previously described (Cameron et al., 1987a; Cameron et al., 1987b), 0.25% trypsin (Sigma) and 0.1% collagenase (Sigma, type V) ( Decapsulated rat testes were subjected to continuous enzyme treatment at 37 ° C. using Cameron et al., 1987a; Cameron et al., 1987b). The resulting Sertoli cell aggregates were evenly distributed in a 20 ml volume of incubation medium in a 75 cm ² tissue culture flask (Costar). The coated Sertoli aggregates were incubated at 39 ° C. in 5% CO ₂ -95% air for 48 hours, after which the cells were hypotonic treated with sterile 0.5 mM Tris-HCl buffer (Galdieri et al.) For 1 minute. , 1981). After washing twice with incubation medium, and re-supply the incubation medium 20ml flask were returned to CO ₂ injection incubator at 37 ℃ 5% CO ₂ -95% air. The resulting pre-treated Sertoli-rich monoculture contained greater than 95% Sertoli cells. The application density (<2.0 × 10 ⁶ Sertoli cells / cm ² ) did not result in a confluent monolayer of cells. (2) Cell transplantation The transplantation protocol follows the procedure already described (Pakzaban et al., 1993). Animal surgery was performed under aseptic conditions. All animals were initially anesthetized with 0.6 ml / kg sodium pentobarbital and then placed in a Koph stereotaxic device. Anterior-posterior = +1.2, medial-lateral = +/- 2.8, dorsal and ventral = 6.0, 5.9, and 5.8 (based on Paxinos and Watson, 1984 atlas), unilateral line using coordinates Striatum transplantation was performed. Sertoli cells were implanted in the ipsilateral striatum in the affected substantia nigra. Each striatum receives a total volume of 3 μL Sertoli cell suspension. One microliter of the Sertoli cell suspension was infused into the dorsoventral site for 1 minute. Controls received medium only. An additional 5 minutes was allowed after reaching the last dorsoventral site before withdrawing the needle. After surgery, animals were allowed to recover by placing them on a heating pad. Animals receive a short course of immunosuppression using cyclosporin A (20 mg / kg / d, ip) immediately after surgery and after transplantation. However, subsequent studies demonstrated that this short course of cyclosporin A was not required (FIGS. 5A-B). Sertoli cells are transplanted into various neurodegenerative disease animal models with stereotactic coordinates defined for the particular disease, as shown in the example of Parkinson's disease, and then the animal model is systematically restored for function by special techniques. To evaluate. This study used 8-week-old male SD rats (n = 12) with 6-OHDA-induced hemiparkinsonism. Three weeks after the lesion, the animals were subjected to behavioral tests including apomorphine-induced rotational and rocking behavior. Baseline data showed significant apomorphine-induced rotation behavior (opposite to the lesion side of the CNS) in all these animals (at least 200 rotations in 30 minutes). Using the elevated body sway test (EBST), significant right-biased sway activity (greater than 70%) was also observed. Three weeks after the lesion, one group of animals (n = 6) received Sertoli cells and one group (n = 6) underwent the same surgical procedure, but received only medium (DMEM without serum) as a control. Received. All animals received cyclosporine (20 mg / kg) two days after transplantation. The animals were re-introduced to the behavioral test at 1, 1.5 and 2 months after transplantation. Animals receiving Sertoli cells showed a significant decrease in rotation (average 50 rotations in 30 minutes), while animals receiving medium alone had pre-transplant rotation levels (FIG. 1). Normalization of rolling behavior persisted over a two month test period. Right-biased rocking activity already demonstrated by Sertoli cell transplanted animals was also significantly reduced during the post-transplant study period (FIG. 2). Animals receiving medium did not show a significant decrease in their right-biased rocking response. At necropsy, brains were removed from the animals and fixed for vibratome incision at 40-80 μm. After staining, there was a marked decrease in glial cells activated at the site of entry (ie, the site of lesion) in rats implanted with Sertoli cells when compared to the site of entry in diseased animals where Sertoli cells were not implanted. Example 2 Growth Incubation Medium for Neurons and Sertoli Cell Preconditioning Medium The incubation medium used for Sertoli cell culture and co-culture was 3 mg / ml L-glutamine (Sigma, brand III), 0.01 cc / ml insulin. Transferrin-Selenium (ITS, Collaborative Research), supplemented with 50 ng / ml retinol (Sigma), 19 μl / ml lactic acid (Sigma) and 0.01 cc / ml gentamicin sulfate (Gibco), 1: 1 Dulbecco's minimum essential medium: Hams F12 nutrient medium (Wittaker Bioproducts) mixed with After the first 48 hours of incubation of the isolated Sertoli cells, the medium was harvested and centrifuged at 1500 rpm for 5 minutes. The supernatant was collected and immediately frozen in a sterile test tube. This medium was identified as Sertoli preconditioned medium (SCM). Isolation and incubation of fetal brain cells Fetal brain cells (FBC) were recovered from the ventral midbrain of fetal rats (15-17 days pregnant). Fetal brain tissue was suspended in medium and initially dispersed by passing it through a series of continuously decreasing size hypodermic needles (18-26 gauge). The resulting suspension was treated with 0.1% trypsin for 5 minutes, followed by treatment with 0.1% trypsin inhibitor for 2 minutes. The suspended FBC was washed three times, resuspended in the incubation medium and applied to a poly-L-lysine coated culture vessel. Cells from fetal rat ventral midbrain (VM) were isolated and cultured for 7 days in control medium (CM) or Sertoli cell preconditioned medium (SCM), as shown in FIG. 3A. VM cells incubated in CM showed no evidence of cell stimulation or differentiation. Referring to FIG. 3B, VM cells incubated in SCM were highly stimulated. FIG. 3C shows that, at high magnification, VM cells incubated in SCM show neurite outgrowth in response to Sertoli cell dispersal trophic factor. Example 3: Identification of Sertoli cells Contamination of latex beads : Sertoli cells were isolated and prepared for incubation as described. Before transplantation (about 12 hours), sterile 1 μm latex beads (10 μg / ml medium, Perco, Tustin, CA) were added to the incubation medium. Sertoli cells rapidly phagocytosed the beads. Immediately prior to implantation, the beaded Sertoli cells were washed (3 times) and resuspended in 1 ml of incubation medium. Referring to FIG. 4A, Sertoli cells were implanted into the striatum of the brain, and the figure shows the invasion path (arrow) and the site of Sertoli cell implantation. At high magnification as shown in FIG. 4B, Sertoli cells (arrows) were easily identified for contamination with 1 μl of latex beads loaded on Sertoli cells prior to implantation. Example 4: Effect of Cyclosporin A (CSA) on Survival of Transplanted Sertoli Cells Fluorescent Cell Labeling : Immediately prior to transplantation (about 2 hours), cell cultures of Sertoli cells were subjected to cell tracking (100 μl stock / ml medium) Treated with CM-Dil fluorescent dye at 37 ° C for 7 minutes for Molecular Probes (Eugen, OR), then placed at 4 ° C for an additional 15 minutes. Fluorescent "labeled" Sertoli cells were washed (3 times) and resuspended in 1 ml of incubation medium. The effect of cyclosporine A on the survival of Sertoli cells implanted in situ was examined. The transplanted Sertoli cells were labeled with a fluorescent label (Dil) before transplantation into the striatum of the brain. The tissue was recovered one month after transplantation. Referring to FIG. 5A, viable fluorescent Sertoli cells were seen in rat hosts that did not receive immunosuppressive treatment with cyclosporin A. Referring to FIG. 5B, viable fluorescent Sertoli cells are shown in a rat host that did not receive cyclosporin A immunosuppressive treatment. This example demonstrates that cyclosporin A is not required for the survival of Sertoli cells implanted in the brain. Example 5: Prophylactic Effect of Sertoli Cells Sertoli cell transplantation is neuroprotective when transplanted before inducing brain lesions. This protective effect of Sertoli cells was demonstrated in an animal model of Huntington's disease (HD). This model is generated by systemic administration of the mitochondrial inhibitor, 3-nitroproproic acid (3NP). It has been demonstrated by Sanberg and colleagues (Koutouzis et al., 1994; Borlongan et al., 1995) and others that injection of 3NP produces specific lesions in the striatum that mimic those seen in Huntington's disease. In this experiment, eight rats were implanted with rat Sertoli cells (as described above) on one side in one striatum of normal rats. Therefore, one side of the brain had Sertoli cells and the other side did not. One month later, the animals were injected with 3NP to induce HD as described elsewhere (Koutouzis et al., 1994; Borlongan et al., 1995). Normal rats when injected with 3NP show left and right damage to the striatum of the brain and have equal behavioral defects on both sides of the body (Koutouzis et al., 1994; Borlongan et al., 1995). One month after 3NP administration, the animals exhibited a unilateral behavioral defect. This was seen by demonstration of apomorphine-induced rotation after lesions in Sertoli-transplanted animals but not controls (number of turns; control = 0.25 ± 0.6; Sertoli-transplantation = 197 ± 31.9, p <0.0001). This asymmetric rolling behavior indicated lesions on the side of the brain that had not been transplanted with Sertoli cells. Thus, Sertoli cell transplants have neuroprotective and preventive effects on subsequent brain lesions with respect to nutritional mechanisms. This provides evidence that Sertoli transplantation may also be beneficial in treating neurodegenerative diseases early, before significant injury is present. Together, these results indicate that Sertoli cells restore the behavioral and functional deficits of animal models of Parkinson's disease and Huntington's disease. The mechanism involved is probably the secretion of Sertoli cell-derived growth factors, as demonstrated by neural tissue growth as shown in Example 2, and regulators that promote the recovery and prolonged support of related neural tissue. is there. In addition, Sertoli cells can protect nervous tissue in the brain by suppressing glial cell activation at the lesion site and promote nervous tissue recovery. These results also demonstrate the in situ survival of the transplanted Sertoli cells. In this application, various publications are referenced by reference or number. Full citations for the publications are listed below. The full disclosures of these publications are incorporated herein by reference to this application in order to more fully describe the state of the art to which this invention pertains. It is to be understood that the invention has been described in an illustrative manner and that the terms used are 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 other than as specifically described.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AU, BB, BG , BR, CA, CN, CZ, EE, FI, GE, HU, IS, JP, KG, KP, KR, LK, LR, LT, L V, MD, MG, MK, MN, MX, NO, NZ, PL , RO, SG, SI, SK, TR, TT, UA, US, UZ, VN (72) Inventor Cameron Don F             United States Florida 33549 Le             Two Clear Lake Drive             18206 (72) Inventor Bolongan Cesario V             United States Florida 33549 Le             Z Jamestown 17802 A

Claims (1)

[Claims] 1. Sertoli cells, which produce trophic factors in situ, are converted into mammalian nutrients A method for generating trophic factors in situ by transplanting them into tissues that require the factors. 2. 2. The method according to claim 1, wherein the tissue requiring a trophic factor is the central nervous system of a mammal. the method of. 3. The mammal has a neurological disorder, including a neurodegenerative disorder, wherein the method further comprises a secretory trophic factor. Repairing the behavioral and functional deficits caused by the disease by the action of 3. The method of claim 2 wherein the method comprises: 4. 2. The method according to claim 1, wherein the Sertoli cells are porcine Sertoli cells. . 5. The transplantation step is further specified as protecting the central nervous system from degenerative diseases. The method according to claim 2. 6. The transplantation process is further identified as restoring damaged central nervous system tissue. 3. The method according to claim 2, wherein the method comprises: 7. Neurological or neurodegenerative diseases include epilepsy, stroke, Huntington's disease, head trauma, spinal cord Trauma, pain, Parkinson's disease, myelin deficiency, neuromuscular disease, neuralgia, amyotrophic side 4. The method according to claim 3, which includes cord sclerosis, Alzheimer's disease, and emotional disorders of the brain. Method. 8. Epilepsy, stroke, Huntington's disease, head injury, spinal cord injury, pain, Parkinson's disease , Myelin deficiency, neuromuscular disease, neuralgia, amyotrophic lateral sclerosis, Alzheimer's disease, And porcine Sertoli cells (the cells) to treat neurological disorders, including emotional disorders of the brain and Vesicles secrete trophic factors in situ) into the subject's central nervous system. A method of producing trophic factor production in situ. 9. 9. The method according to claim 8, wherein the subject is a human. Ten. Sertoli cells (these secretory cells secrete trophic factors in situ) How to produce in situ trophic factor production by transplanting to the area of tissue damage in the subject Law. 11． Sertoli cells, which produce trophic factors in situ, are converted into mammalian nutrients A method for generating trophic factors in situ by transplanting them into tissues that require them. Use of Rutoli cells. 12． 2. The method according to claim 1, wherein the tissue requiring a trophic factor is the central nervous system of a mammal. Use of. 13. The mammal has a neurological disease, including a neurodegenerative disease, and the use is dependent on the production of secretory trophic factors. Claim 2 for recovering the behavioral and functional deficits caused by the disease by use Use as described in. 14. 2. Use according to claim 1, wherein the Sertoli cells are porcine Sertoli cells. . 15． A contract wherein the transplant is further identified as protecting the central nervous system from degenerative disease 3. Use according to claim 2 of the claim. 16． The transplant is further specified as restoring damaged central nervous system tissue Use according to claim 2. 17． Neurological or neurodegenerative diseases include epilepsy, stroke, Huntington's disease, head trauma, spinal cord Trauma, pain, Parkinson's disease, myelin deficiency, neuromuscular disease, neuralgia, amyotrophic side 4. The method according to claim 3, which includes cord sclerosis, Alzheimer's disease, and emotional disorders of the brain. use. 18． Epilepsy, stroke, Huntington's disease, head injury, spinal cord injury, pain, Parkinson's disease , Myelin deficiency, neuromuscular disease, neuralgia, amyotrophic lateral sclerosis, Alzheimer's disease, And porcine Sertoli cells (their) to treat neurological disorders, including emotional disorders of the brain and Cells secrete trophic factors in situ) into the subject's central nervous system. Sertoli cells that are more beneficial in producing trophic factor production in situ. 19. The in-situ trophic factor production according to claim 18, wherein the subject is a human. Sertoli cells that are useful for 20. Sertoli cells (these secretory cells secrete trophic factors in situ) Implantation in the area of tissue damage in a subject results in in situ trophic factor production Sertoli cells that are beneficial to.