MXPA97007027A - Sertolia cells as auxiliary of transplantation for the transplantation of celu - Google Patents

Abstract

A method to produce a localized immunosuppressive effect sustained in localized tissues is achieved by transplanting Sertoli cells near the tissue.

Description

SERTO I CELLS AS TRANSPLANTATION AUXILIARY FOR THE TRANSPLANTATION OF CELLS DESCRIPTION OF THE INVENTION The present invention relates to methods for transplanting cells to create an immunosuppressive and trophic effect located in the tissue. The transplantation of cells and tissues has been used therapeutically in a wide variety of disorders including, but not limited to, cystic fibrosis (lungs), lung failure, degenerative diseases of the heart to neurodegenerative disorders. As an example, the central nervous system (CNS) (brain and spinal cord) has a poor regenerative capacity, which is illustrated in a number of neurodegenerative disorders. An example of such a disorder is Parkinson's disease. The preferred pharmacotherapy for Parkinson's disease is L-dopa, which helps the symptoms of this disease in humans. However, neuropathological damage and progression of weakness is not reversed by this treatment protocol. Laboratory and clinical studies have shown the transplantation of cells to the CNS which is a potentially significant alternative therapeutic modality for neurodegenerative disorders such as Parkinson's disease (Wictorin et al., 1990; Lindvall et al., 1990; Sanberg et al., 1994; Bjorklund and Stenevi, 1985; Freeman et al., 1994). In some cases, the transplanted neural tissue can survive and form connections with the CNS of the receiver (ie, the host) (Wictorin et al., 1990). When the host is successfully accepted, the transplanted tissue (ie, the graft) has been shown to reduce the behavioral deficits associated with the disorder (Sanberg et al., 1994). The mandatory stage for the success of this type of treatment is the prevention of graft rejection (ie graft acceptance). Currently, fetal neural tissue is the main source of graft for neural transplantation (Lindvall et al., 1990, Bjorklund, 1992, Isacson et al., 1986, Sanberg et al., 1994). Other sources of grafting available include adrenal chromaffin cells and various cell types that secrete neural growth factors and trophic factors. The field of neural tissue transplantation as a productive treatment protocol for neurodegenerative disorders has received much attention resulting in the progression of clinical analyzes. Preliminary results and clinical observations sor. promising, although the phenomenon of graft rejection remains a problem. Recently, studies have suggested that Sertoli cells, when transplanted simultaneously with the pancreatic islet cell to the diabetic rat, act as an effective local immunosuppressant in the host tissue (Selawry and Cameron, 1993). As a result, the graft is not rejected and the islets remain viable allowing the transplanted β cells to function normally and produce insulin for an indefinite period. As a result, the accepted graft overcomes the primary physiological malfunction of hyperglycemia, thus relieving the related complications of this endocrine disorder. The cell transplant protocol is achieved without prolonged systematic immunosuppression, otherwise necessary when the islets are transplanted into Sertoli cells. In general, systemic immunosuppression is necessary without successful transplantation will be achieved in humans. Immunosuppression of the whole body (ie, systematic) may result, finally, acceptance of the graft. However, it is acquired by placing the individual at a medical risk making the same immunosuppressive therapy more available than a benefit in some cases. For a lack of a better immunosuppressive treatment, systemic immunosuppressants, with C-cyclosporin-A (CsA) as the choice of treatment, have been used as an auxiliary therapy in neural transplantation protocols and other protocols (Sanberg et al., 1994; Freeman et al., 1994; Borlongan et al., 1935). Disputably, treatment with systemic CsA may be counterproductive to the successful acceptance of the graft in the CNS, due to its systemic effect and since CsA itself has been shown to cause harmful side effects and may, in fact, be cytotoxic to neural tissues. (Berden et al., 1985; de Groen et al., 1984). It may be desirable to improve the productive cell transplantation techniques already used for neurodegenerative disorders, such as Parkinson's disease, in ways that could be effectively slower in the process of neurodegenerative disease, to more actively promote the re-establishment of the physiology of normal neural tissue and better alleviate functional disabilities associated with the malfunction of neural tissue. Likewise, it might be desirable to avoid systemic immunosuppression with the ability to immunosuppress locally (ie, at the graft site) through an immunosuppressant, which is biologically tolerated by the host. Sertoli cells can provide this desired option, since it is evident from diabetic studies, as summarized in the above, that co-transplantation with Sertoli cells will provide an immunosuppressant. local and will promote, therefore, an efficient acceptance of the graft and a functional restoration of tissue-related malfunction.

In accordance with the present invention, there is provided a method for producing a sustained local immunosuppressive effect and a trophic effect in tissues by transplanting Sertoli cells close to the tissue. BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as they are better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings in which: Figures 1A-B show photomicrographs of two sections of representative brain, in which the sections were stained with Lectin, the left side of each brain corresponds to the chromaffin cells transplanted on one side, while the right side of each brain corresponds to the chromaffin cells with Sertoli cells on the side - transplanted; Figures 2A-D show a greater amplification of the photomicrograph of Figure 1 of the transplant sites of the brain sections illustrated in Figure 1; panels A and C show the chromaffin cells transplanted on one side, while panels B and D show the co-transplanted lateral chromaffin cells with Sertoli cells; Figures 3A-C are light micrographs illustrating cells from the ventral mesencephalon of fetal rats (MV) isolated and cultured for seven days in control medium (CM) or a preconditioned Sertoli cell (SCM) medium and photographed with contrast optics of interference, darkfield, in which (A) represents VM cells incubated in CM that show no evidence of stimulation or differentiation, (B) represents VM cells incubated in SCM that appear highly stimulated, and (C) at a higher amplification , represents VM cells incubated in SCM exhibiting neurite growth; Figures 4A-B are electron micrographs illustrating the cellular structure and tissue architecture of Sertoli cell co-grafts / chromaffin cells in the brain striatum, in which (A) represents dense electronic chromaffin cells (arrows ) easily identified due to the inclusion of unique secretory granules to these cells and, (B) shows the area in a box in (A) At higher amplification, with higher resolution, Sertoli cells (arrows) are immediately adjacent to the dense electronic chromaffin cells; Figures 5A-B are electronic micrographs illustrating (A) the striatum of the brain showing the penetration tract (arrows) and the Sertoli cell transplantation site, and (B) showing the area in a box in (A) a A larger amplification, with a higher resolution, Sertoli cells (arrows) are easily identified due to 1μ latex bead inclusions, which were loaded onto the cells before transplantation; and Figures 6A-B are two light micrographs illustrating Sertoli cells grafted in if marked with a fluorescent label (Dil) before their transplantation into the striatum of the brain, wherein (A) represents fluorescent Sertoli cells, viable in a host of rat that has not received immunosuppressive therapy with Cyclosporin A (CsA), and (B) shows fluorescent Sertoli cells, viable in the rat host that has received the immunosuppression therapy of cyclosporin A. The present invention provides a method for producing a localized immunosuppressive effect sustained and a trophic effect on the tissue. This is achieved by the general stage of transplantation of Sertoli cells near the tissue. The sustained localized immunosuppressive effect means that the transplanted Sertoli cells will suppress the immune response ordinarily placed by the host tissue to the intrusion of foreign entities such as transplanted cells and that immunosuppression will occur at the (local) graft site instead of immunosuppression generalized (whole systemic) body which occurs with the ordinary methods of immunosuppression through agents such as CsA. By tissue, is meant any form of tissue, including, but not limited to, cells, blood, organs and dissociated cells. In a preferred embodiment, the transplanted cells (which are intended to replace malfunctioning cells or in some way alleviate tissue malfunction) can avoid being rejected and thus survive and functionally integrate into the host tissue. In the CNS, this will promote the restoration of normal neural tissue function and thus reduce the behavioral and functional deficits associated with the neurological and / or neurodegenerative disorder being treated. However, the method of the present invention can also be used with other transplantable cells or tissues instead of tissue / neural cells such as endocrine cells, muscle cells and other cells, using techniques similar to those described for neural cells. In addition, the method of the present invention can be used to improve the advantages of tissue and organ transplantation, such as lung transplantation, by providing localized immunosuppression. That is, Sertoli cells are used to facilitate the survival of the transplant and the graft function of the cells that are being transplanted. With local immunosuppression by an immunosuppressive agent derived from Sertoli cells (which is now partially characterized), there could be no successful antibody or cellular immune attack undertaken against the transplanted cells, including the same Sertoli cells. In addition, since immunosuppression is local and through a biologically tolerable agent, the side effects associated with both systemic immunosuppression and the cytotoxicity of agents such as CsA could be avoided. In this way, the Sertoli cell transplantation method provides a significant improvement over the use of systemic immunosuppression with CsA as the auxiliary therapy necessary for transplantation, as shown in the following example. Immunosuppression localized by an immunosuppressive agent derived from Sertoli cell can facilitate the survival of both xenoinjertoe and allografts. With allografts, co-transplantation with Sertoli cells should provide localized immunosuppression in order to eliminate the need for systemic immunosuppression. With xenografts, co-transplantation with Sertoli cells can provide sufficient local immunosuppression in order to eliminate the need for systemic immunosuppression or Sertoli cells that can be used in combination with a systemic immunosuppressant to prevent rejection thus reducing the dose of systemic immunosuppressant required. When co-transplanted, Sertoli cells not only provide immunosuppression, but also provide regulatory, nutritional, and other factors designated as a trophic support to the co-transplanted tissue. Therefore, Sertoli cells will not only provide inhibition of the immune response, but will also allow for the increased growth and viability of xenograft allografts through concomitant trophic support. The source of Sertoli cells is through the primary cell isolation of the mammalian test. The protocol for harvesting the cells is well defined (Cameron and Muffly, 1991; Griswold, 1992) and is considered a routine methodology. In most published reports of co-transplantation of Sertoli cells, the cells are derived from the rat (Selawry and Cameron, 1993). Although used, rat Sertoli cells in the following examples will be contained. that the method of the present invention can be used with Sertoli cells from any suitable mammalian source. A preferred source of Sertoli cells for use with mammals, such as humans, are Porcine Sertoli cells. However, if available and adequate, human Sertoli cells can be used. In one embodiment, Sertoli cells are co-transplanted with the selected neural tissue or other appropriate tissue in the CNS through intracranial infusion (Sanberg et al., 1995). Close to the tissue, it means that the Sertoli cells are placed in general close to the selected tissue such as the neural tissue. Generally, this means that Sertoli cells can be infused or transplanted into any mammal in order to be located close to the selected tissue. For example, the location can be any site where there is a neural tissue such as CNS, PNS or fluids which bathe neural tissue such as cerebral spinal fluid and blood, blood vessels, or other tissues which are enervated such as tissue endothelial, muscle tissues, end organs, etc. The closeness of Sertoli cells to neural tissue is determined by specific neural cells and their function seeks to be restored in a given transplant. Additionally, in transplants involving non-neurai tissue, the closeness of Sertoli cells is determined by the specific tissue that is being transplanted. The source of neural cells for transplantation depends on the neurological disorder to be treated. For example, Parkinson's disease is treated with ventral mesencephalic tissue (Lindvall et al., 1990) or chromaffin cells (Lindvall et al., 1987), Huntington's disease is treated with striatal lateral eminence cells (Isacson et al. ., 1986) and neurological pain is treated with adrenal chromaffin cells (Sagen et al., 1993). Other tissue types experimentally transplanted into specific animal models of neurodegenerative disorders in humans are summarized elsewhere (Dunnett and Bjorklund, 1994) and provide detailed descriptions of cell isolation and transplantation methods. Other non-neural tissues that have been transplanted are generally reviewed elsewhere (Sanberg, 1992) and provide detailed descriptions of cell isolation and transplantation methods. The present invention also teaches a method for producing a localized immunosuppressive effect sustained in a target tissue by transplanting Porcine Sertoli cells into the tissue. For example, transplanting Porcine Sertoli cells. the central nervous system (CNS) of a subject, the cells that secrete trophic factors in itself, to treat neurological disorders including epilepsy, stroke, Huntington's disease, head injury, spinal damage, pain, Parkinson's disease, deficiency myelin, muscular dystrophy and other neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease and affective disorders of the brain. The following examples demonstrate the methods of use of the present invention as well as the efficacy to produce a sustained localized immunosuppressive effect. EXAMPLE 1 Materials and Methods Six-week-old male Sprague-Dawley rats were used, which were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The animals were stored in individual Plexiglas cages in a room with controlled humidity and temperature and under a 12-hour light-dark cycle. Food and water were freely provided ad lib. Cell Culture Bovine chromaffin cells were obtained from Dr. Jaqueline Sagen's laboratory at the University of Chicago in Illinois. After arrival, the cells were plated using DMEM-F12 flasks with serum. The cells were counted using the tripano blue method and revealed a total of 8 X 10"6 per ml of surviving cells.A 95% availability of chromaffin cells were measured on the day of arrival and on the day of transplantation. Half of the chromaffin cell solution was co-cultured overnight with Sertoli cells Sertoli cell preparation is done according to the method described by Selawry &; Cameron (Selawry and Cameron, 1993), which is incorporated herein by reference. Specifically, the testes were removed, shredded into several pieces and placed in a 50 ml conical tube containing 50 ml of Ham's F12 / DMEM medium. The pieces were washed once by centrifugation at 800 X g for two minutes. The supernatant was aspirated, and the tissue resuspended in 40 ml of the medium containing 40 mg of trypsin and 0.8 mg of DNase in a sterile 250 ml Erlenmeyer flask. The flask was placed in an oscillating incubator at 37 ° C at 60-90 ose / minutes for 30 minutes. This stage removed the Leydig cells. The tubules were then transferred to a 50 ml conical tube and centrifuged at 800 X g for two minutes. The supernatant fraction was aspirated and the pellet resuspended in 40 ml of 1 M glycine, 2 mM EDTA containing 0.01% of soybean trypsin inhibitor and 0.8 mg of DNase, and incubated at room temperature for 10 minutes. This stage lysed any of the residual Leydig cells. The cells were washed through centrifugation for two minutes, and the stage was repeated twice, or until the medium was no longer cloudy. The pellet is resuspended through moderate homogenization with a glass Paeteur pipette -in 40 ml of the medium containing 20 mg of collagenase in an Erlenmeyer flask, and incubated at 37 ° C for five minutes with 60-90 ose / minutes . The cell suspension was centrifuged at 800 xg for two minutes and the pellet resuspended through moderate homogenization with a Pasteur pipette in a 40 mg collagenase medium and 0.2 mg DNase and incubated in an Erlenmeyer flask at 37 ° C. ° C for 30 minutes with 60-90 ose / minutes. The cells were then washed by centrifugation for two minutes, and the process was repeated at least three times to remove the peritubular cells. The cells are resuspended by moderate homogenization with a Pasteur pipette in a 40 ml medium containing 40 mg of hyaluronidase and 0.2 mg of DNase and incubated at 37 ° C for 30 minutes with 60-90 ose / minutes. The cells were pelleted through gentle centrifugation for two minutes and washed at least five times to eliminate the germ cells. The fraction enriched with cells = resulting ertoli are resuspended in 0.25 ml of the medie with the chromaffin cells for at least 24 hours before transplantation. On the day of transplantation, the solution containing the fraction enriched with Sertoli cells and the chromaffin cells is resuspended using a Pasteur pipette, then sucked through a Hamilton syringe with a 20 gauge spinal needle. Surgery Surgical procedures they were performed under sterile conditions, as is well known in the art (Pakzaban et al., 1993). All animals were initially anesthetized with 0.60 ml / kg of sodium pentobarbital, then placed in a Kopf stereotaxic instrument. Bilateral striatal transplants were performed with a fixation of coordinates in: anterioposterior = +1.2, mediolateral = +/- 2.8; dorsoventral - 6.0, 5.9 and 5.8 (based on the atlas of Paxinos and Watson, 1984). The right hemisphere of the brain was transplanted with bovine chromaffin cells, while the left hemisphere received chromaffin cells plus Sertoli cells. Each side received a total volume of 3 μl of the cell cocktail solution (1 μl per DV site). After the surgery the animals were placed on heating pads until they recovered. The animals received a short course of immunosuppression using Ciclospcpr.a-A (20 mg / kg / d, i.p.) immediately after surgery and the day after transplantation. All the animals were sacrificed one month after the transplant. Histology Animals were anesthetized with 0.70 ml / kg sodium pentobarbitol, then disseminated with 500 ml of 0.9% isotonic saline and 500 ml of paraformaldehyde. The animals were then decapitated, and the brain was removed and post-fixed overnight in 40% paraformaldehyde and 30% sucrose in PBS. The next day, sections of the brain were cut into 30 micras using Vibroslice (Campden Instrument, UK). The immune response of host tissue was analyzed using the Lectin method (see below). Three experiments conduct independent qualitative comparisons of the left and right sides of the brain in a random-blind fashion. Lectin Method After sectioning of the vibrotome, the sections of the brain were placed in 0.1% Triton X-100 for 15-30 minutes. The sections were then washed in 0.1 M cationic PBS (pH 7.2), containing 0.1 mM CaCl 2, 0.1 mM MgCl 2. Incubation of the sections was performed in 20.0 μg / ml of lectin made in cationic PBS at 4 ° C for hours. Rinsing three times in PBS was performed before incubation with DAB. The DAB supply solution was made by dissolving 10 mg of DAB in 20 ml of phosphate buffer (0.1 M, pH 7.2) and adding 0.5 ml of 1% CoCl2 to the DAB solution while stirring. The sections were first preincubated in the DAB solution for 15 minutes.

After adding 0.6 ml of 3% H202 to 20.5 ml of the DAB solution, the incubation lasted five to ten minutes or until an appropriate reaction was achieved. The sections were once again rinsed three times in PBS. Finally, the sections were removed from the distilled water to remove the salts. RESULTS Histological analyzes of both transplanted sites revealed that the site transplanted with chromaffin cells alone had a higher glial infiltration and a larger number of macrophages than the site transplanted with chromaffin cells plus Sertoli cells (Figures 1 and 2). No significant difference in glial response was observed between animals treated with or without cyclosporin. Two conclusions were made from the data: 1) a localized effect of the Sertoli cells, which appeared to suppress the immune response of the host tissue to the transplanted cells, and 2) a sustained immunosuppression, with or without a short course of CsA administration, can be obtained with simultaneous cell transplantation of cro-afma with Sertcl cells (Figure 4). Therefore, it can be concluded that Sertoli cells can provide an immunologically privileged site in the CNS by direct intracranial infusion. Furthermore, with the significant immunosuppression obtained following the xenografts in this study, Sertoli cells can provide superior beneficial effects by creating immunologically privileged sites following the allografts, and improving the survival and integration of the transplant through immunological and trophic support. EXAMPLE 2 Specific Protocol: The protocol involves three basic steps, isolation of Sertoli cells, co-culture with specific neural cells or other appropriate cells and transplantation from co-culture to the CNS (for details consider cell isolation, see Selawry and Cameron ( 1993) and for details regarding cell transplantation see (Paksaban et al., 1993) Isolation of Sertoli cell The isolation procedure follows a well-defined method as described in the reference (Selawry and Cameron, 1993). The cell culture medium used in all the isolation stages and in which they were incubated was DMEM: Hams F12 supplemented with retinol, STIs and gentamicin sulfate (Cameron and Muffly, 1991). The tests were surgically collected from sixteen-day-old male Sprague-Dawley rats. The tests were decapsulated and prepared for enzymatic digestion to separate other types of testicular cells from Sertoli cells. The enzymatic procedure using collagenase (0.1%), hyaluronidase (0.1%) and trypsin (0.25%) was routinely used in many cell isolation protocols. After the sequential enzymatic digestion, the Sertoli cell isolate was washed with the culture medium, transferred to sterile cell culture vessels and placed in an air tissue culture incubator with 95% Co2 at 5%, moistened After forty-eight hours of preincubation in an incubator at 39 ° C, the Sertoli cells were washed to remove any contaminating waste. The fraction enriched with resulting Sertoli cells was resuspended to 0.25 ml of DMEM / F12 medium, and incubated at 37 ° C for at least 24 hours. The Sertoli cells were released from the floor of the vessel with 0.01% trypsin, transferred to a sterile conical test tube and repeatedly washed by centrifugation and then treated with a trypsin inhibitor to stop the enzymatic action of trypsin. On the day of transplantation, fractions enriched with Sertoli cells were resuspended and suctioned through a Kamilton syringe with a 20 gauge spinal needle. IB. Isolation and Pretreatment of Sertoli Cells As previously described (Cameron and Muffly, 1991) the decapsulated rat tests were subjected to sequential enzymatic treatment at 37 ° C using 0.25% trypsin (Sigma) and 0.1% collagenase (Sigma, type V ) (Cameron and Muffly, 1991). The resulting Sertoli cell aggregates were equally distributed in a 20 ml medium volume of incubation in 75 cm2 tissue culture flasks (Costar). Sertoli aggregates in plates were incubated at 39 ° C in 5% C02-95% air for 48 hours, after which the cells were subjected to hypotonic treatment with 0.5 mM Tris-HCl buffer for one minute (Galdieri et al. al., 1981) to accelerate the removal of the germ cells from contamination. After two washes with the incubation medium, the flasks were filled with 20 ml of the incubation medium and returned to the incubator injected with C02 at 37 ° C and 5% C02-95% air. The resulting pre-treated Sertoli-enriched monocultures contained more than 95% Sertoli cells. Plate density (<; 2.0 X 106 Sertoli cells / cm2) did not result in a confluent monolayer of cells. 2. Co-culture with specific neural cells Lae Sertol cells and the neural cell specific for transplantation for the neurodegenerative model were suspended in trypsin (C.01%), washed three times with medium and placed in a cell culture vessel sterile twenty-four hours before the transplant. The resulting co-culture was placed in an incubator with 5% C02-95% air at 37 ° C until used for transplantation. 3. Transplantation of the co-culture in the CNS The transplant protocol follows the procedure previously described by Paksaban et al., (1993). The surgery of the animal was performed under sterile conditions. All animals were initially anesthetized with 0.60 ml / kg of sodium pentobarbital, then placed in a Kopf stereotaxic instrument. For the Parkinson's disease model, striatal unilateralee transplants were performed with a fixation of coordinates in: anterioposterior - +1.2, mediolateral - +/- 2.9, dorsoventral - 6.0, 5.9 and 5.8 (based on the Paxinos and Watson atlas) ( 1984) . Different coordinates were used for the different neurodegenerative animal models also based on Paxinos and Watson (1984). The striatum ipsilateral to the nigra Leeionada eilance was transplanted with Sertoli cells or with co-culture of Sertoli cells. Each striatum receives a total volume of 3 μl of Sertoli cell or co-culture suspension. One microliter of the cell suspension was penetrated for one minute by the dorsoventral site. Then, five minutes were allowed to pass until reaching the last dorsoventral site before retracting the needle. After surgery, the animals were placed on heating pads until they recovered. Animals received a short course of immunosuppressive therapy using Cyclosporin-A (20 mg / kg / d, i.p.) immediately after surgery and the day after transplantation. Sertoli cell suspensions and / or co-culture were transplanted into animal models of several neurodegenerative disorders by the stereotaxic coordinates defined for the specific traetorno, as illustrated in the example of Parkinson's disease. All animals were systematically tested for functional recovery through techniques specific to that animal model. EXAMPLE 3 Figure 4A illustrates that dense electron chromaffin cells (arrows) were easily identified, due to the inclusion of unique secretory granules to the cells. Figure 4B shows the area in the box in Figure 4A at a higher amplification, where at higher resolution, Sertoli cells (arrows) immediately adjacent to the dense electron chromaffin cells were detected. This demonstrates the survival of the adrenal chromaffin cells grafted with Sertoli cells in the brain. (1) Neural Cell Growth Incubation Medium and Sertoli Cell Pre-conditioning Medium: The incubation medium used for Sertoli cell culture and co-culture was the Minimum Essential Medium of Dulbeco: Nutrient Medium Ha s F12 (Whittaker Bioproducts ) mixed with 1: 1 and supplemented with 3 mg / ml of L-glutamine (Sigma, grade III), O.Occ / ml insulin-transferin-selenium (ITS, Collaborative Reeearch, Inc.), 50 ng / ml retinol (Sigma), 19 μl / ml lactic acid (Sigma) and O.Occid / ml gentamicin sulfate (Gibco). After the first 48 hours of the incubation period of the isolated Sertoli cells, the media was collected and centrifuged at 1500 rpm for 5 minutes. The copolymer was collected and immediately frozen in sterile test tubes. This medium was identified as the Sertoli pre-conditioning medium (SCM). Isolation and Incubation of Fetal Brain Cell Fetal brain cells (FBC) of ventral mesencephalon were collected from fetal rats (15-17 days of gestation). The fetal brain tissue was suspended in a medium and initially dispersed by passing it through a series of hypodermic needles that were desirably reduced (caliber 18-26). The resulting euepension was treated with 0.1% trypsin for five minutes and followed by 0.1% trypsin inhibitor for two minutes. The suspended BCF was washed (three times), resuspended in an incubation medium and plated in culture vessels coated with poly-L-lysine.

Lae ventral mesencephalon cells from fetal rats (VM) were isolated and cultured for seven days in a control medium (CM) or in a preconditioned medium of Sertoli cells (SCM) as shown in Figure 3A, VM cells were incubated in CM did not show any evidence of cell stimulation or differentiation. Referring to Figure 3B, the VM cells that were incubated in SCM were highly stimulated. Figure 3C illustrates that at higher amplification, VM cells incubated in SCM exhibited an exaggerated growth of neurites. EXAMPLE 4 Incorporation of latex beads: Sertoli cells were isolated and prepared for incubation as described. Before transplantation (approximately 12 hours), sterile latex beads of 1 μm (10 μl / ml medium, Peleo, Tuetin, CA) were added to the incubation medium. Sertoli cells rapidly phagocytosed the beads. Immediately before transplantation, the Sertoli cells in the bead were washed (three times) and resuspended in ml of the incubation medium. Referring to Fure 5A, the Sertoli cells were suspended in the middle of the brain, where the penetration tract (arrows) and the Sertoli cell transplantation site are shown. At a larger amplification as shown in Figure 5B, the Sertoli cells (arrow) were easily identified due to the inclusion of lμ of latex beads, which were loaded on the Sertoli cells before transplantation. EXAMPLE 5 Effects of Cyclosporin A (CeA) on the survival of transplanted Sertoli cells Fluorescent cell labeling: Immediately before transplantation (approximately two hours), Sertoli cell monoculture was treated with fluorescent dye CM-Dil to screen the cells (lOOμl of supply / ml of medium; Molecular Probes, Inc., Eugene, OR) for 7 minutes at 37 ° C and then placed in a refrigerator (4 ° C) for an additional 15 minutes. Sertoli "labeled" fluorescent cells were washed (three times) and resuspended in 1 ml of the incubation medium. The effect of cyclosporin A on the survival of Sertoli cells grafted in situ was examined. The grafted Sertoli cells were labeled with a fluorescent label (Dil) before transplantation to the striatum of the brain. The tissue was collected one month after the transplant. Referring to Figure 6A, Sertoli fluorescent cells viablee are seen in a rat huéeped that did not receive immunoepression therapy with cyclin, osporin A. Referring to Figure 6B, viable fluorescent Sertoli cells are shown in a rat "host" receiving the immunosuppression therapy of cyclosporin A. This example demonstrates that cyclosporin A is not necessary for the survival of Sertoli cells transplanted to the brain. Through the requestSeveral publications are pre-scheduled by appointment. All citations for the publication are later. The description of these publications in totality is hereby incorporated by reference in this application so as to fully determine the nature of the technique to which this invention pertains. The invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of the description rather than the limitation. Obviously, many modifications and variations of the present invention are possible in view of the foregoing teachings. Therefore, it should be understood that within the scope of the appended claims, the invention may be practiced in a manner other than that specifically described. REFERENCES REFERENCES Berden et al., "Severe central nervous eyetem toxicity associated with cyclosporine" Lance 26: 219-220 (1585).

Bjorklund and Stenevi, "Intracerebral neural grafting: a historical perspective" in Bjorklund, A. and U. Stenevi, ede. Neural qrafting in the mammalian CNS, Ameterdam: Eleevier, 3-11 (1985). Bjorklund, "Dopaminergic traneplants in experimental Parkinsonism: Cellular mechanisms of graft-induced functional recovery" Current Biology, 2: 683-689 (1992). Borlongan et al., "Cycloeporine-A increaees spontaneous and dopamine agonist-induced locomotor behavior in normal rate" Cell Traneplant. , 4: 65-73 (1995). Borlongan et al. "PR: Systemic 3-nitropropionic acid: Behavioral deficits and striatal damage in rats", Brain Research Bulletin, 36: 549-556 (1995). Cameron et al., "Succeseful ielet / abdominal teetis transplantation does not regu- late Leydig cells" Transplantation. 50: 649-653 (1990). Cameron and Muffly, "Hormonal regulation of spermated binding to Sertoli celle in vitro" J.Cell Sci. , 100: 532-533 (1991). de Groen et al., "Central nervous syete toxicity after liver traneplantation" N. Ensl. J. Med. 14: 861-866 (1984).

Dunnett and Bjorklund, Functional Neural Transplantation, Advances in Neuroscience. Volume 2, Raven Press, New York. Freeman et al., "The USF protocol for fetal nigral transplantation in Parkinson 'e dieeaee" Experimental Neurology., 129: 6-7 (1994). Griswold, "Protein secretion by Sertoli cells: general consideratione" in Rueeel, L.D. and M.D. Griswold, eds. The Sertoli Cell, Cache River Prese, Clearwater, FL., 195-200. Isacson et al., "Graft-induced behavioral recovery in an animal model of Huntingtons die" Proc. Nati 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. Neuro1. , 22: 457-468 (1987). Lindvall et al., "Graft of fetal dopamine neurons survive and improve motor function in Parkinecn'e disease" Science. 247: 574-577 (1990). Pakzaban et al., "Increased proportion of Ache-rich zones and improved morphological integration in host striatum of fetal grafte derived from the lateral but not the medial gangli nic eminence" Exp. Brain Res., 97: 13-22 (1993). Paxinos and Watson, "The rat brain in stereotactic coordinatee" Sydney, Academic Prees (1984). Sagen et al., "Transplants of immunologically isolated xenogeneic chromaffin cells provide a long-term source of pain-reducing neuroactive substance" J. Neurosci. 13: 2415-2423 (1993). Sanberg, PR. (Editor-in-chief) "Cell Transplantation", Elservie Science Publiehere. New York, 1992-Present Sanberg et al., "Cell transplantation for Huntington's disease" R.G. Landee Co. , Boca Raton, FL, pp. 19-21 (1994). Sanberg et al., "Sertoli celle induces immunoreactivity and functional recovery following tranaplantation into the striatum of 6-OHDA reagent rate (in preparation) (1995) Selawry and Cameron," Sertoli cell-enriched fraction in euccessful islet cell transplantation "Cell Transplant .. 2: 123-129 Ir93). Wictorin et al., "Reformation of long axon pathways in adult rat CNS by human forebrain neuroblasts" Nature, 347: 556-558 (1990).

Claims (18)

1. CLAIMS 1. A method to produce a sustained localized immunosuppressive effect and a trophic effect on a target tissue by transplanting Sertoli cells near the target tissue.
2. 2. The method, in accordance with the claim 1, characterized in that the transplantation step is further defined as co-bringing back the Sertoli cells and transplanting the tissue of the same type as the target tissue in the target tissue.
3. 3. The method, in accordance with the claim 2, characterized in that Sertoli cells are co-transplanted with neural cells in a mammalian central nervous system.
4. 4. The method, in accordance with the claim 3, characterized in that the neural cells co-transplanted with the Sertoli cells are first co-cultured with the Sertoli cells and the co-cultured cells are co-transplanted together.
5. 5. The method, in accordance with the claim 1, characterized in that the transplant is also defined as a direct infusion of the Sertoli cells in the central nervous system.
6. 6. The method, according to claim 1, characterized in that the Sertoli cells are Sertoli porcine cells.
7. The method, according to claim 1, characterized in that the mammal suffers from neurological disorder or neural degeneration disorder including epilepsy, attack, Huntington's disease, head injury, spinal damage, pain, Parkinson's disease, deficiency of myelin, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective traetornoe of the brain.
8. 8. A method for producing an immunosuppressive, localized and sustained effect on a target tissue by planting Porcine Sertoli cells in the central nervous system of a subject, cells also secreting trophic factors in situ, to treat neurological disorders, including epilepsy, attack, Huntington's disease, head injury, eepinal damage, pain, Parkineon's disease, myelin deficiencies, dystrophy, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective disorders of the brain.
9. 9. The method, according to claim 8, characterized in that the subject is a human being.
10. 10. The use of Sertoli cells to produce a localized immunosuppressive effect and a trophic effect on target tissue by transplanting Sertoli cells near the target tissue.
11. 11. The use, in accordance with the claim 10, in which the transplant is defined as the co-transplant of Sertoli cells and transplant tissue of the same type as the target tissue in the target tissue.
12. 12. The use according to claim 11, wherein the Sertoli cells are co-transplanted with neural cells in a mammalian central nervous system.
13. The use, according to claim 12, wherein the neural cells co-transplanted with Sertoli cells are first co-cultured with the Sertoli cells and the co-cultured cells are co-transplanted together.
14. 14. The use according to claim 10, wherein the transplant is further defined as a direct infusion of the Sertoli cells in the central seventh nerve.
15. 15. The ueo, according to claim 10, wherein the Sertoli cells are Sertoli porcine cells.
16. 16. The use, according to claim 11, wherein the mammal suffers from a neurological disorder or traetorno of neural degeneration including epilepeia, attack, Huntington's disease, head injury, eepinal damage, pain, Parkineon's disease, myelin deficiency, neuromuscular disorders, neurological pain, amyotrophic lateral sclerosis, Alzheimer's disease, and affective disorders of the brain.
17. 17. A use of Sertoli cells to produce a sustained localized immunosuppressive effect and a trophic effect on a target tissue by transplanting Porcine Sertoli cells into the central seven nerve of a stem, lae cells also secreting trophic factors in itself, to treat neurological disorders including epilepsy, attack, Huntington's disease, head injury, spinal damage, pain, Parkinson's disease, myelin deficiencies, muscular dystrophy, neurological pain, lateral sclerosis amyotrophic, Alzheimer's disease, and affective traetornoe of the brain.
18. 18. The word, according to claim 17, wherein the object is a human eer.