JP3699727B2 - Sertoli cells as nerve recovery-inducing cells for neurodegenerative diseases - Google Patents

Description

TECHNICAL FIELD The present invention relates generally to cell transplantation, and more particularly to a method for transplanting cells that recovers behavioral and functional deficits associated with neurological disease after transplantation into the central nervous system (CNS).
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) includes sputum, stroke, Huntington's disease, head trauma, spinal cord trauma, pain, Parkinson's disease, myelin deficiency, neuromuscular disease, neuralgia, muscle It is becoming another treatment for neurological and neurological diseases, including atrophic lateral sclerosis, Alzheimer's disease, and brain affective disorders. Preclinical data and clinical data show that transplanted cells (grafts) used in cell transplantation protocols for these types of neurodegenerative diseases survive, coalesce with host tissues, and provide 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 graft source in Parkinson's disease (Lindvall et al., 1990; Bjorklund, 1992). Similarly, fetal striatal tissue has been used successfully as a graft material in Huntington's disease (Isacson et al., 1986; Sanberg et al., 1994).
Nerve dysfunctional animals had been transplanted with non-fetal cells and non-neuronal cells / tissues. For example, chromaffin cells from adult donors have been used to treat Parkinson's disease. A major advantage of this type of transplant protocol is that the graft source is not a fetal source, thereby avoiding ethical and logical problems associated with acquiring fetal tissue. Normalization of behavior is observed using the chromaffin cell protocol. However, functional recovery of this behavior is temporary, and 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 the animal's normal behavioral activity in Parkinson's disease models prematurely applies the clinical application of other treatment therapies as well as this protocol.
The administration of growth factors as a means of treating neurological and neurodegenerative diseases was intended in the art. However, delivering these drugs to the brain still involves a number of difficulties that should be resolved successfully. In general, these drugs cannot be administered systemically, and injection into the brain is an impractical and incomplete solution. Although it has been suggested to manipulate cells to deliver specific monotrophic factors when transplanted into the brain, stable transfection and survival of cells when transplanted into the brain is a constant problem. Furthermore, it is becoming increasingly recognized that multitrophic factors acting in concert are likely necessary for the successful treatment of neurological and neurodegenerative symptoms.
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 is clear that the efficacy of the treatment is due, in part, to their known immunosuppressive secreted factors due to the presence of Sertoli cells (Selawry and Cameron, 1993; Cameron et al., 1990). Sertoli cells are also known to secrete several important vegetative growth factors.
Therefore, it would be desirable to use Sertoli cells alone as a source for diseases in which damaged tissue growth factor and trophic factor support is beneficial. Examples include neurological diseases including wound healing and neurodegenerative diseases. Sertoli cells can serve as an in situ factory for trophic factors, thereby speeding wound healing and restoring functional and behavioral deficits associated with neurological and neurodegenerative diseases.
SUMMARY OF THE INVENTION In accordance with the present invention, a method is provided for producing in situ trophic factor production by transplanting Sertoli cells, which cells secrete trophic factors in situ, into a mammal.
[Brief description of the drawings]
Other advantages of the present invention will be readily understood and become 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 rotational behavior, showing> 7 rotations per minute when animals from both groups were challenged with apomorphine pre-transplantation, or at least a total of 210 rotations over 30 minutes (lesions) In contrast, animals receiving media alone continued to show significant rotation during the post-transplant period, in contrast, animals receiving Sertoli cells had a marked decrease in their rotational behavior over the post-transplant period ( Greater than 60%).
FIG. 2 is a graph showing biased rocking behavior, with> 80% biased rocking activity (opposite to lesion) as evidenced by an elevated body rocking test with animals from both groups. In the post-transplant period, animals receiving medium alone continued to exhibit significant biased rocking activity, in contrast, animals receiving Sertoli cells were biased rocking behavior over the post-transplant period. Did not show.
FIGS. 3A-C show the ventral mesencephalon of fetal rats isolated and cultured for 7 days in control medium (CM) or Sertoli cell conditioned medium (SCM) and photographed with dark field, interference contrast optics ( Photomicrograph showing cells from (VM), (A) shows VM cells incubated in CM showing no evidence of stimulation or differentiation, and (B) is clearly highly stimulated (C) shows VM cells incubated in SCM showing neurite outgrowth as a result of Sertoli secretory trophic factor at high magnification.
4A-B are electron micrographs showing the invasion path (arrow) and the striae of the brain showing the site of Sertoli cell transplantation (A), (B) is a box shape in (A) at high magnification and high resolution. Regions are shown and Sertoli cells (arrows) are easily identified for 1μ latex bead inclusions, which were placed in the cells prior to transplantation.
Figures 5A-B are two light micrographs showing transplanted Sertoli cells labeled in situ with a fluorescent label (DiI) prior to transplantation into the striatum of the brain, (A) is cyclosporin A (CsA). (B) shows viable fluorescent Sertoli cells in a rat host that received cyclosporin A immunosuppressive treatment.
DETAILED DESCRIPTION OF THE INVENTION Generally, the present invention provides a method for promoting the recovery, protection, and support of dysfunctional tissues by a mechanism that includes in situ production of Sertoli cell-derived growth factors and regulators, commonly referred to as trophic factors . Furthermore, 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 important advantage of utilizing Sertoli cells as an in situ factory for producing trophic factors is that Sertoli cells have been shown to have an effective immunosuppressive effect. Therefore, no concurrent additional therapy is required to produce immunosuppression. In other words, Sertoli cells can be used as a source of trophic factors and also provide a self-induced local immunosuppressive effect.
As trophic factors secreted by Sertoli cells, Sertoli cell-derived growth factors and regulators such as insulin-like growth factors I and II, epidermal growth factors, transforming growth factors α and β, and interleukin 1α (Griswold, 1992) Is mentioned. See Table 1 for a more extensive list of Sertoli cell secretory factors. Such factors have been shown to have a healing 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 takes advantage of the phenomenon that Sertoli cells can produce a growth-supporting liquid microenvironment that is rich in nutrients at the site of cellular 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 transplant protocol used in the diabetes model, the method of the present invention uses only one type of cell, namely Sertoli cells, thereby allowing two different cell types to be transferred to a single host site. Considerably reduces the inherent logical and operational problems in 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. Furthermore, in a preferred embodiment of the present invention, porcine Sertoli cells can be transplanted into a mammal such as a human. Further, veterinary use of the present invention is contemplated and syngeneic Sertoli cells will be selected for transplantation into the desired mammalian host.
As demonstrated in the experimental part below, the present invention may be used as a treatment to recover behavioral and functional deficits associated with neurodegenerative diseases such as Huntington's disease and Parkinson's disease. This can be done without the side effects associated with conventionally used immunosuppressive adjuvant therapy, such as the chronic use of cyclosporin A. Sertoli cells provide both secretory factor secretion and immunosuppressive effects.
As shown in the examples below, transplantation of Sertoli cells prior to the 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-type disease provided both neuroprotective and prophylactic effects for subsequent brain lesions. Therefore, early Sertoli cell transplantation after diagnosis of a neurodegenerative disease can provide beneficial treatment, prevention or reduction of the disease. In addition, Sertoli cells can be transplanted in other types of CNS trauma, such as head lesions, to treat, prevent and prophylactically reduce the effects of CNS injury.
The following examples demonstrate the ability of the present invention to recover the behavioral deficits associated with neurodegenerative diseases.
Example 1: Sertoli cell transplantation
Special protocol:
Protocols generally involve two basic steps, (1) Sertoli cell isolation and (2) cell transplantation, both of which are described briefly below (for details on cell isolation, see Selawry and Cameron (1993)). Also, see Pakzaban et al. (1993) for details on cell transplantation, both of which are included for reference).
(1A) Sertoli cell isolation Isolation procedures are routinely utilized according to well-described methods, Selawry and Cameron (1993). The cell 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 collected from 16-day-old male SD rats by surgery. Testes were decapsulated and prepared for enzymatic digestion to separate other testicular cell types from Sertoli cells. Enzymatic manipulation is a typical manipulation used in many cell separation protocols, using collagenase (0.1%), hyaluronidase (0.1%), and trypsin (0.25%). After continuous enzyme digestion, the Sertoli cell isolate was washed with media, transferred to a sterile culture vessel, and placed in a humidified 5% CO ₂ -95% air tissue culture incubator. After 48 hours of preincubation in a 39 ° C. incubator, Sertoli cells were washed to remove contaminating debris. The resulting Sertoli cell rich 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 container floor with trypsin, transferred to a sterile conical tube, washed repeatedly by centrifugation, and treated with trypsin inhibitor to stop the trypsin enzymatic action. During the day of implantation, the Sertoli cell rich 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, Using type V) (Cameron et al., 1987a; Cameron et al., 1987b), the delaminated rat testicles were subjected to continuous enzyme treatment at 37 ° C. The resulting Sertoli cell aggregate was 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 for 48 hours at 39 ° C. in 5% CO ₂ -95% air, after which the cells were hypotonicized with sterile 0.5 mM Tris-HCl buffer (Galdieri et al. 1981) promoted removal of contaminating germ cells. After washing twice with incubation medium, the flask was again refilled with 20 ml of incubation medium and returned to the CO ₂ injection incubator at 37 ° C. in 5% CO ₂ -95% air. The resulting pretreated sertoli rich single culture contained more than 95% sertoli cells. The coating 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 surgical device. Unilateral line using coordinates set in anteroposterior direction = +1.2, medial lateral = +/- 2.8, dorsal ventral = 6.0, 5.9, and 5.8 (based on Paxinos and Watson, 1984 atlas) A striatum transplant was performed. Sertoli cells were transplanted into the ipsilateral striatum of the affected substantia nigra. Each filament receives a Sertoli cell suspension with a total volume of 3 μL. One microliter of Sertoli cell suspension was injected over the dorsoventral region for 1 minute. The control received medium only. An additional 5 minutes was allowed after reaching the last dorsoventral region before the needle was retracted. After surgery, the animals were placed on a heating pad and allowed to recover. Animals receive a short course of immunosuppression using cyclosporin A (20 mg / kg / d, ip) immediately after surgery and on the day after transplantation. However, subsequent studies have demonstrated that this short course of cyclosporin A is not required (FIGS. 5A-B).
Sertoli cells are transplanted into animal models of various neurodegenerative diseases with stereotactic surgical coordinates defined for a specific disease, as shown in the example of Parkinson's disease, and the animal model is then systematically restored to 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, animals were subjected to behavioral tests including apomorphine-induced rotational and rocking behavior. Baseline data showed significant apomorphine-induced rotational behavior (opposite to lesion side of CNS) in all these animals (at least 200 rotations in 30 minutes). Significant right-biased rocking activity (greater than 70%) was also observed using the elevated biological rocking test (EBST).
Three weeks after the lesion, one group of animals (n = 6) received Sertoli cells and one group (n = 6) was subjected to the same surgical procedure, but only medium (DMEM without serum) as a control. I received it. All animals received cyclosporine (20 mg / kg) after the first 2 days of transplantation. The animals were reintroduced into the behavior 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 were at the pre-transplant rotation level (FIG. 1). Normalization of rotational behavior persisted over a 2 month test period. The right-biased rocking activity already demonstrated by Sertoli cell transplanted animals was also significantly reduced in the test period after transplantation (FIG. 2). Animals receiving medium did not show a significant decrease in their right-biased rocking response.
At necropsy, the 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 invasion site (ie, lesion site) in Sertoli cell transplanted rats when compared to the invasion site in lesioned animals where the Sertoli cells were not transplanted.
Example 2: Growth of nerve cells
Incubation medium and Sertoli cell preconditioned 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), Dulbecco mixed 1: 1, supplemented with 50 ng / ml retinol (Sigma), 19 μl / ml lactic acid (Sigma) and 0.01 cc / ml gentamicin sulfate (Gibco) Minimum essential medium: Hams F12 nutrient medium (Wittaker Bioproducts).
After the first 48 hours of incubation of isolated Sertoli cells, the medium was collected 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 harvested from the ventral midbrain of fetal rats (15-17 gestation). The fetal tissue was first dispersed by suspending it in the medium and passing it through a series of successively decreasing size hypodermic needles (18-26 gauge). The resulting suspension was treated with 0.1% trypsin for 5 minutes followed by 0.1% trypsin inhibitor for 2 minutes. The suspended FBC was washed three times, resuspended in incubation medium and applied to poly-L-lysine coated culture vessels.
Cells from the ventral midbrain (VM) of fetal rats were isolated and cultured for 7 days in control medium (CM) or Sertoli cell conditioned 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 VM cells incubated in SCM at high magnification show neurite outgrowth as a response to Sertoli cell-dispersing trophic factor.
Example 3: Identification of Sertoli cells
Latex beads contamination :
Sertoli cells were isolated and prepared for incubation as described. Prior to implantation (approximately 12 hours), sterile 1 μm latex beads (10 μg / ml medium, Perco, Tastin, CA) were added to the incubation medium. Sertoli cells phagocytosed the beads quickly. Just prior to transplantation, beaded Sertoli cells were washed (3 times) and resuspended in 1 ml of incubation medium.
Referring to FIG. 4A, Sertoli cells are transplanted into the striatum of the brain, and the entry path (arrow) and the site of Sertoli cell transplantation are shown. At high magnification as shown in FIG. 4B, Sertoli cells (arrows) were readily identified due to contamination of 1 μ latex beads loaded into Sertoli cells prior to transplantation.
Example 4: Effect of cyclosporin A (CSA) on survival of transplanted Sertoli cells
Fluorescent cell labeling :
Immediately prior to transplantation (approximately 2 hours), Sertoli cell single cultures were subjected to CM-DiI fluorescence for 7 minutes at 37 ° C. for cell tracking (100 μl stock / ml medium; Molecular Probes (Eugen, OR)). Treated with dye and then left 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 cyclosporin A on the survival of Sertoli cells transplanted in situ was investigated. The transplanted Sertoli cells were labeled with a fluorescent label (DiI) prior to transplantation into the striatum of the brain. The tissue was collected 1 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, live 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 transplanted into the brain.
Example 5: Prophylactic effect of Sertoli cells Sertoli cell transplantation is neuroprotective when transplanted prior to inducing brain lesions. This preventive effect of Sertoli cells was demonstrated in an animal model of Huntington's disease (HD). This model is generated by systemic administration of a 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 3NP injections produce special lesions in the striatum that mimic those found in Huntington's disease.
In this experiment, rat Sertoli cells (as described above) were transplanted on one side into one normal striatum in 8 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 as described elsewhere (Koutouzis et al., 1994; Borlongan et al., 1995) to induce HD. 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 organism (Koutouzis et al., 1994; Borlongan et al., 1995).
One month after administration of 3NP, the animals showed unilateral behavioral deficits. This was seen by demonstration of apomorphine-induced rotation after lesions in Sertoli transplanted animals, not controls (number of rotations; control = 0.25 ± 0.6; Sertoli transplant = 197 ± 31.9, p <0.0001). This asymmetric rotational behavior indicated a lesion on the side of the brain that was not transplanted with Sertoli cells. Therefore, Sertoli cell transplants have a neuroprotective and preventive effect on subsequent brain lesions with respect to trophic mechanisms. This provides evidence that Sertoli transplantation may be beneficial for early treatment or benefit of neurodegenerative disease before significant damage is present.
These results indicate that when combined, 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 factor, as demonstrated by neural tissue growth as shown in Example 2, and regulatory factors that promote the recovery and prolonged support of the associated neural tissue. is there. Furthermore, Sertoli cells can protect nerve tissue in the brain by inhibiting glial cell activation at the lesion site and promote nerve tissue recovery. These results also demonstrate the in situ survival of transplanted Sertoli cells.
Within this application, various publications are referenced by citation or number. Full citations for the publication are listed below. In order to more fully describe the state of the art to which this invention pertains, the entire disclosures of these publications are incorporated herein by reference to this application.
It is to be understood that the present invention is described in an exemplary manner and the terminology used is intended to be illustrative and not limiting.
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.

Claims (4)

A pharmaceutical composition comprising Sertoli cells, nerve cells and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1, wherein the nerve cell is first co-cultured with the Sertoli cell. The pharmaceutical composition according to claim 1, wherein the Sertoli cell is a porcine Sertoli cell. A method of producing trophic factor production in situ in the central nervous system,
Transplanting Sertoli cells to the central nervous system of the subject excluding the human body,
A method wherein the Sertoli cells secrete trophic factors in situ.

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