Suppression of Kindled Seizures by Paracrine ... - Wiley Online Library

Epilepsia, 46(8):1162–1169, 2005 Blackwell Publishing, Inc. C 2005 International League Against Epilepsy

Laboratory Research

Suppression of Kindled Seizures by Paracrine Adenosine Release from Stem Cell–Derived Brain Implants ∗ †Martin Güttinger, ∗ †Denise Fedele, ‡Peter Koch, §Vivianne Padrun, §William F. Pralong, ‡Oliver Brüstle, and ∗ †Detlev Boison ∗ Institute of Pharmacology and Toxicology, University of Zürich, and †Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Federal Institute of Technology (ETH), Zürich, Switzerland; ‡Institute of Reconstructive Neurobiology, University of Bonn Medical Center and Hertie Foundation, Bonn, Germany; and §Institute of Neurosciences, Federal Institute of Technology (EPF), Lausanne, Switzerland

Summary: Purpose: Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Previously, adenosine-releasing embryonic stem (ES) cells have been generated by disruption of both alleles of adenosine kinase (Adk−/− ). Lack of ADK did not compromise the cells’ differentiation potential into embryoid bodies or glial precursor cells. The aim of the present study was to investigate the potential of differentiated Adk−/− ES cell progeny for seizure suppression by paracrine adenosine release. Methods: To isolate paracrine effects of stem cell–derived implants from effects caused by network integration, ES cell– derived embryoid bodies and glial precursor cells were encapsu-

lated into semipermeable polymer membranes and grafted into the lateral brain ventricles of kindled rats. Results: While seizure activity in kindled rats with wildtype Adk+/+ implants remained unaltered, rats with adenosinereleasing Adk−/− ES cell–derived implants displayed transient protection from convulsive seizures and a profound reduction of afterdischarge activity in EEG recordings. Long-term seizure suppression was precluded by limited viability of the encapsulated cells. Conclusions: We thereby provide a proof-of-principle that Adk−/− ES cell–derived brain implants can suppress seizure activity by a paracrine mode of action. Adk-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders. Key Words: Epilepsy—Adenosine—ES cells —Cell therapy—Seizure suppression.

The most straightforward application of cell-based therapies is the local delivery of therapeutic compounds by engineered cells at the site of transplantation. With this strategy, a steady and potentially more physiologic concentration of the compound may be achieved without the complication of systemic side effects (1,2). The antiepileptic potential of adenosine can thus be exploited by intracerebral implants of cells engineered to release adenosine. This was recently accomplished by a tumor cell line [baby hamster kidney (BHK) cells] engineered to lack adenosine kinase (3). The local release of adenosine from these encapsulated engineered cells was demonstrated to suppress seizures in kindled rats. Long-term studies were precluded

by the limited viability of encapsulated fibroblasts (3). To achieve long-term cell survival and potentially direct integration of therapeutic cells into affected host tissue, stem cell–derived brain implants should constitute a superior source for cell grafting. Because embryonic stem (ES) cells can be maintained and expanded in an undifferentiated state, it is possible to generate virtually unlimited numbers of cells for transplantation. However, direct grafting of undifferentiated ES cells is restricted by the formation of teratomas and low graft survival (4); therefore a protocol has been established that permits the efficient in vitro generation of precursors for oligodendrocytes and astrocytes. Such wild-type cells were not associated with tumor growth or nonneural differentiation when implanted into the brain and spinal chord of a neonatal myelin disease model (5). Because of such potential benefits of stem cell–derived implants, in the present study, we evaluated ES cell

Accepted April 5, 2005. Address correspondence and reprint requests to Dr. D. Boison at Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland. E-mail: boison@ pharma.unizh.ch

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ADENOSINE STEM CELL THERAPY derivatives as a new cellular source for the local therapeutic delivery of adenosine. In a previous study, a stem cell–based delivery system for adenosine was generated by disruption of both alleles of adenosine kinase (ADK) in mouse ES cells (6). These Adk−/− ES cells were differentiated into glial precursor cells and released significant amounts of adenosine (6). However, it has never been demonstrated that adenosine-releasing stem cell–derived brain implants are actually able to suppress seizure activity. Before direct cell implantations can be attempted, where a combination of paracrine and network effects may influence seizure activity, it is essential to ensure that paracrine effects by adenosine alone are sufficient to prevent seizure activity. We therefore set out to study whether ES cells differentiated into embryoid bodies (EBs) and glial precursors might qualify for this application by determination of the anticonvulsant effect in an established model of temporal lobe epilepsy. To distinguish paracrine effects of Adk−/− donor cells from indirect effects due to direct cell transplantation and tissue integration, the cells were encapsulated into semipermeable polymer membranes and implanted into the lateral brain ventricles of kindled rats. In addition, the discrimination of the anticonvulsant action of adenosine, specifically, independent of other factors potentially released from the encapsulated ES cell progeny, was accomplished by comparing the results with respective wild-type Adk+/+ ES cell–derived implants. MATERIALS AND METHODS Cell culture The wild-type and genetically altered ES cells (Adk+/+ and Adk−/− , respectively) used in these experiments were derived from the mEMS32 cell line (7). ES cells were cultured at 37◦ C under 5% CO2 on a feeder layer of irradiated mouse embryonic fibroblasts in Dulbecco’s minimal essential medium (DMEM) knockout medium (Life Technologies, Rockville, MD, U.S.A.) containing 15% fetal calf serum (FCS; Life Technologies) and supplemented with L-glutamine (200 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), nonessential amino acids (all from Life Technologies), β-mercaptoethanol (Sigma), and leukemia inhibitor factor (LIF, Chemicon, Temecula, CA, U.S.A.). This medium is referred to as stem cell medium. EBs were generated by first plating Adk−/− and Adk+/+ ES cells on gelatin-coated dishes without feeders until ∼90% confluency. The ES cell cultures were then removed from the plate by incubation with trypsinethylenediaminetetraacetic acid (EDTA; Life Technologies) at 37◦ C for 5 min. The ES cells were pelleted, resuspended in ES cell medium without LIF, and cultured for 4 days in bacteriology dishes (Sterilin, U.K.). Glial precursor cells were generated from Adk−/− and Adk+/+ ES cells by using a stepwise differentiation

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protocol, as described (5,6). The glial precursors were routinely cultured on polyornithine-coated dishes in N3 medium, which is based on a 1:1 mixture of DMEM with Ham’s F12 supplemented with insulin (25 µg/ml), human apo-transferrin (100 µg/ml), progesterone (20 nM), putrescine (100 µM), sodium selenite (30 nM), penicillin (100 U/ml), and streptomycin (100 µg/ml). In addition, laminin (1 µg/ml) was added when plating the cells. To keep the cells in a proliferative state, the growth factors, fibroblast growth factor 2 (FGF-2; 10 ng/ml) and epidermal growth factor (EGF) (20 ng/ml), were added daily. The N3 medium plus EGF and FGF-2 is referred to as N3EFL medium. On growth factor withdrawal, these cells differentiate primarily into astrocytes, and a smaller fraction, into oligodendrocytes (6). Cell encapsulation Adk−/− and Adk+/+ EBs were encapsulated (24-hold EBs corresponding to 1.5 × 105 ES cells per capsule) into semipermeable polyethersulfone polymer hollow fiber (Akzo Nobel AG, Wupperthal, Germany) membranes (5 mm long, 0.5 mm inner diameter; wall thickness of 50 µm) without a supporting matrix, because EBs form three-dimensional spherical structures in culture. Adk−/− and Adk+/+ glial precursor cells were encapsulated (3 × 105 cells per capsule) into identical membranes containing a polyornithine-coated polyvinyl alcohol (PVA) matrix (Rippey Corporation, El Dorado Hills, CA, U.S.A.) as previously described (3). Capsules were sealed by photopolymerization of an acrylate-based glue. The encapsulated EBs were maintained at 37◦ C for 5 days in ES cell medium without LIF to allow the attachment of the encapsulated cells to the capsule wall before implantation. Encapsulated glial precursor cells were maintained at 37◦ C for 5 days in N3EFL medium to keep the precursors initially proliferating, which is considered to be an advantage when using capsules with matrix material. Adenosine release The amount of adenosine released from ES cell–derived EBs was assessed from plated and encapsulated cells. ES cell–derived EBs were suspended in 10 ml of ES cell medium without LIF at a density corresponding to 1–2 × 105 ES cells/ml. For sample collection, the medium was replaced with fresh medium 24 h after suspension, and 2 h later, 300 µl of medium was collected. These aliquots were analyzed in an enzyme-coupled bioluminescence assay, as described (6). To analyze the amount of adenosine released from encapsulated ES cell–derived EBs, four capsules of each genotype were made and incubated in pairs in 500 µL of medium. Two hours after changing the medium, 200 µl of the culturing medium was collected for adenosine analysis. Epilepsia, Vol. 46, No. 8, 2005

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Animals and surgery All animal experiments were conducted in accordance to guidelines of the local animal welfare authorities. Male Sprague–Dawley rats (Harlan Netherland, Horst, The Netherlands) were obtained at a body weight of 220 to 240 g. All rats were allowed to become acclimated for ≥1 week before being used in the experiments. The rats were housed (two rats per cage) under 12-h light/dark cycle (lights on from 8:00 a.m.) with food and water provided ad libitum. Under general pentobarbital anesthesia (50 mg/kg, i.p.), rats (n = 20) were placed in a Kopf stereotactic frame, and bipolar, coated, stainless-steel electrodes (0.20 mm in diameter; Bilaney Consultants, Düsseldorf, Germany) were implanted bilaterally into the hippocampus and fixed with a pedestal of dental acrylate. Coordinates for hippocampal electrodes were (toothbar at 0) 5.0 mm caudal to bregma, 4.8 mm lateral to midline, and 7.0 mm ventral to dura. Kindling and cell grafting One week after electrode implantation, the animals were stimulated unilaterally ≤12 times daily with a Grass S-88 stimulator (1-ms square-wave pulses of 50 V at 10Hz frequency for 10 s, 5-min interval between stimulations). Stimulations were maintained for a total period of ≤15 days until all animals reacted reproducibly with a grade 5 seizure after the first daily test stimulus. Behavioral seizures were scored according to the scale of Racine (8). With this strict kindling paradigm, ∼30% of kindled rats did not respond to conventional antiepileptic drugs (AEDs) (3). These pharmacoresistant animals were identified by resistance to phenytoin (PHT; in 50% polyethyleneglycol 400 in saline; 60 mg/kg, i.p.) and were excluded from this study to be used in an ongoing project on pharmacoresistance in the rat kindling model of epilepsy. Under stereotactic guidance, Adk−/− or wild-type Adk+/+ ES cell–derived implants were grafted into the lateral brain ventricles of kindled rats ipsilateral to the site of stimulation. Coordinates for capsule implantation were (toothbar at −8.3): 1.8 mm caudal to bregma, 1.4 mm lateral to midline, and 8.5 mm ventral to dura. Assessment of seizure suppression Beginning 3 days after grafting, the anticonvulsant efficacy of encapsulated Adk−/− ES cell–derived implants or their wild-type Adk+/+ counterparts was tested by the delivery of intrahippocampal test stimulations (1-ms squarewave pulses of 50 V at 10-Hz frequency for 10 s) every other day. Convulsions were visually scored according to the scale of Racine (8). The electroencephalogram (EEG) was recorded for periods of 1 min before and 4 min after application of the stimulating pulse by using a Human Scoring registration/software unit (B. Geehring, Institute of Pharmacology and Toxicology, University of Zurich, Switzerland). Epilepsia, Vol. 46, No. 8, 2005

Histology Two and 7 days after grafting, rats containing Adk+/+ and Adk−/− EB-derived implants were killed and their capsules (n = 2–5 each) retrieved. Capsules retrieved after 2 days were derived from control animals at a time point when cell viability was expected to be high, whereas capsules retrieved after 7 days were obtained from experimental animals shortly after the delivery of the last test stimulus. Capsules were fixed in 4% paraformaldehyde (EMS, Hatfield, PA, U.S.A.) with 1% glutaraldehyde (Fluka, Buchs, Switzerland) in PBS for 3 h, rinsed in phosphate-buffered saline (PBS), and dehydrated through graded alcohol washes. In addition, to check for the in vitro viability of encapsulated cells, two capsules containing EBs from each genotype were fixed 12 days after encapsulation corresponding to day 7 of the in vivo study. To cut micrometer sections, the capsules were embedded with a commercial glycol methacrylate kit (Fluka). Procedures were performed according to the manufacturer. After hardening of the embedded capsules at 60◦ C for 3 days, sections of 1.0 µm thickness (Supercut microtome 2065; Reichert) were mounted on glass slides and stained with hematoxylin (Papanicolaou; Merck, Darmstadt, Germany) and eosin (Fluka). After retrieval of capsules from rat brain, 25-µm coronal slices of the host brain were cut on a microtome and stained with cresyl violet, as described previously (9), and analyzed for implant location and surrounding tissue reactions. RESULTS Adenosine release by Adk−/− EBs In a previous study, the amounts of adenosine released from undifferentiated ES cells and from glial precursor cells were determined (6). In that study, only minimal amounts of adenosine were released from Adk−/− ES cells (2.6 ± 0.4 ng adenosine per 105 cells per hour), whereas undifferentiated Adk−/− N3EFL glial precursor cells released 11.7 ± 1.7 ng adenosine per 105 cells per hour. Because EBs represent an intermediate step in the differentiation of ES cells to glial precursors and therefore constitute a potentially versatile tool for cell encapsulation, in the present study, the amounts of adenosine released by plated and encapsulated Adk+/+ and Adk−/− ES cell–derived EBs were analyzed with an enzyme-coupled bioluminescent assay. Two hours after changing the medium in which floating EBs were cultured, a sample of medium was collected from each of five replicate plates per genotype and analyzed for adenosine content. At the time of sample collection, one plate of EBs per genotype was trypsinized to a single-cell suspension, and the cells were counted to obtain an approximate number of cells per milliliter. The adenosine concentration in the medium from Adk−/− EBs (n = 5) was found to be 21.6 ± 13.2 ng/ml, corresponding to an approximate release of 9.0 ± 5.5 ng

ADENOSINE STEM CELL THERAPY

FIG. 1. Adenosine release from cultured and encapsulated embryoid bodies (EBs). Adenosine levels in the medium of cultured EBs (n = 5 for each genotype) and encapsulated EBs (n = 4 capsules per genotype, cultured in pairs) derived from Adk −/− and Adk +/+ embryonic stem (ES) cells was analyzed by using an enzyme-coupled bioluminescent assay. Adk −/− EBs in culture or encapsulated released significant amounts of adenosine, whereas Adk +/+ EBs did not. Error bars, ±SDs.

adenosine per 105 cells per hour, whereas comparable medium from Adk+/+ EBs (n = 5) was found to contain 4.5 ± 3.3 ng/ml adenosine (Fig. 1), corresponding to ∼3.2 ± 2.3 ng adenosine per 105 cells per hour. Thus the amounts of adenosine released from Adk−/− ES cell–derived EBs are between those released from undifferentiated Adk−/− ES cells (2.6 ± 0.4 ng adenosine per 105 cells per hour) and those released from undifferentiated Adk−/− N3EFL glial precursor cells (11.7 ± 1.7 ng adenosine per 105 cells per hour). The data are thus in agreement with our previous observation (6) that adenosine release increases with ongoing differentiation of Adk−/− ES cells. Because the amounts of adenosine released from cultured cells do not necessarily allow the prediction of the release from encapsulated cells, encapsulated ES cell– derived EBs (n = 4 for each genotype) were cultured in pairs in 500-µL medium, and 2 h after changing the medium, a sample was collected for adenosine analysis. The concentration of adenosine found in the medium from pairs of encapsulated Adk−/− EBs was 19.4 ± 13.2 ng/ml compared with 3.1 ± 0.5 ng/ml adenosine in medium from encapsulated Adk+/+ EBs (Fig. 1). These raw data correspond to an absolute release of 4.8 ± 3.3 ng adenosine per Adk−/− capsule within 2 h and a corresponding release of 0.8 ± 0.1 ng adenosine per Adk+/+ capsule. Although we load each capsule with a specified number of cells (e.g., 3 × 105 glial precursor cells), the actual number of cells within each capsule after 1 week in culture is difficult to predict. Therefore the amounts of released adenosine are given as concentrations (ng/ml) and absolute amounts (ng) and have not been normalized to cell number. We conclude that, compared with wild-type controls, both nonencapsulated and encapsulated EBs derived from Adk−/− ES cells display a three- to sixfold enhancement of adenosine release, which may be of therapeutic value.

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Seizure suppression by adenosine-releasing stem cells To assess the potential for seizure suppression, Adk−/− ES cells were (a) differentiated into EBs and glial precursor cells, (b) encapsulated into semipermeable membranes to study the paracrine effect of adenosine isolated from effects due to cellular engraftment, and (c) subsequently tested for seizure suppression after grafting of a single capsule into the lateral brain ventricles of kindled rats (n = 5 for each cell type). In parallel, a control group of kindled rats was implanted with capsules containing wild-type Adk+/+ EBs and glial precursor cells (n = 5 for each cell type). Three days before implantation, all kindled animals reproducibly responded with a convulsive grade 5 seizure to every test stimulus (n = 20) (Fig. 2). Three, five, and seven days after implantation of a capsule, the rats were subjected to single test stimuli, and the behavioral seizure responses were scored. The experiment was terminated at day 7. In kindled animals that had received control Adk+/+ EB and glial precursor cell implants (n = 5 for each cell type), convulsive grade 5 seizure activity was not reduced as a result of grafting (Fig. 2). In contrast, 3 days after the implantation of Adk−/− EB and glial precursor cell implants, all animals (n = 5 for each cell type) displayed complete protection from any behavioral seizure activity (grade 0) (Fig. 2). Thus Adk−/− EB as well as Adk−/− glial precursor cell implants released amounts of adenosine sufficient to suppress kindled seizures. Control animals implanted with wild type Adk+/+ EB or glial precursor cell grafts continued to react with convulsive grade 5 seizures after each test stimulus (Fig. 2). Five days after implantation of the capsules, two rats grafted with Adk−/− EB implants and three animals grafted with Adk−/− glial precursor cell implants were still completely protected from seizure activity (grade 0), and the remaining three and two rats in the respective groups displayed preimplantation grade 5 seizures (Fig. 2). Seven days after grafting of Adk−/− ES cell–derived implants, all animals had returned to preimplantation grade 5 seizure activity (Fig. 2). It is important to note that the anticonvulsive action of the encapsulated adenosine releasing stem cell–derived cells led to an “allor-nothing” effect, because we observed either complete seizure suppression (grade 0) or, as in the controls, maximal grade 5 seizure activity. We never observed any intermediate seizure grades. Suppression of afterdischarge activity by adenosine-releasing stem cells The antiepileptic effect of Adk−/− ES cell–derived implants was further investigated by analyzing epileptic afterdischarges in bilateral intrahippocampal EEG measurements. During a 10-s stimulus of 50 V at 10 Hz, a stimulation artifact is observed in the EEG, which is variable from rat to rat, but is unchanged before and after implantation of the capsule within the same animal Epilepsia, Vol. 46, No. 8, 2005

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FIG. 2. Seizure suppression by Adk −/− embryonic stem (ES) cell–derived implants. Seizure activity of kindled rats was analyzed after application of a test stimulus 3 days before and 3, 5, and 7 days after implantation of encapsulated Adk −/− or Adk +/+ ES cell–derived implants. Three days before grafting encapsulated cell grafts, all rats (n = 20) displayed convulsive grade 5 seizures. Three days after implantation of encapsulated Adk −/− embryoid bodies (EBs) or glial precursor cells into the lateral brain ventricles of kindled rats (n = 5 for each cell type), behavioral seizures were completely suppressed in all rats. In contrast, in the group that was grafted with control Adk +/+ EB and glial precursor cell implants (n = 5 for each cell type, total n = 10), grade 5 seizure activity was maintained. One animal lost its pedestal after 3 days and was excluded from the experiment (n = 4 from day 5 onward for Adk −/− EB group). Five days after grafting of ES cell–derived EB and glial precursor cell implants, two Adk −/− EB-grafted animals and three Adk −/− glial precursor cell grafted animals displayed complete protection from seizure activity. Seven days after grafting, all rats displayed preimplantation seizure activity.

(Fig. 3). After the stimulation artifact, the responding epileptic discharge of hippocampal neurons is observed as high-frequency, rhythmic spikes ipsalateral and contralateral to the stimulus (3). Three days before grafting, strong electric afterdischarges that accompanied stimuluselicited grade 5 seizures were recorded in all rats used in this study (Fig. 3A–D, first panel). Control Adk+/+ EB and glial precursor cell implants had no influence on this epileptic afterdischarge activity, as shown in EEG recordings taken 5 days after implantation (Fig. 3A and C, second panel). In these animals, grade 5 seizures were consistently elicited by each test stimulus. In contrast, 3 and 5 days after the implantation of adenosine-releasing Adk−/− EB and glial precursor cell implants, seizure suppression was paralleled by a suppression of afterdischarge activity, as became evident in EEG recordings from protected rats (Fig. 3B and D, second panel). It is important to note that suppression of afterdischarge activity always correlated with seizure suppression (grade 0). Thus behavioral seizure suppression was associated with a reduction of epileptic afterdischarge activity. Seizure suppression is limited by cell viability within the capsules To assess the viability of cells within the capsules, a histologic analysis was done on capsules taken from rats at various times after implantation. Capsules loaded with ES Epilepsia, Vol. 46, No. 8, 2005

cell–derived EBs and taken from rats at 2 and 7 days after implantation were fixed and stained with hematoxylin– eosin. Capsules taken at day 2 were derived from control animals to assess the immediate impact of the implantation process on cell survival. Capsules taken at day 7 were retrieved from kindled animals after the delivery of the last test stimulus on the same day. We did not kill protected animals at day 3 or 5 for retrieval of the capsules because we wanted to know how long seizure suppression could be maintained. Viable cells were identified in capsules taken at 2 days after implantation by the positive purple stain of intact nuclei with hematoxylin and pink-stained cytoplasm with eosin (Fig. 4). Tight aggregates of cells with a rounded or triangular morphology were identified in EB-loaded capsules of both genotypes. It is interesting to note that these viable cellular aggregates attached to the capsule walls and frequently stretched throughout the diameter of the capsule with occasional void spaces in the center of the capsules (Fig. 4A). Because the hematoxylin stains only nuclei in viable cells and we have no way of correlating cell debris with a cell number, it is difficult to determine the actual percentage of viable cells within the capsules at this point. However, an estimation of 85– 90% cell viability was made by gross observation of the proportion of hematoxylin-stained nuclei to cell debris in representative sections of stained capsules taken from rats 2 days after implantation. In contrast, in capsules taken

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FIG. 3. Representative EEG recordings after application of test stimulations in kindled rats. Bilateral intrahippocampal EEGs were recorded after application of a test stimulus (end of stimulus is marked by an arrow and vertical line) in kindled rats that had received (A) a control Adk +/+ or (B) an adenosinereleasing Adk −/− embryoid body (EB) capsule, or (C) a control Adk +/+ , or (D) an adenosine-releasing Adk −/− glial precursor cell capsule. Representative traces are shown 3 days before and 5 days after implantation. Three days before grafting of an embryonic stem (ES) cell–derived implant, all animals displayed epileptic afterdischarges (A– D, first panel) associated with grade 5 seizures (boxed). Five days after grafting of control Adk +/+ ES cell–derived implants (A and C, second panel), epileptic afterdischarges remained unchanged, whereas 5 days after implantation of adenosine-releasing (B) Adk −/− EB or (D) Adk −/− glial precursor cell implants, epileptic afterdischarges were drastically reduced, and animals displayed grade 0 behavioral seizures. In each EEG, the upper trace represents the recording of the ipsilateral (i) hippocampus (kindling stimulation and capsule implantation side), whereas the lower trace represents the recording from the contralateral (c) hippocampus. Note that the artifact during stimulation is variable from rat to rat but is unchanged before and after implantation of the capsule within the same animal as previously observed (3).

FIG. 4. Hematoxylin–eosin staining of encapsulated embryonic stem (ES) cell–derived Adk −/− embryoid bodies (EBs). A: Representative capsule retrieved 2 days after implantation shows purple-stained nuclei and pink-stained cytoplasm of viable cells within the capsule. B: Representative capsule retrieved 7 days after implantation shows evidence of cells that have been loaded into the capsule, but none remains viable at this time. Scale bar, 100 µm.

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7 days after implantation, cell debris could be observed but no purple nuclei, indicating that the cells were dead at this time (Fig. 4). At day 7, not a single cell with an intact nucleus was found. By this time, cells had already detached from the capsule walls, and the formerly tight cellular aggregates were broken up. It is important to note that no differences were observed between Adk−/− and Adk+/+ capsules at day 2 or at day 7. Thus as shown earlier (3), the loss of the antiseizure efficacy of grafted Adk−/− implants over time was found to correlate with cell loss within the capsules. In contrast, EB-derived cell capsules maintained in vitro for the same period did not show any significant cell loss. Cresyl violet– stained coronal brain sections of graft recipients did not show any adverse tissue reactions surrounding the implant site (data not shown). DISCUSSION The aim of this study was to demonstrate the feasibility of a stem cell–based treatment approach for pharmacoresistant focal epilepsies. The rationale for targeting the adenosinergic system for epilepsy therapy is supported by the following recent findings: (a) a dysregulation of the adenosinergic neuromodulatory system was shown to be associated with chronic seizure activity (10,11), (b) seizure suppression was achieved by activation of adenosine A1 receptors in a mouse model of pharmacoresistant epilepsy (12), and (c) kindled seizures were suppressed by the local release of adenosine from encapsulated Adk-deficient fibroblasts (3). In the present contribution, ES cell–derived Adk−/− EBs and Adk−/− glial precursor cells were encapsulated in semipermeable polymer membranes and grafted into the lateral brain ventricles of kindled rats. This approach allowed the evaluation of the paracrine effect of adenosine itself on seizure suppression, independent of effects caused by network integration or by released trophic factors from host cells in response to direct grafting into the tissue. Additionally, this study addresses the capability of ES cell–derived adenosine-releasing implants in a model that is known to be treatable by local adenosine release (3). Both Adk−/− EBs and Adk−/− glial precursor cells suppressed seizure activity in this model, whereas the respective control Adk+/+ implants did not. Seizure suppression was confirmed by a reduction of behavioral seizure activity (Fig. 2), which was reproducibly accompanied by a reduction of epileptiform afterdischarges (Fig. 3B and D, second panel). These results suggest that Adk−/− ES cell–derived implants release sufficient amounts of adenosine to suppress seizure activity, and the in vitro analysis of adenosine release from cultured and encapsulated EBs confirms that enhanced amounts of adenosine are released as a consequence of the ADK knockout (Fig. 1). The amounts of adenosine released from cultured Adk−/− Epilepsia, Vol. 46, No. 8, 2005

EBs (9.0 ± 5.5 ng adenosine per 105 cells per hour) is essentially equivalent to the amounts of adenosine released from cultured ADA-O cells (8.4 ± 2.6 ng adenosine per 105 cells per hour), a cell line that previously suppressed kindled seizures in rats (3). To estimate the amount of adenosine released from each cell capsule, we determined adenosine concentrations in supernatants from cultured cell capsules. The absolute release of 4.8 ± 3.3 ng adenosine per Adk−/− capsule within 2 h also is comparable to the adenosine release from individual ADA-O capsules (3 to 7 ng in 24 h), which suppressed kindled seizures in rats (3). Taken together, these results suggest that Adk−/− EBs and N3EFL cells release amounts of adenosine considered to be sufficient for the seizure suppression observed in our present experiments. Because ADK is involved in a salvage pathway for nucleotide synthesis and is not essential for the de novo synthesis of purines, it is unlikely that other anticonvulsive molecules apart from adenosine may accumulate in and be released from ADK-deficient cells. Indeed, in previous pharmacologic control experiments using the A1 receptor– selective antagonist, DPCPX, it was demonstrated that the anticonvulsive effect of encapsulated Adk−/− cells is mediated by adenosine (3,13). Because of the short duration of seizure suppression in the present study, a comparable control experiment was not possible. Although seizure suppression from Adk−/− ES cellderived implants was apparent, it lasted only for ≤5 days. This was most likely due to a lack of cell survival in the capsules. The histologic analysis of capsules grafted into the lateral brain ventricle of naive rats revealed viable cells after 2 days in vivo, whereas after 7 days in vivo, almost no viable cells could be detected (Fig. 4). To date, data for encapsulation of ES cells and their progeny are lacking, and reasons for the cell death within the capsule are only speculative. Because the encapsulated ES cell progeny were cultured in vitro before grafting, they were thus adapted to an enriched environment with nutrients and growth factors. Thereafter, it is possible that the ventricular environment could not support the cells. This notion is supported by the fact that encapsulated ES-derived cells survived, when kept for a corresponding time in vitro under optimized tissue-culture conditions. In addition, because the cells were maintained in a proliferative state, possibly an overgrowth of cells within the limited space of the capsule added stress to the cells by decreasing the availability of the cell surface to supporting factors in the environment. Because of the lack of adverse tissue reactions surrounding the implant site, host-mediated effects leading to the death of the encapsulated cells are unlikely. In a recent study, seizure suppression by encapsulated adenosine-releasing myoblasts was maintained for ≤8 weeks, a time span during which adenosine A1 receptors remained responsive to pharmacologic inhibition or activation (13). Because of these findings, a downregulation of the A1 receptor

ADENOSINE STEM CELL THERAPY appears to be an unlikely explanation for the transient seizure-suppressive effect of the grafts described here. Direct stem cell–derived grafts might provide a potential strategy for long-term seizure suppression in chronic epilepsy. To assess the therapeutic value of direct grafting of adenosine-releasing stem cell derivatives, epilepsy is considered an ideal model because the integration of Adk−/− cells might directly counteract the overexpression of ADK, which has been described in epileptic hippocampus of kainic acid–treated mice (10), and thus restore the equilibrium of adenosine in an epileptic focus. A recent study provided evidence that ES cell–derived glia and neurons can functionally integrate on the single-cell level into host brain, as demonstrated after injection into the cerebral ventricle of embryonic rats (14,15). Thus it is possible that a direct implantation of adenosine-releasing cells into an epileptic focus may lead to functional three-dimensional integration within a critical region, which could potentially support long-term survival of the grafts by locally released factors. The feasibility of the strategy to graft therapeutic cells directly near an epileptic focus was indicated by recent studies in which a higher seizure threshold or delayed development of kindled seizures was confirmed after the grafting of therapeutic cells (16,17). Along these lines, grafts of γ -aminobutyric acid (GABA)-producing immortalized mouse cortical neurons implanted bilaterally into the anterior portion of the substantia nigra were found to suppress the frequency of spontaneous seizures in epileptic rats (18). Furthermore, ES cell–derived neural precursors have been shown to survive, differentiate, and display functional activity after implantation into the rat pilocarpine model of epilepsy (19). In conclusion, we demonstrated that adenosinereleasing stem cell–derived brain implants are able to suppress seizure activity in kindled rats by a paracrine mode of action. The local delivery of adenosine to an epileptic focus by Adk−/− ES cell–derived implants therefore emerges as a promising strategy for the long-term control of seizure activity in chronic epilepsy. Acknowledgment: We thank Rachel Buschwald and Michaela Segschneider for their outstanding technical support. This work was supported by the Swiss National Science Foundation grants 3100A0–100841 (to D.B.) and 510540 (PNR 46; to Patrick Aebischer and W.F.P.) and by the National Center of Competence in Research (NCCR) on Neural Plasticity and Repair (to D.B.), the Hertie Foundation (to O.B.), and the

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Deutsche Forschungsgemeinschaft (TR-SFB 3; to O.B. and P.K.).

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Epilepsia, Vol. 46, No. 8, 2005