THE JOURNAL OF COMPARATIVE NEUROLOGY 349148-164 (1994)

Implants of Polymer-Encapsulated Human NGF-Secreting Cells in the Nonhuman

Primate: Rescue and Sprouting of Degenerating Cholinergic

Basal Forebrain Neurons

DWAINE F. EMERICH, SHELLEY R. WINN, JAMES HARPER, JOSEPH P. HAMMANG, E. EDWARD BAETGE, AND JEFFREY H. KORDOWER

CytoTherapeutics, Inc., Providence, Rhode Island 02906 (D.F.E., S.R.W., J.P.H., E.E.B.); Division of Biologv and Medicine, Brown University, Providence, Rhode Island 02906 (J.H.);

and Department of Neurological Sciences and Rush Alzheimer’s Disease Center, Rush Presbyterian Medical Center, Chicago, Illinois 60612 (J.H.K.)

ABSTRACT Baby hamster kidney (BHK) cells were genetically modified to secrete high levels of human

nerve growth factor (BHK-hNGF). Following polymer encapsulation, these cells were im- planted into the lateral ventricle of four cynomolgus monkeys immediately following a unilateral transectioniaspiration of the fornix. Three control monkeys received identical implants, with the exception that the BHK cells were not genetically modified to secrete hNGF and thus differed only by the hNGF construct. One monkey received a fornix transection only. All monkeys displayed complete transections of the fornix as revealed by a comprehensive loss of acetylcholinesterase-containing fibers within the hippocampus ipsilateral to the lesion. Control monkeys that were either unimplanted or received BHK-control (non-NGF secreting) cell implants did not differ from each other and displayed extensive losses of choline acetyltransferase and p75 NGF receptor (NGFrI-immunoreactive neurons within the medial septum (MS; 53 and 5410, respectively) and vertical limb of the diagonal band (VLDB; 21 and 30%, respectively) ipsilateral to the lesion. In contrast, monkeys receiving implants of BHK-hNGF cells exhibited a only a modest loss of cholinergic neurons within the septum (19 and 2010, respectively) and VLDB (7%). Furthermore, only implants of hNGF-secreting cells induced a dense sprouting of cholinergic fibers within the septum, which ramified against the ependymal lining of the ventricle adjacent to the transplant site. Examination of the capsules retreived from monkeys just prior to their death revealed an abundance of cells that produced detectable levels of hNGF in a sufficient concentration to differentiate PCl2A cells in culture. These findings support the use of polymer-encapsulated cell therapy as a potential treatment for neurodegenerative diseases such as Alzheimer disease where basal forebrain degeneration is a consistent pathological feature. Moreover, this encapsulated xenogeneic system may provide therapeutically effective levels of a number of neurotrophic factors, alone or in combination, to select populations of neurons within the central nervous system. c 1994 Wiley-Liss, Inc.

Key words: encapsulation, NGF, cholinergic neurons

Basal forebrain cholinergic neurons degenerate in several human dementing illnesses, including Alzheimer disease (AD; Whitehouse et al., 1982; Arendt et al., 1983; Candy et al., 1983; Allen et al., 1988). The loss of cortical cholinergic neurotransmission resulting from the degeneration of these neurons could play an integral role in the cognitive decline observed in AD (Bartus et al., 1982; Coyle et al., 1983). At present, treatments are ineffective for slowing or prevent-

ing the loss of cholinergic neurons or the associated memory deficits. Converging lines of evidence indicate that nerve growth factor (NGF) has potent target-derived trophic and tropic effects upon cholinergic basal forebrain neurons. The

Accepted May 27, 1994. Address reprint requests to Dr. Dwaine F. Emerich, Ph.D., Cyto Therapeu-

tics Inc., Providence, RI, 02906.

o 1994 WILEY-LISS, JNC.

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 149

highest levels of NGF protein and mRNA are found in the target regions of basal forebrain cholinergic neurons (Kor- sching et al., 1985; Sheldon and Reichardt, 1986). Radiola- beled NGF is taken up by cholinergic terminals and specifi- cally transported in a retrograde fashion to cholinergic basal forebrain perikarya (Schwab et al., 1979; Seiler and Schwab, 1984), further indicating that these neurons are a primary site of NGF action in the central nervous system (CNS). Target-derived NGF binds to both low- (p75) and high- (trh A) affinity NGF receptors, which in the adult rat, nonhuman primate, and human brain are found within cholinergic neurons of the basal forebrain (Hefti et al., 1986; Kordower et al., 1988, 1989b; Batchelor et al., 1989; Koh et al., 1989; Mufson et al., 1989a; Steininger et a]., 1993, Kordower et al., in press). Chronic intraventricular administration of NGF stimulates the expression of choline acetyltransferase (ChAT) and NGF receptor mRNAs. Addi- tionally, NGF administration in rodents and nonhuman primates prevents the loss of basal forebrain cholinergic neurons following axotomy (Hefti, 1986; Williams et al., 1986; Fischer et al., 1987; Kromer, 1987; Montero and Hefti, 1988; Tuszynski et al., 1990; 1991; Koliatsos et al., 1991a,b). The effects of NGF appear to be physiological because both the memory deficits and basal forebrain atrophy displayed by a subpopulation of aged rats can be reversed by intraventricular NGF infusions (Fischer et al., 1987). Together, these data indicate that the use of NGF may represent a useful treatment strategy for AD and/or other diseases characterized by basal forebrain-mediated cholinergic deficits. Accordingly, clinical trials employing intraventricular mouse NGF for the treatment of AD have been initiated (Olson et al., 1992).

Prior to wide-scale human clinical trials with NGF, several criteria should be met, including a consistent, reliable, and well-characterized source of human NGF (hNGF; Phelps et al., 1989). The hNGF should be biologi- cally active and affect basal forebrain cholinergic neurons in experimental animals including nonhuman primates (Tuszynski et al., 1990, 1991; Koliatsos et al., 1991a,b). Transplantation of cells that have been genetically modified to secrete high levels of hNGF is one means of meeting these criteria (Gage et al., 1987; Rosenberg et al., 1988; Breakefield, 1989; Ernfors et al., 1989; Stromberg et al., 1990; Kawaja et al., 1992; Maysinger et al., 1992; Hoffman et al., 1993; Winn et al., 1994). Recently, we modified baby hamster kidney (BHK) fibroblasts to produce high and stable levels of hNGF (Winn et al., 1994). These BHK cells had been encapsulated within semipermeable polymer mem- branes and had been shown to survive implantation for extended periods of time while continuing to release biologi- cally relevant levels of hNGF (Winn et al., 1994). Together with the inherent advantages of using a genetically engi- neered cell line, polymer encapsulation permits molecules of interest to diffuse from the capsule into the surrounding host tissue (for review, see Emerich et al., 1992). Polymer encapsulation also prevents elements of the immune sys- tem from entering the capsule, thereby protecting the encapsulated cells from immune destruction. This tech- nique prevents the inherent risk of tumor formation and allows the use of unmatched human or even animal cells, without immunosuppressing the recipient. Additionally, site-specific delivery of protein factors from encapsulated engineered cells may help overcome some of the difficulties in drug delivery associated with the blood-brain barrier. The utilization of these types of polymeric devices also

enables retrieval of the encapsulated cells if necessary or desired. Thus, encapsulating neurotrophic factor-secreting cells prior to implantation might provide a safe and practi- cal means of chronically delivering compounds to discrete CNS targets as treatments for a variety of progressive neurodegenerative disorders.

We recently demonstrated that implants of encapsulated hNGF-secreting BHK cells prevent the loss of cholinergic neurons in rodents following fimbria/fornix transections (Winn et al., 1994) in a manner similar to that seen following intraventricular infusions of NGF (Hefti, 1986; Kromer, 1987; Montero and Hefti, 1988; Koliatsos et al., 1991a). To extend these findings to the primate order, the present study determined whether implants of polymer- encapsulated hNGF-secreting cells prevent the degenera- tion of primate cholinergic basal forebrain neurons, which normally occurs following axotomy.

MATERIALS AND METHODS BHK-hNGF cell line production

Two human genomic clones (php N8D8, php N8B9) coding for the 5' and 3' ends of the p-hNGF gene were obtained from the American Type Culture Collection. A 440-bp 5' Scal-EcoR1 fragment from php N8D8 was ligated to a 3' 2.0-kb EcoRl fragment isolated from php N8B9. The hNGF genomic sequence contained 37 bp of the 3' end of the first intron, the double ATG sequence believed to be the protein translation start for pre-pro-hNGF and the complete coding sequence and entire 3' untranslated region of the human gene. The combined 2.51-kb P-hNGF fragment was subcloned into the dihydrofolate reductase- based pNUT expression vector immediately downstream from the mouse metallothionein-I promoter (-650 to +7; Hoyle et al., 1993) and the first intron of the rat insulin I1 gene. The pNUT-B-hNGF construct was introduced into BHK cells by using a standard calcium-phosphate-mediated transfection method (Baetge et al., 1986). Further details concerning the hNGF construct and the transfection method can be found in Winn et al. (1994). BHK cells were grown in DMEM, 10% fetal bovine serum, antibiotic/antimycotic, and L-glutamine (Gibco) in 5% COP and at 37°C. Trans- fected BHK cells were selected in medium containing 200 pM methotrexate (Sigma) for 3-4 weeks, and resistant cells were maintained as a polyclonal population either with or without 200 KM methotrexate. BHK cells treated identi- cally as above except for the insertion of the hNGF con- struct served as controls in these experiments.

Encapsulation procedure Asymmetric hollow fibers of poly (acrylonitrile-co-vinyl

chloride) copolymer (PAN-PVC) were fabricated by a dry- wet (jet) spinning technique according to Cabasso (1980). Encapsulation devices were manufactured by mounting approximately a 1-cm length of dry hollow fiber onto hubs with a septal fixture at the proximal end that has loading access for cells to be injected into the lumen of the device. BHK cells -C hNGF were removed from the culture flasks by using Ca++ and Mg++-free HBSS and trypsinlEDTA and prepared as a single-cell suspension. The BHK cell suspen- sions at a density of 2 x lo7 cells/ml were mixed with Vitrogen (Celtrix, Palo Alto CA) and loaded into the encap- sulation devices as described elsewhere (Winn et al., 1994). After infusion of the cellular suspensions, the hub was sealed and the cell-loaded devices were maintained in

150

serum-free medium 2-3 days prior to hNGF analysis by enzyme-linked immunosorbent assay (ELISA). For hNGF analysis by ELISA, the cell-loaded capsules were rinsed in HBSS, placed in 1 ml of fresh PC-1 medium, and the medium was collected after 20 hours’ assay time. The capsules were subsequently “tethered” by placing a silastic tube over the acrylic hub. The cell-loaded devices were transferred into sterile polypropylene tubes containing serum-free medium and were maintained in an incubator at 5% COZ and 37°C for 1-2 days prior to implantation.

Surgical procedures Young adult cynomolgus (Mucucu fusciculuris) monkeys

of both sexes (N = 8; 4-6 kg) were used in this study. Animals were sedated with ketamine HCL (10 mg/kg, i.m.1 and intravenous lines were secured for fluid administra- tion. The animals were intubated by the orotracheal method, and anesthesia was maintained throughout the procedure with isoflurane (1.5-2.0%)+ Animals were placed on a heating pad to maintain body temperature, and electrocar- diogram leads were placed to monitor heart rate and rhythm. To facilitate relaxation of the brain and minimize trauma due to retraction pressure, mannitol(O.250 mg/kg, i.v.) was administered immediately prior to the craniotomy. Unilateral transections of the left fornix were performed by using an open microsurgical approach developed by Kor- dower and Fiandaca (1990). After securing the animals in a Kopf stereotaxic headframe, a midline incision was made in the scalp and the skin retracted laterally. The medial attachment of the temporalis muscle was mobilized and a surgical drill used to create a parasagittal bone flap (size = 1.5 cm x 4.0 cm) that exposed the frontal superior sagittal sinus. The dura was retracted and a self-retaining retractor used to expose the interhemispheric fissure. The parasagit- tal bridging veins were coagulated where needed to facili- tate retraction of the cerebral hemisphere. With the aid of a surgical microscope, arachnoid adhesions were divided. When necessary, veins overlying the corpus callosum were coagulated. The corpus callosum was incised longitudinally, exposing medial subcortical structures from the septum and head of the caudate rostrally through the foramen of Monro caudally. At the caudal level of the foramen of Monro, the fornix is easily visualized as a discrete 2-3-mm wide white fiber bundle. The fornix was initially transected by using a ball dissector; then the cut ends of the fornix were suctioned to ensure completeness of the lesion (Fig. 1).

Following the transection of the fornix, individual BHK cell-containing capsules were manually placed within the lateral ventricle with fine forceps between the head of the caudate and the septal nucleus (Fig. 1). A total of five capsules were implanted in each animal and oriented in a row in the rostrocaudal direction. The capsules abutted the caudate and septum, remained upright, and did not require further securing. Four animals received BHK-hNGF cap- sules, three received BHK-control cell-loaded capsules, and one monkey received a fornix transection but no transplant. With hemostasis achieved, the dura was reapproximated, the bone flap was sutured back in place, and the galea and skin were sutured by using routine methods. All animals received antibiotics (Cefotaxime, 50 mgikg, i.m.) preopera- tively and for 4 days postoperatively.

Preparation of tissues Twenty-three or 28 days following surgery, animals were

anesthetized as described above. Equal numbers of BHK-

D.F EMERICH ET AL.

Fig. 1. Drawing of dorsal aspect of primate brain illustrates transec- tion of the fornix and placement of polymer capsules containing baby hamster kidney (BHK) cells. Following a craniotomy, the cerebral hemisphere is retracted by using a modified spatula, and the fornix is unilaterally transected by using a microdissector. The polymer capsules are then placed within the lateral ventricle located between the caudate (left) and septal nucleus (right) by using microforceps. The upper portion of the capsules consists of a silastic tether and is clipped to permit closure of the dura.

hNGF and BHK-control animals were killed at each time point, and no differences in any result were obtained based upon the two postgrafting time points. Two to three milliliters of cerebrospinal fluid (CSF) was obtained from either the lumbar region (N = 1 BHK-control, 2 BHK- hNGF) or cisterna magna (N = 2 BHK-control, 2 BHK- hNGF) to assay hNGF levels. Animals were then placed into the stereotaxic frame, the previously prepared bone flap was removed, the cerebral hemisphere retracted, and the BHK cell-loaded capsules removed. Immediately follow- ing removal of the capsules, animals were transcardially perfused by using a peristaltic pump with 1 liter of phos- phate-buffered saline (PBS; pH 7.4) containing 1 ml of Heparin followed by fixation with 3.5 liters of 4% parafor- maldehyde. The brains were blocked in the transverse plane following fixation, stored in 25% buffered sucrose (pH 7.4), and refrigerated for 5-7 days. Frozen sections were cut (30 Fm) on a sliding knife microtome and stored in a cryopro- tectant solution.

Immunohistochemistry. Every seventh section through the septalidiagonal band complex was processed immunocy- tochemically for ChAT and the low-affinity p75 NGFr. Immunocytochemical labeling was conducted according to modifications of previously published protocols. Briefly, the immunohistochemical procedure was conducted as follows: (1) overnight incubation in PBS containing 0.4% Triton + 2% normal serum, (2) 48-hour incubation in the primary ChAT polyclonal antibody (Chemicon; 1: 10,000) or NGFr

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 151

monoclonal antibody (generously provided by Dr. Mark Bothwell; 1:20,000), (3) overnight rinse in PBS + 0.2% Triton, (4) 6 x 5-minute rinses in PBS followed by 1.5 hours’ incubation in the appropriate biotinylated secondary IgG antibody (Vector; 1:100), (5 ) 6 x 5-minute rinses in PBS + Triton, (6) incubation with “Elite” avidin-biotin complex (Vector, 1:1,000) for 1.5 hours, (7) 3 x 10-minute rinses in PBS, and (8) incubation in the chromagen solution containing 0.05% 3,3’ diaminobenzidine and 2.5% nickel ammonium sulfate dissolved in 0.1% Tris buffer for 5 minutes, followed by hydrogen peroxide (0.01%) for 5 minutes. The reaction was terminated by 3 x 1-minute rinses in PBS. Sections were mounted, dehydrated in alcohol, and coverslipped. Control sections were processed in an identical manner except the primary antibody solvent or an irrelevant IgG was substituted for the primary antibody. Even though staining was eliminated in sections in which the primary antibody was deleted or an irrelevant IgG substituted, the potential for antiserum to react with structurally related proteins cannot be excluded. Thus, a degree of caution that is inherent to immunohistochemical procedures is warranted. In this regard, in this study the terms ChAT- or p75 NGF receptor-immunoreactivity refers to “like” immunoreactivity. Adjacent sections were stained with hematoxylin and eosin (H&E) to aid in cytoarchitech- tonic delineation.

To verify the completeness of the lesion, sections through the hippocampus were processed histochemically for the visualization of acetylcholinesterase (AChE; Hedreen et al., 1985). Sections were incubated for 1 hour in a solution (pH 5.0) containing 100 mM sodium acetate (65 ml), 50 mg acetythiocholine iodide, 100 mM sodium citrate (5 ml), 30 mM copper sulfate (10 ml), 15 ml distilled H20, 5 mM potassium ferricyanide (4 ml), and 0.001M tetraisopropylpyrophoramide. After 3 x 10-minute washes in sodium acetate buffer, the sections were incu- bated for 1 minute in 4% ammonium sulfide. After 5 x 10-minute washes in sodium nitrite, the sections were incubated for 1 minute in a 0.05% silver nitrate solution. After 5 x 10-minute washes in sodium nitrate, sections were mounted, dehydrated, and coverslipped as before. For control, sections were processed in an identical manner except that acetylthiocholine iodide was omitted from the incubation medium.

For quantification of cholinergic cell loss, the number of ChAT and p75 NGFr-positive neurons were manually counted within the MS and VLDB at a total magnification of l ox . Neurons on the midline were ex- cluded from this analysis. Representative sections (4/brain/ histological stain) located approximately 200 pm apart from each other were used for this analysis. For statistical analysis, the numbers of neurons ipsilateral to the lesion were expressed as a percentage of neurons ipsilateral to the lesionitransplant relative to the intact contralateral side. A repeated-measures analysis of variance (ANOVA), with the experimental manipulation as the main factor and the level of section as the repeated measure, was used to determine differences between the BHK-control and BHK-hNGF groups. Acceptable statistical significance was established at P < 0.05.

Acetylcholinesterase.

Quantitation.

Activity of NGF Biological activity. Conditioned media (CM) from en-

capsulated BHK-control and BHK-hNGF cells were passed through a 0.2-pn filter and added to PCl2A cells grown on

standard tissue culture (6- or 24-well plates at a concentra- tion of 50-100,000 cellsiml) to test for the presence of hNGF in the CM. All medium conditioning and neurite outgrowth assays were performed in 5% COz and at 37°C. As a positive control, 2.55 mouse NGF was added to some of the wells to induce neuritic extensions (50 ng/ml). The PCl2A cells were scored for neurite processes that were 2 3 times the length of the cell body diameter after a period of 1-4 days. In addition, the rate of neurite induction and the stability of the neurites were observed, and a comparison was made between the culture conditions.

Quantitation of hNGF released from en- capsulated BHK-hNGF cells was performed as follows, and all of the reagents were obtained from Boehringer- Mannheim Biochemicals unless otherwise noted. Nunc- Immuno Maxisorp ELISA plates were coated with 150 pl/well of anti-mouse-p (2.5s) NGF at 1 ngiml in coating buffer (1 x PBS without CaClz or MgC12/0.1% sodium azide; pH 9.6). The coated plates were incubated at 37°C for at least 2 hours or, alternatively, at 4°C overnight. The coating solution was withdrawn from the wells, and the wells were washed with 3 x 300-pl of wash buffer (50 mM Tris-HC1/200 mM NaC12/ 1% Triton X-100/0.1% sodium azide; pH 7.0). The wells were then blocked with 300 p1 of coating solution containing 10 mglml of BSA at room temperature for 30 minutes, followed by further washes (3 x 300-p1 wash buffer). CM samples were diluted 1:l twice in sample buffer (the sample buffer is the same as wash buffer, only with 2% BSA), with 10 yl of the prepared samples loaded into the wells. The plates were covered and incu- bated for at least 2 hours at 37°C or overnight at 4°C. The solutions were removed from the wells by suction and washed 3 x 300 pl of wash buffer. To each well, 100 ~1 of 4 Uiml of anti-mouse+ (2.5s) NGF-P-galactosidase conju- gate was added. The plates were incubated at 37°C for at least 1 hour. The solutions were removed from the wells by suction and washed 3 x 300 p1 of wash buffer. Finally, 200 p1 of chlorophenol red-j3-D-galactopyranoside substrate so- lution (40 mg CPRG in 100 mM Hepesil50 mM NaC1/2 mM MgClZ/0.1% sodium azide/l% BSA; pH 7.0) were added to the wells, incubated at 37°C for 30 minutes to 1 hour or after the color development was sufficient for photometric determination at 570 nm; the samples were analyzed on a plate reader and measured against recombinant NGF pro- tein standards.

NGF ELISA.

RESULTS Animals rapidly recovered from surgery and survived the

duration of the experiment. Animals receiving BHK-hNGF- containing capsules were lethargic and exhibited decreased appetite for several days following surgery. One of these animals also exhibited multiple seizures beginning approxi- mately 2 days following surgery and dissipating within 5 days following surgery. No such complications were noted in any of the animals receiving BHK-control cell implants, and no differences were observed between the BHK-hNGF and BHK-control animals after the first postsurgical week.

The polymer capsules were left in situ from one animal receiving BHK-control implants (Fig. 3). In all other mon- keys, the BHK cell-loaded devices were retrieved from the lateral ventricles 23-28 days following implantation with little to no host tissue adhering to the capsules. The level of hNGF produced by the capsules within prior to implanta- tion was 21.4 5 2.0 ng/capsule/24 hours and 8.5 f 1.2

152 D.F EMERICH ET AL.

Fig. 2. PCLBA neurite outgrowth bioassay for human nerve growth factor (hNGF). Phase-contrast photomicrographs of PC12Acells treated for 3 days with conditioned medium from BHK-control (A) or BHK- hNGF (B) cell-loaded devices that have been retrieved from grafted

monkeys 1 month following transplantation. Virtually all of the PC12A cells exhibit extensive neurite processes, whereas no processes are evident in the cells treated with RHK-control conditioned medium. Scale bar = 25km.

nglcapsule/24 hours in the retrieved capsules as measured by the NGF ELISA. Because each monkey was grafted with five capsules, the total amount of NGF produced per animal was 107 ngi24 hours and 41.5 ngl24 hours prior to grafting and 1 month following grafting, respectively. The BHK- control capsules produced no detectable hNGF (assay sensi- tive to 25 pg NGF/ml). Conditioned medium from BHK- hNGF cells induced a marked outgrowth of neurite processes from PClZA cells (Fig. 2). Addition of 50 ng/ml of pure mouse PNGF to identically plated cells elicited neurite outgrowth that was indistinguishable from hNGF in CM in terms of the speed of neuritogenesis and the stability of the neurites over a 4-day exposure period. Neurite outgrowth induced by the BHK-hNGF CM was blocked when a monoclonal antibody to NGF (Boehringer-Mannheim) was added to the cultures (data not shown).

Based upon visual inspection at the time of capsule removal, all capsules were located within the lateral ven- tricle and abutted both the head of the caudate and the lateral septum. All capsules were removed intact, and there was no evidence that any capsule broke either in situ or during the retrieval procedure. The cell-loaded devices were left in situ in 1 BHK-control animal to demonstrate place- ment of the devices and assess the host tissue response (Fig. 3A,B). The host response to the capsules in this animal and all others was minimal. There was a relative paucity of reactive glia, which, if observed at all, was only seen at circumscribed locations at the graft-host interface (Fig. 3B). Furthermore, other non-neuronal cells such as macro- phages were not observed within the perigraft region. Directly adjacent to the implant, a proliferation of small to moderate sized blood vessels was occasionally observed.

Fig. 3. Low- (A) and (B) medium-power photomicrographs through the septum of hemotoxylin and eosin (FEE)-stained sections illustrate the placement of two polymer capsules within the lateral ventricle. The capsules were originally oriented vertically. During histological process- ing, they became displaced, and the cells contained within them were lost. (Bi Arrows point to the limited infiltration of non-neuronal cells in the perigraft region. (C, D) Photomicrographs from glycol methacrylate-

embedded sections from capsules retrieved prior to perfusion. (C) Numerous H&E-stained cells were found within the full length of the capsule 1 month following transplantation. (D) These cells displayed a heaithy morphology, and numerous mitotic figures were observed. Cd, caudate nucleus; LV, lateral ventricle; S, septum. Scale bar = 500 km for A, 100 Fm for B, 150 km for C, 50 km for D.

154 D.F EMERICH ET AL.

This appeared to be unassociated with the number of viable cells within the implanted capsules. The number and caliber of vessels within the perigraft region appeared normal within just a few microns of the implant.

The morphology of BHK-hNGF cells within a retrieved capsule is shown in Figure 3C. Few adhering host cells were found on the capsule wall, and a large number of viable BHK cells, evenly distributed at high density, were present within the polymeric device. Numerous mitotic figures were observed throughout all of the cell-loaded capsules (Fig. 3D). Morphologic analysis of H&E-stained acrylate sections revealed that the viability of encapsulated cells was equiva- lent between the BHK-control and BHK-hNGF cell-loaded capsules.

AChE-stained sections revealed a dense pattern of AChE- containing neurons and fibers in many brain regions includ- ing the basal forebrain, striatum, cerebral cortex, and hippocampus in a pattern similar to that described for the localization of AChE-containing elements in the monkey brain (e.g., 46). Positive staining was completely eliminated in sections incubated without the acetycholine thioiodide substrate. In all animals, histological examination revealed that the left fornix was completely transected at a compa- rable neuroanatomical level, whereas the contralateral fornix remained intact. This lesion resulted in the compre- hensive loss ofAChE-containing fibers within the hippocam- pus ipsilateral to the lesion (Fig. 4B,D) relative to the contralateral side (Fig. 4A,C). All hippocampal subfields displayed an almost complete loss of AChE-containing fibers ipsilateral to the lesion with just a few scattered AChE-containingfibers seen coursing within the hippocam- pus for short distances (Fig. 4D). The one exception to this was the rim of AChE-containing fibers, which was observed within the inner third of the molecular layer of the dentate gyrus. This was seen in all animals.

NGFr- and ChAT-immunoreactive stained sections re- vealed a general pattern of labeled perikarya and fibers within the monkey forebrain consistent with previous observations (e.g., Kordower et al., 1988, 1989a). No differ- ences in the extent of the fornix lesion or the loss of cholinergic neurons were observed between the animal that received no transplant and those receiving BHK-control cells. Accordingly, the data from these groups were com- bined. In these animals, a significant reduction was ob- served in the number of cholinergic neurons ipsilateral to the lesion. NGFr-positive neurons were decreased 54% (Figs. 5, 6) and ChAT-immunoreactive neurons were de- creased 53% (Figs. 5, 7) within the MS ipsilateral to the lesioniimplant relative to the contralateral side. Control implanted monkeys displayed a 30% reduction of p75 NGFr-immunoreactive neurons and 2 1% loss of ChAT- immunoreactive neurons within the VLDB (Figs. 8, 9). In contrast, monkeys receiving implants of the BHK-hNGF cells displayed only a modest loss of p75 NGFr- (19%) and ChAT-imunoreactive (20%) neurons within the MS ipsilat- era1 to the implant/lesion relative to the contralateral side (Figs. 5-7). Similarly, only a 7% loss of p75 NGFr- and ChAT-immunoreactive neurons was observed within the VLDB in monkeys receiving implants of BHK-hNGF (Figs. 8, 9). Qualitatively, it appeared that cholinergic neurons within the MS of hNGF-treated animals were larger (Fig. 101, more intensely labeled, and elaborated more extensive proximal dendrites than those displayed by BHK-control

animals (Fig. 6). This was especially true for NGFr- immunostained sections.

In addition to effects on cell viability, BHK-hNGF im- plants induced a robust sprouting of cholinergic fibers proximal to the implant. All monkeys receiving BHK-hNGF implants displayed dense collections of p75 NGFr-immuno- reactive fibers (Fig. 11A,C) throughout the entire dorsal- ventral extent of the lateral septum. This effect was unilat- eral as the septum contralateral to the implant displayed only a few cholinergic fibers (Fig. 11A) in a manner similar to that seen in control-implanted monkeys. The cholinergic nature of this sprouting effect was confirmed by an identical pattern of fibers that were both ChAT immunoreactive (Fig. 11D) and AChE positive (data not shown). These fibers ramified against the ependymal lining of the ventricle adjacent to the transplant site and were particularly promi- nent within the dorsolateral quadrant of the septum corre- sponding to the normal course of the fornix. Many of these fibers were highly varicose, of thin CNS-type caliber, and easily distinguishable from the the course degenerating ChAT- and NGFr-immunoreactivity, which is usually ob- served within the dorsolateral quadrant of the septum following a fornix transection. Adjacent to the ventricle, the fibers coalesced into a dense terminal-like pattern (Fig. 1 lC,D). Interestingly, this fiber plexus could not be directly traced to its cells of origin. These fibers did not appear to originate in the septum because processes emanating from cholinergic neurons in the septal complex could not be traced to this region of dense cholinergic sprouting. This effect is unrelated to any potential surgical damage to the septal region or any other nonspecific factors because all monkeys receiving identical BHK-control implants lacking only the hNGF gene failed to display a sprouting of cholinergic fibers (Fig. 11B). Despite the trophic and tropic effects of BHK-hNGF implants upon primate cholinergic basal forebrain neurons, hNGF was not detected within CSF taken from either lumbar and cisterna magna taps.

DISCUSSION The present studies indicate that hNGF released from

polymer-encapsulated BHK cells has marked effects on axotomized basal forebrain cholinergic neurons in nonhu- man primates. Intraventricular implants of BHK-hNGF cells significantly attenuated the loss of cholinergic neurons in the MS and VLDB and produced a robust sprouting of cholinergic fibers within the lateral septum adjacent to the implant. Monkeys receiving encapsulated hNGF-secreting implants sustained only a 20% loss of septal neurons, which was significantly less than the 54% loss displayed by monkeys receiving control implants. This represents a 63% savings of cholinergic neurons in monkeys receiving the BHK-hNGF implants. The inability to maintain 100% of ChAT- and NGFr-immunoreactive neurons following hNGF treatment in the present study is similar to other studies. Tuszynski et al. (1991) reported that infusions through osmotic pumps of 180 p,g of either mouse or hNGF over a 4-week period resulted in sparing of cholinergic neurons, similar to that observed in the present study. In contrast, Koliatsos et al. (1991b) reported that infusions of high levels of hNGF (625 kg of mouse or hNGF through a ventricular cannula every 2 days for 2 weeks; 5 mg hNGF total) completely prevented the loss of degenerating cholin- ergic neurons following fornix transections in primates. In the present study, each monkey received approximately 105

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1 - 2 3 4 - 5 6 7 8 FFX Control hNGF Only Capsules Capsules

Fig. 5. Quantification of NGFr- and choline acetylesterase (ChATb immunoreactive neurons within the medial septum following fornix transections. Monkeys underwent unilateral fornix lesions, followed by no implant (FFX) or implantation of either encapsulated BHK-control or BHK-hNGF-secreting cells (N = 4/group). This figure shows the

percent (1-8) of remaining cholinergic neurons ipsilateral to the lesion for each individual monkey. The loss of cholinergic neurons ipsilateral to the fornix transection was significantly attenuated (P < 0.05) by implantation of BHK-hNGF cells. See text for details.

ng/day of hNGF at the time of implantation and 41.5 ngiday a t the time of death 23-28 days later. Thus, it is conceivable that higher levels of hNGF would have pro- duced a greater sparing of cholinergic basal forebrain neurons. It is interesting that hNGF was not detectable within cisterna magna or lumbar CSF. This is in contrast to Tuszynski et al. (1991) who were able to detect hNGF within CSF following infusions of 180 kg over a 4-week period. It is important to note, however, that hNGF had potent effects upon the viability of primate cholinergic basal forebrain neurons and ability of NGF-responsive cells to sprout new processes. These data suggest that the optimal dose to obtain the trophic effects of hNGF may be in the nanogram range if the neurotrophin is secreted over a greater surface area, as in the present study, by using the polymer capsules rather than a finite point source, which occurs following a cannula infusion. Although the demon- stration that hNGF promotes the survival of axotomized cholinergic neurons in nonhuman primates is an important milestone in the development of this approach in clinical settings, to date, no studies have evaluated the long-term effects of hNGF administration in nonhuman primates. It would be useful to determine whether the short-term beneficial effects observed in the present study, as well as other studies, are maintained with long-term administra- tion of hNGF in fornix lesioned primates. In addition, the structural and functional consequences in aged nonhuman primates should be studied. Toward this end, preliminary data suggest that encapsulated BHK cell implants provide trophic and tropic influences to degenerating basal fore- brain neurons in aged Rhesus monkeys (Kordower et al., 1994).

In addition to the potent effects of hNGF-secreting implants on the viability of degenerating cholinergic basal forebrain neurons, these implants stimulated the growth of fibers from intact cholinergic basal forebrain cells. The

sprouting of cholinergic fibers in the lateral septum in response to NGF-secreting transplants is similar to that observed in both rodents and primates following intraven- tricular NGF infusions (Williams et al., 1986; Koliatsos et al., l990,1991a,b; Tuszynski et al., 1990). The source of the cholinergic sprouting observed here remains to be eluci- dated. Cholinergic fibers could not be traced from the medial septum to the lateral septum, where the sprouting effect was most vigorous, and thus does not appear to be the source of this phenomenon. Only a few fibers from the VLDB could be observed coursing dorsally and contributing to this dense fiber plexus. Because this sprouting response was both C U T - and p75 NGFr-immunoreactive, these data suggest that it originates within neurons of the nucleus basalis, the only other forebrain region that ex- presses both C U T and p75 NGF receptors (Kordower et al., 1988). Large collections of cholinergicip75 NGFr- containing neurons reside within the anteromedial subdivi- sion of the nucleus basalis. These neurons lie just caudal to the plexus of sprouting fibers and potentially could contrib- ute, in large part, to its formation. Tract-tracing experi- ments would aid in determining the source of the sprouting fibers. Furthermore, dose-response studies are needed to establish the levels of hNGF that would be efficacious in sustaining neuron viability while minimizing unintended effects such as the sproutingof cholinergic fibers toward the source of exogenously delivered hNGF. However, it is likely that any dose of hNGF that restores the phenotype of damaged cholinergic neurons would also produce some degree of hypertrophy and/or sprouting of intact neurons.

The effects of the hypertrophy andlor sprouting of normal or partially damaged neurons is currently un- known. Mobley et al. (1988) proposed that chronic NGF treatment might actually result in aberrant sprouting of neurites in the brain and produce associated deleterious effects, and Butcher and Woolf (1989) suggested that

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 157

Fig. 6. Low- (A, B) and high- (C, D) power photomicrographs through the septa1 diagonal band complex stained for the p75 NGF receptor from fornix-transected cynomolgus monkeys receiving polymer- encapsulated implants of either (A, C) a BHK-control transplant or (B) a BHK-hNGF transplant. (A, C) Note the extensive loss of p75 andD.

NGFr-immunoreactive neurons ipsilateral to the lesion (right) in the control implanted monkey. B,D: In contrast, numerous NGFr-positive neurons were observed in a fornix-transected monkey receiving the BHK-hNGF implant. Scale bar = 500 pm for A and B, 100 pm for C

anti-NGF should be administered to AD patients to prevent pathological sprouting of cholinergic fibers. Crutcher and Saffran (1990) also proposed that NGF could remodel

cholinergic axons within the site of delivery, resulting in the selective survival and sprouting of neighboring neurons. Indeed, monkeys receiving hNGF in the present study

158 D.F EMERICH ET AL.

Fig. 7. A. A fornix-transected monkey receiving BHK-control cell implants displays an extensive loss of CUT-immunoreactive neurons within the medial septum (MS). B: This loss was attenuated in monkeys receiving BHK-hNGF implants. Scale bar = 150 pm.

Fig. 8. Quantification of NGFr- and CUT-immunoreactive neu- rons within the vertical limb of the diagonal band in response to fornix transection followed by no implant (FFX) or implantation of either encapsulated BHK-control or BHK-hNGF-secretingcells (N = 4igroup ). This figure shows the percent of remaining cholinergic neurons ipsilat-

eral to the lesion for each individual monkey (1-8). The loss of cholinergic neurons ipsilateral to the fornix transection was signifi- cantly attenuated (P < 0.05) by implantation of BHK-hNGF cells. See text for details.

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 159

Fig. 9. NGFr-immunoreactive stained section through the vertical limb of the diagonal band from a fornix-transected monkey receiving a BHK-hNGF transplant. Note the similar pattern of NGFr-immunoreac- tive stained neurons (A) contralateral and (B) ipsilateral to the lesioniimplant. In contrast, some fornix-transectedicontrol-implanted

monkeys displayed an extensive loss of cholinergic neurons in this region (D) ipsilateral to the implant relative to the contralateral side (C). It is important that an extreme case of cell loss was chosen for illustration in the BHK-control condition; see Figure 8 for mean neuronal changes. Scale bar = 100 km.

appeared lethargic in their home cages for the duration of the experiment. Although a systematic analysis was not carried out, these animals appeared qualitatively different from control grafted cohorts. Thus, a comprehensive behav- ioral assessment following hNGF is warranted. Because NGF treatment would likely be most effective early in the course of AD, long-term administration of NGF would be required. Accordingly, a careful examination of the positive and potentially negative effects of long-term administration of NGF in aged nonhuman primates will be critical. It is important to note, however, that the functional effects of NGF-mediated aberrant sprouting on the function of the organism are still unknown, although empirical data has demonstrated that infusions of NGF induce sprouting in the aged rat and still enhance cognitive performance (Fischer et al., 1987). These data support the use of NGF for the treatment of AD.

The hypertrophy of residual cholinergic neurons could also be beneficial when considered in the context of a chronic neurodegenerative disease such as AD. The de-

creases in cholinergic parameters within the cortex and hippocampus of AD patients could be partially or com- pletely compensated for by either increased neurochemical activity of remaining cholinergic neurons or by sprouting of cholinergic terminals, which could reinnervate their nor- mal target structures. This compensation could underlie the recovery of cognitive performance in young rodents following excitotoxic lesions of the nucleus basalis (Unger and Schmidt, 1992) as well as in nonlesioned, aged rodents (Fischer et al., 1987) following NGF treatment.

The use of genetically modified cells to deliver hNGF is a very powerful experimental tool (Gage et al., 1987; Rosen- berg et al., 1988; Breakefield, 1989; Ernfors et al., 1989; Stromberg et al., 1990; Kawaja et al., 1992; Maysinger et al., 1992; Hoffman et al., 1993; Winn et al., 1994). The treatments received by animals receiving hNGF-secreting implants and animals receiving control implants only differ by the hNGF construct. Therefore, the effects obtained can be ascribed to this hNGF with a high degree of certainty. However, it remains possible for a factor or factors secreted

Fig. 10. High-power photomicrographs illustrate the morphology of p7.5 NGF receptor-immunoreactive neurons within the medial septum of monkeys receiving (A-E) BHK-hNGF implants or (F-H) BHK- control implants. All neurons were located within the medial septum ipsilateral to the lesioniimplants. A-E: Note the enlarged perikarya and extensive neuritic arbor displayed by monkeys receiving the

BHK-hNGF implants. In particular, the neuron in panel E displays an atypical “chandelier-like” morphology. F-H: In contrast, many neu- rons within the medial septum ipsilateral to the IesioniBHK-control implants appeared atrophic with a stunted neuritic branching pattern. Scale bar = 50 pm.

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 161

Fig. 11. Sprouting of cholinergic fibers in BHK-hNGF animals. A: Low-power photomicrograph of NGFr-immunostained sections illus- trates a dense plexus of cholinergic fibers seen on the side of hNGF treatment along the dorsoventral extent of the septum (arrows). This plexus was most extensive in the dorsal quadrant. B: In contrast, minimal fiber staining is seen in this area in monkeys receiving BHK-control cell implants. C: High-power photomicrograph illustrates

the morphology of the NGFr-immunoreactive fibers that coalesce as a dense bundle adjacent to the ventricle. D C U T immunocytochernistry visualizes the same plexus of fibers that ramified against the ependyrnal lining of the ventricle, forming a terminal-like pattern adjacent to the transplant site. LV, lateral ventricle. Scale bar = 500 pm in A, 400 pm inB,50 pminCandD.

162

by the fibroblasts to have synergistic effects with hNGF. Using cell lines also provides an unlimited and reliable source of de novo synthesized hNGF that can be delivered directly to discrete CNS sites. Cell lines may also be banked, cloned, and tested for viral contamination or the presence of other adventitious agents providing additional safety incen- tives. The long-term and stable expression of high levels of hNGF from genetically engineered cell lines should be verified for clinical use. The BHK polyclonal cell line used in the present study can maintain high levels of hNGF secretion for a t least 20 passages in vitro without continued drug selection and from cell-loaded capsules maintained for 3 and 6 months in vivo (Winn et al., 1994). In the present study, BHK cells reduced their production of hNGF to approximately one-third of preimplant levels. However, the hNGF titers produced in vivo a t the time the monkeys were killed were still quite high and sufficient to induce morpho- logical differentiation of PCl2A cells in a manner indistin- guishable from that seen following administration of 50 ng of mouse NGF. Previous transplantation studies using rat fibroblasts genetically modified to secrete mouse NGF demonstrated markedly lower levels of NGF secretion in vitro and no data from cells maintained in vivo for greater than 1 month (Rosenberg et al., 1988; Schumacher et al., 1991; Frim et al., 1992; Kawaja et al., 1992; Hoffman et al., 1993).

Delivery of growth factorb) to CNS structures remains a challenge in the development of potential therapies for chronic degenerative disease(s). Trophic factors do not readily cross the blood-brain barrier, and most studies have administered neurotrophins directly into the CNS either by infusions or the implantation of cells genetically engineered to produce NGF (Hefti, 1986; Williams et al., 1986; Fischer et al., 1987; Kromer. 1987; Montero and Hefti, 1988; Tuszynski et al., 1990, 1991; Koliatsos et al., 1991a,b). The latter strategy may have some limitations because suspen- sions of genetically modified cells cannot easily be secured within the ventricle and require implanting directly to brain parenchyma in proximity to NGF-responsive cells. In AD, the nucleus basalis degenerates more extensively than the septohippocampal complex (Mufson et al., 1989b). Within each hemisphere of the human brain, the large nucleus basalis measures up to 2 cm in the rostrocaudal direction and up to 1.5 cm in the medial-lateral direction. Thus, the use of unencapsulated cells may require multiple needle passes to provide adequate trophic support to this dispersed and heterogeneously distributed group of neu- rons. This may be especially problematic because the nucleus basalis is located in the ventral aspect of the brain, and needle penetrations would pierce through the cortex, striatum, and globus pallidus. Recent studies have demon- strated that polymer-encapsulated cells may provide a means for providing the CNS with a chronic source of protein delivery such as NGF and may be an efficacious procedure for the treatment of a number of human disor- ders including AD (Hoffman et al., 1993; Winn et al., 1994), Parkinson disease (Emerich et al., 1992; Aebischer et al., 1991a,b; Tresco et al., 1992), diabetes (Lacy et al., 19911, and chronic pain (Sagen et al., 1993). These capsules surround cells of interest with a semipermeable and immu- noprotective barrier allowing nutrients to enter the cap- sule, so the encapsulated cells will survive and function, and hNGF and other molecules below a certain molecular weight cut off to diffuse out the capsule (for review, see Emerich et al., 1992). The capsule can be implanted directly

D.F EMERICH ET AL.

into the ventricles or parenchyma, where hNGF will diffuse into and act on the surrounding tissue. The membrane prevents elements of the immune system from entering the capsule, thereby protecting the encapsulated cells from immune destruction. The technique, therefore, allows use of unmatched human cells (allografts), or even animal cells (xenografts), without immunosuppression of the recipient.

Degenerative changes in cholinergic neurons are a criti- cal and consistent component in AD and may be respon- sible, in part, for the observed cognitive deficits. Although a number of transmitter systems and neuronal populations are affected in AD, the degree of cholinergic degeneration has been correlated with the onset of memory deficits (Sims et al., 1981) and the duration of the dementia that charac- terizes AD (Wilcock et al., 1982). In animal models of AD, cholinergic agonists improve, whereas antagonists impair, performance on a variety of learning and memory tasks (Drachman et al., 1980; Bartus et al., 1985). Unfortunately, treatments that augment cholinergic function such as the use of cholinesterases and acetylcholine precursors have produced only mild improvements in certain features of AD dementia. However, the pharmacokinetics of most choliner- gic drug treatments have limited their utility, and the high degree of variability between subjects has hindered their evaluation (Bartus, 1986).

The relative selectivity of NGF combined with the ability to support the viability of cholinergic neurons has led to the suggestion that using NGF may prove more efficacious than cholinergic agonists or cholinomimetics in augmenting cholinergic forebrain function. In this regard, NGF admin- istration can rescue cholinergic neurons destined to degen- erate and/or die following axotomy (Hefti, 1986; Williams et al., 1986; Fischer et al., 1987; Kromer, 1987; Montero and Hefti, 1988; Tuszynski et al., 1990, 1991; Koliatsos et al., 1991a,b) or a number of other experimental procedures (Schumacher et al., 1991; Frim et al., 1992; Unger and Schmidt, 1992; Liberini et al., 1993). NGF may partially reverse the age-related atrophy of basal forebrain choliner- gic neurons and the associated cognitive deficits that occur in subpopulations of aged rodents (Fischer et al., 1987). The relationship between cognition, cholinergic neurons, and NGF suggests that the use of NGF may represent a useful primary or adjunct treatment strategy for AD and/or other diseases characterized by cholinergic dysfunctions.

In the current study, we have reversed degeneration of primate cholinergic basal forebrain neurons that occurs following fornix transections by implants of polymer cap- sules that contain cells that have been genetically modified to secrete high levels of hNGF. This strategy for providing a chronic source of hNGF to the brain may provide a means of treating neurological disorders that are in part character- ized by a degeneration of NGF-responsive cell groups.

ACKNOWLEDGMENTS This research was partially supported by NS29585,

NS25655, and AGO9466 (J.H.K.). We thank Dr. Mark Bothwell for generously providing the NGF receptor anti- body used in this study. We thank the polymer engineering group at CytoTherapeutics for the production and charac- terization of the hollow fiber material. We also thank the cell and molecular biology and histology personnel at CytoTherapeutics for their contributions. Thanks to Louise Finnegan for assistance on the manuscript.

ENCAPSULATED NGF-SECRETING CELLS IN PRIMATES 163

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