Neuronal Progenitor Cells of the Neonatal Subventricular Zone Differentiate and Disperse Following Transplantation Into the Adult Rat Striatum

Cell Transplantation, Vol. 7, No. 2, pp. 137–156, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0963-6897/98 $19.00 1 .00

PII S0963-6897(98)00009-8

Original Contribution NEURONAL PROGENITOR CELLS OF THE NEONATAL SUBVENTRICULAR ZONE DIFFERENTIATE AND DISPERSE FOLLOWING TRANSPLANTATION INTO THE ADULT RAT STRIATUM TANJA ZIGOVA,* VIORICA PENCEA,* RANJITA BETARBET,*† STANLEY J. WIEGAND,‡ CHARLIE ALEXANDER,‡ ROY A. E. BAKAY,† AND MARLA B. LUSKIN*1 *Department of Cell Biology, †Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA 30322 and ‡Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591

M Abstract — We have investigated the suitability of a recently identified and characterized population of neuronal progenitor cells for their potential use in the replacement of degenerating or damaged neurons in the mammalian brain. The unique population of neuronal progenitor cells is situated in a well-delineated region of the anterior part of the neonatal subventricular zone (referred to as SVZa). This region can be separated from the remaining proliferative, gliogenic, subventricular zone encircling the lateral ventricles of the forebrain. Because the neurons arising from the highly enriched neurogenic progenitor cell population of the SVZa ordinarily migrate considerable distances and ultimately express the neurotransmitters GABA and dopamine, we have examined whether they could serve as an alternative source of tissue for neural transplantation. SVZa cells from postnatal day 0 –2 rats, prelabeled by intraperitoneal injections of the cell proliferation marker BrdU, were implanted into the striatum of adult rats approximately 1 mo after unilateral denervation by 6-OHDA. To examine the spatio-temporal distribution and phenotype of the transplanted SVZa cells, the experimental recipients were perfused at short (less than 1 wk), intermediate (2–3 wk) and long (5 mo) postimplantation times. The host brains were sectioned and stained with an antibody to BrdU and one of several cell-type specific markers to determine the phenotypic characteristics of the transplanted SVZa cells. To identify neurons we used the neuron-specific antibody TuJ1, or antimembrane-associated protein 2 (MAP-2), and anti-GFAP was used to identify astrocytic glia. At all studied intervals the majority of the surviving SVZa cells exhibited a neuronal phenotype. Moreover, morphologically they could be distinguished from the cells of the host striatum because they resembled the intrinsic granule cells of the olfactory bulb, their usual fate. At longer times, a greater number of the transplanted SVZa cells had migrated from their site of implantation,

often towards an outlying blood vessel, and the density of cells within the core of the transplant was reduced. Furthermore, there were rarely signs of transplant rejection or a glial scar surrounding the transplant. In the core of the transplant there were low numbers of GFAP-positive cells, indicating that the transplanted SVZa cells, predominantly TuJ1-positive/MAP2-positive, express a neuronal phenotype. Collectively, the propensity of the SVZa cells to express a neuronal phenotype and to survive and integrate in the striatal environment suggest that they may be useful in the reconstruction of the brain following CNS injury or disease. © 1998 Elsevier Science Inc.

ACCEPTED 1/7/98. 1 Correspondence should be addressed to Marla B. Luskin,

Ph.D., Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.

M Keywords — Neuronal progenitor cells; Cell suspension graft; Striatum; Survival; Migration. INTRODUCTION

Cell transplantation is an emerging and promising strategy for the treatment of some neurological diseases and disorders, including Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and injuries. Many of the cell types previously used for neurotransplantation— embryonic ventral mesencephalic tissue (4,10,25), polymer-encapsulated (19), adrenal medullary (17), other nonneuronal (20,47), or genetically modified (3,15,20, 22,52) cells— have produced encouraging results, although for various reasons, none are fully satisfactory. Consequently, there is an ongoing search for a way to improve the efficacy of the existing candidates, as well as to identify alternative sources of donor tissue. We are beginning to examine the potential usefulness of a recently identified and unique population of neuro-

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nal progenitor cells that are situated in the subventricular zone, surrounding the lateral ventricles of the postnatal forebrain (38). Both in vitro and in vivo studies have established that, in the subventricular zone, it is the most anterior portion, or SVZa, which contains an enriched population of neuronal progenitor cells (38,41). The subventricular zone posterior to the SVZa is predominantly a source of glia (23,39). Thus, one of the advantages of adapting the SVZa cells for transplantation purposes is that most or all the grafted cells should be neurons, if their identity is not altered by the transplantation. Such an outcome has been sought, because transplants of conditionally immortalized neural progenitors and neural ‘‘stem-like’’ cells do not always result in the desired ratio of neurons to glia (43,54). In addition to the neuronal phenotype, SVZa cells have a number of characteristics that may make them particularly suitable for transplantation to the injured or diseased brain. Notably, in vivo and following homotopic transplantation, SVZa-derived cells migrate substantial distances. Ordinarily they migrate along an extended pathway to reach the olfactory bulb that begins at the anterior– dorsal tip of the lateral ventricle (32). Furthermore, the migration of the SVZa-derived cells does not appear to involve a radial glia system of fibers (48) thought to be indispensable for the migration of many neuronal cell types (50), including those of the striatum (24,53). Another unusual characteristic of migrating SVZa-derived cells is that they undergo division while en route to the olfactory bulb (45), suggesting they retain features that are associated with immature neurons, shown to fare better following transplantation (9). This property of migration may allow for a more uniform distribution of transplanted cells in their new environment. After reaching their destination in the olfactory bulb, the SVZa cells differentiate into granule and periglomerular cells, the interneurons of the olfactory bulb, a subset of which are dopaminergic (5). Because SVZa cells generate progeny that express dopamine as a neurotransmitter, it is reasonable to consider them a potential replacement for the neurons of the substantia nigra pars compacta, known to supply the dopaminergic innervation to the striatum, and which are lost in Parkinson’s disease. As a first step in determining the potential utility of SVZa cells, this study was designed to evaluate key features of the neonatal subventricular zone progenitor cells following their heterotopic transplantation into the denervated striatum of adult rats. We sought to determine whether the heterotopically transplanted cells survive following engraftment into the striatum and whether they retain a neuronal phenotype. Another aim of the study was to determine whether the SVZa cells engrafted into the striatum become encased in a glial scar, as unfortu-

nately occurs for some cell types after transplantation (4). Not only was there little to no glial scar evident in the striatum following the transplantation of SVZa cells, but furthermore, there was a dispersion of SVZa cells away from their site of implantation, indicating that transplanted SVZa cells have the capacity to integrate into the denervated striatum. Preliminary reports of these findings have been given elsewhere (7,64). MATERIALS AND METHODS

Animals Neonatal and adult Sprague–Dawley rats were used in the experiments described. The neonatal rats used to obtain donor SVZa progenitor cells for transplantation were obtained from a breeding colony maintained at Emory University. The adult Sprague–Dawley rats were obtained from two sources. Some of the host animals (n 5 18, 300 – 400 g) were obtained from the breeding colony housed at Emory University, while others (n 5 14, 360 –530 g) were purchased from Zivic-Miller by Regeneron Pharmaceuticals, Inc. Our convention was to designate the day on which a vaginal plug was detected as embryonic day 0 (E0); birth usually occurs at E22, also considered to be postnatal day 0 (P0). Both neonatal and adult rats were maintained on a 12 L:12 D cycle and had a continuous supply of food and water. All experimental procedures were conducted in compliance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals. Pretransplantation Procedures 6-Hydroxydopamine Lesions. A standard procedure was followed for the unilateral elimination of the dopaminergic cells of the substantia nigra (57). Adult rats, anesthetized by an intraperitoneal injection (i.p.) of Equithesin (0.3 mL/100 g body weight) were secured in a stereotaxic frame prior to surgery. An incision was made through the skin overlying the sagittal suture to expose the skull. A single hole was drilled through the skull of each rat, centered around 4.5 mm anterior to bregma and 1.5 mm lateral to the sagittal suture. To chemically destroy the dopaminergic cells of the substantia nigra, each adult rat received a single injection, using a 10-mL Hamilton syringe of 3– 4 mL of 6-hydroxydopamine hydrobromide (6-OHDA HBr; Sigma; 2.86 mg 6-OHDA HBr/mL in 0.2 mg/mL L-ascorbatesaline) into their right ascending mesostriatal dopaminergic pathway. The position of the substantia nigra in the adult rat and the 6-OHDA release site was deduced from the stereotaxic atlas of Paxinos and Watson (49). The anterior–posterior (A-P) coordinates were either 24.3 or 24.5 mm relative to bregma. The mediolateral (M-L)

Grafting of neuronal progenitors into the striatum ● T. ZIGOVA

coordinates were either 21.2 or 21.5 mm relative to the midline, and the dorsoventral (D-V) coordinates were either 28.0 or 27.1 mm relative to the pial surface with the tooth-bar of the stereotaxic apparatus set at 3.3 or 3.5 mm below the interaural line. The rate of the delivery of 6-OHDA was approximately 0.5 mL per minute, and the cannula was left in place for an additional 1–2 min before it was retracted, thereby allowing the released toxin to diffuse locally, and reducing backflow along the cannula tract. In the rat, the dopaminergic projection to the ipsilateral striatum has been reported to degenerate within a week following 6-OHDA treatment (35), thereby producing a hemiparkinson condition. Labeling and Isolation of Donor SVZa cells. To label SVZa neuronal progenitor cells prior to their transplantation, we followed a procedure described by Zigova et al. (62), with a few modifications. To label dividing SVZa cells in vivo (i.e., before harvesting) P0 –P2 rat pups were intraperitoneally injected with the thymidine analog bromodeoxyuridine (BrdU; 5 mg of BrdU/mL of 0.007 N NaOH in 0.9% saline, 0.3 mL/pup, Sigma). The pups were injected twice—the night before and 1 h before cell harvesting. The SVZa’s of the rat pups were removed by microdissection and dissociated into a single cell suspension following the procedure described by Zigova et al. (62). The cell density of the SVZa cell suspension ranged from 7.5– 8.9 3 107 cells/mL and the cell viability was 90 –95%, as determined by a rapid double-staining procedure using fluorescein diacetate to detect living cells and propidium iodide to detect dead or dying cells (31). Assessment of the Percentage of BrdU-Labeled SVZa Cells. Because the labeling of all dividing SVZa progenitor cells cannot be achieved by the administration of only two injections of BrdU, a procedure was devised to determine the percentage of SVZa donor cells that were BrdU labeled prior to their transplantation. SVZa cells were labeled as described, isolated following the second BrdU injection, dissociated, and then plated at 5.8 3 105 cells/cm2 on poly-D-lysine coated dishes for 24 h (41). Subsequently, the cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and then rinsed in cold 0.1 M phosphate-buffered saline (PBS). To fragment the DNA, the cells were treated with 1 N HCl at 45°C for 30 min, neutralized by a 5-min rinse in 0.1 M borate buffer (pH 8.5) at room temperature, and washed again with PBS. The cultures were treated with 5% normal goat serum in PBS for 30 min and then incubated overnight in the primary antibody, a rat monoclonal antibody to BrdU (Accurate, NY) at a 1:500 dilution. The next day the cultures were rinsed in 0.1 M PBS and incubated for 2 h in the secondary antibody, goat antirat IgG conjugated to

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Table 1. Incorporation of BrdU by SVZa cells prior to transplantation Experiment No. 1 2

No. of fields counted

No. of BrdU(1) cells

Total No. of SVZa cells

Percentage of BrdU(1) cells

10 10

150 139

620 674

24.0 20.6

In each experiment 13 neonatal rats received two intraperitoneal injections of BrdU, 24 and 1 h prior to perfusion. Following the second BrdU injection the region of the SVZa was harvested, dissociated, and then cultured for 24 h (for details see Materials and Methods). After fixation the SVZa cells were processed for BrdU immunohistochemistry and visualized by either light or fluorescence microscopy (Fig. 4). We counted the number of BrdU(1) cells vs. the total number of the cells in 10 fields per experiment.

rhodamine (Jackson ImmunoResearch, PA) at a 1:200 dilution. After the secondary antibody was removed, the cultures were rinsed three times with PBS and coverslipped with VectaShield (Vector, CA). The slides of the cultured SVZa cells were viewed on a Zeiss Axiophot microscope equipped with fluorescence and Nomarski optics. The BrdU-labeled cell nuclei were visualized by their red rhodamine fluorescence, and unlabeled cells were detected using Nomarski optics. The BrdU-labeled and all SVZa cells were counted in 10 random fields per experiment and their percentage was calculated (Table 1). Transplantation Procedure Transplantation of SVZa Cells. Approximately 1 mo (30 –34 days) after the substantia nigra of the adult rats was lesioned by injection of 6-OHDA, the animals were anesthetized by an intraperitoneal injection of Equithesin (0.3 mL/100 g body weight) and placed in a stereotaxic apparatus. An incision was made through the skin overlying the sagittal suture to expose the skull. A 10-mL Hamilton syringe was used to inject stereotaxically (Tables 2 and 3) 2.5– 4.0 mL of a suspension of labeled SVZa cells into striatum on the 6-OHDA–lesioned side (Fig. 1). The syringe needle was left in place for 2 minutes following each injection and then withdrawn. The skull was wiped dry with a surgical swab, checked for any possible bleeding, and the overlying skin was repositioned and closed with surgical clips. After surgery the animals were placed under an infrared heat lamp to recover and subsequently returned to their cages. Tissue Processing and Immunohistochemistry After transplantation the host animals were allowed to survive for a short (3 days), intermediate (14 –21 days), or relatively long time period (140 –152 days). Subsequently, the animals were anesthetized with an overdose

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Table 2. Results characterizing SVZa transplants in the adult striatum 3 days postimplantation (a) Rat number 1 2 3

(b) Injection site coordinates (mm)

(c)

(d)

(e)

BrdU(1) cells Total # of cells

Density of SVZa cells within the graft

663/1829

1111

89.7 6 2.1 (47.3–152.3)

435/1406

111

—

111

128.3 6 3.3 (120.9–250.4) NA

A-P

M-L

Depth

Amount injected (mL)

1.2 1.2 0.2 1.2 1.2 0.2 1.2

2.5 2.5 2.5 2.5 2.5 2.5 2.5

5.0 6.0 5.5 5.0 6.0 5.5 5.0

3.0 3.0 3.0 3.0 3.0 3.0 3.0

(f) Distance (mm) of SVZa cells from graft midline

NA 5 not applicable. This table summarizes the results obtained after transplantation of SVZa cells into the adult striatum. (a) Adult rats received transplants of BrdU-labeled SVZa cells. (b) The reference points for the injection site coordinates were as follows: distance anterior to bregma for the anterior–posterior (A-P) dimension, distance lateral to the sagittal sinus for the medial–lateral (M-L) dimension, and distance below the pial surface for depth. (c) The volume of each injection was 3 mL. The suspension contained 7.5– 8.9 3 107 cells/mL and the cell viability was 90 –95%. (d) The level of BrdU incorporation is expressed as the ratio of the number of BrdU-positive cells relative to the total number of (labeled 1 unlabeled) cells. (e) The density of SVZa cells in the graft was scored as 1111 for 31– 40 cells/square (1640 mm2), 111 for 21–30 cells/square, 11 for 11–20 cells/square, and 1 when less than 11 cells/square were found (for details, see Materials and Methods). (f) To evaluate the distance that SVZa cells moved away from the main mass of the transplant into the surrounding host parenchyma, we measured the distances of individual cells from the graft midline. The values are expressed as the mean 6 SEM. The range of values is given in parentheses.

of chloral hydrate (5 mL/kg body weight) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Their brains were removed and postfixed for at least 1 h in the same fixative, washed with 0.1 M PBS, and then transferred to a 20% sucrose solution in 0.1 M PBS. The following day the brains were embedded in O.C.T. (Miles Inc., IN) and frozen using liquid nitrogen. Eighteen micron-thick coronal or sagittal cryostat sections were thaw mounted onto Superfrost microscope slides (Fisher Scientific, PA). Immunohistochemistry was used to reveal transplanted, BrdU-labeled SVZa cells in the host striatum. The cryostat sections were washed in 0.1 M PBS, twice treated with 2 N HCl at 60°C for 15 min to fragment the DNA, and subsequently twice neutralized in 0.1 M borate buffer (pH 8.3) for 15 min. After a thorough wash in 0.1 M PBS the sections were incubated in 10% normal goat serum in PBS for 30 min and then treated overnight in rat monoclonal antibody to BrdU (Accurate, NY) at a dilution 1:500, as described above. The next day the sections were rinsed in 0.1 M PBS and incubated for 2 h in goat antirat IgG conjugated to biotin (Jackson ImmunoResearch, PA) at a dilution 1:200. Next, the slides were incubated in an avidin– biotin solution (Vectastain Elite, Vector, CA) for 30 min at room temperature, rinsed in PBS and incubated for a few minutes in a solution containing diaminobenzidine (DAB, Vector, CA) to produce a dark-brown precipitate in the nuclei of BrdU-labeled cells. These sections were usually counterstained with either 0.4% Gill’s hematoxylin

(Polysciences, PA), or 0.5% cresyl violet (Roboz Surgical Instrument Co., Inc., MD) and then dehydrated in ethanol and coverslipped with D.P.X. (BDH Limited, UK). We used a number of cell-type specific antibodies in conjunction with anti-BrdU to determine the phenotype of the transplanted SVZa cells. In some cases, however, sections were stained with a single cell-type specific antibody. To visualize cell-type specific labeling together with anti-BrdU labeling on the same section, the tissue was first reacted to reveal anti-BrdU staining and then to show cell-type specific immunoreactivity. To identify astrocytes (8), the sections were treated overnight with rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; Dako) at a dilution 1:500. To label neurons, the sections were incubated with TuJ1, a neuron-specific antibody recognizing class III b-tubulin (34) supplied by Dr. A. Frankfurter, at a dilution 1:500. In addition, some sections were incubated overnight in a mouse monoclonal antibody to microtubule-associated protein 2, MAP-2 (Sigma, St. Louis, MO), which recognizes differentiating neurons (30) and was used at a dilution 1:500. Sections were treated for 1 h with one of the secondary antibodies: for GFAP, FITC-conjugated goat antirabbit (Jackson ImmunoResearch, PA) was used at a dilution 1:200; and for TuJ1 or MAP-2 FITC-conjugated goat antimouse antibody (Jackson ImmunoResearch, PA) was used at a dilution 1:100. Then the sections were washed with PBS and coverslipped either with 90% glycerol or Vectashield (Vec-

Grafting of neuronal progenitors into the striatum ● T. ZIGOVA

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Table 3. Coordinates for implantation of SVZa cells and density of SVZa cells in the graft (a) Rat Number

(b)

(c) Injection site coordinates (mm)

(d)

1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 0.2 1.2 1.2 0.2 1.2 1.2 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2

3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 4.0 3.0 3.0 3.0 3.0 3.0 3.0 2.5 2.5 4.0 3.0 3.0

(e)

(a)

(b)

(c) Injection site coordinates (mm)

(d)

1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.3 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2 1.2 0.7 0.2

4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0 4.0 3.0 3.0

(e)

Amount Density of Amount Density of Survival injected SVZa cells Rat Survival injected SVZa cells (days) A-P M-L Depth (mL) in graft Number (days) A-P M-L Depth (mL) in graft

1

14

2

14

3

14

4

14

5

17

6

17

7

17

8

17

9

17

10

17

11

17

2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5

5.0 6.0 5.5 5.0 6.0 5.5 5.0 6.0 5.5 5.0 6.0 5.5 5.0 6.0 5.5 5.0 6.0 5.5 5.0 6.0 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5

111

12

17

11

13

19

NA

14

19

11

15

20

111

16

20

11

17

21

11

18

21

11

19

21

11

20

140

11

21

152

NA

22

152

2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.5 2.6 2.5 2.7 2.9 2.5 2.7 2.9 2.5 2.7 2.5 2.5 2.7 2.5 2.5 2.7 2.5

5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.0 5.5 5.0 4.7 5.5 5.0 5.3 5.5 5.0 5.3 5.5 5.0 5.3 5.5

1 11 11 11 11 11 11 11 11 11 11

Coordinates for implantation of SVZa cells and density of SVZa cells in the graft. (a and b) The brains of adult animals (previously injected with 6-OHDA) received implants of BrdU-labeled SVZa cells into their right striatum, and the density of the transplanted cells was determined following perfusion at various time points posttransplantation. (c) The coordinates are given using the position of bregma as the A-P reference point, the sagittal sinus as the M-L reference point and the pial surface as the depth reference point. (d) The volume of each injection was 3 mL. (e) To estimate the density of transplanted SVZa cells in the host brain we used the same scoring procedure as described in Table 2 (also see Materials and Methods).

tor, CA). For control sections the primary antibody was omitted. To evaluate the extent of the degeneration of the dopaminergic cell bodies and axons in the substantia nigra, some sections were processed for tyrosine hydroxylase (TH) immunohistochemistry. Slides were washed in 0.1 M PBS, incubated for 1 h in 10% normal goat serum in PBS and then overnight in rabbit polyclonal antibody to TH (Eugene Tech, NJ), at a dilution 1:1000. The following day the sections were rinsed in 0.1 M PBS and incubated for 1 h in the secondary antibody, goat antirabbit IgG conjugated to biotin (Jackson ImmunoResearch, PA) at a dilution 1:200. The sections were then

rinsed again and incubated in an avidin– biotin solution (Vectastain Elite, Vector, CA) for 30 min at room temperature. After a final rinse in 0.1 M PBS the sections were incubated for a few minutes in a solution containing diaminobenzidine (DAB, Vector, CA) and later on coverslipped using 90% glycerol. Following examination of the slides, selected sections were counterstained with cresyl violet, dehydrated in ethanol, and coverslipped with D.P.X. The immunohistochemically stained sections were examined by epifluorescence microscopy on a Zeiss Axiophot microscope using single rhodamine and fluorescein filters as well as a dual filter set. Camera lucida drawings of representative sections showing the

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Fig. 1. Diagram illustrating the temporal sequence of the procedures used to study the outcome of transplanting SVZa neuronal progenitor cells into the denervated striatum of adult rats. The striatum of adult rats was implanted with SVZa progenitor cells 30 –34 days after receiving a unilateral 6-OHDA lesion of the nigrostriatal pathway. The animals were subsequently perfused at one of the three times indicated.

distribution of transplanted SVZa-derived cells after a short survival period were prepared on a Leitz Laborlux 12 microscope. Quantitative Analysis of the Grafted Tissue Composition of the Graft. The cellular composition of the SVZa cell grafts within the host striatum was evaluated in several ways (see below). First, we determined the number of BrdU-positive cells, and second, we calculated the density of SVZa cells within the main mass of the transplant. To determine the number of BrdU-positive SVZa cells after transplantation, cell counts were made from the 18 mm coronal sections viewed at a total magnification of 312.53 with a 253 objective and 103 ocular. A rectangular square 317 3 317 mm grid was inserted into the 103 ocular. The numbers of BrdU-labeled and unlabeled SVZa cells were counted in 27–30 squares (4,015 mm2/square) of the grid per section in every studied animal. Displacement and Density of Transplanted SVZa Cells. Procedures were developed for measuring the density of the transplanted cells and the extent to which they migrated away from their site of implantation. The distance the transplanted cells migrated away from the graft was defined as the shortest length between the midline of the main mass of the transplant and the displaced SVZa cell within the striatal parenchyma. Because our preliminary experiments demonstrated that the transplanted SVZa cells could be distinguished morphologically from the cells of the striatum, for measurement purposes we either identified the transplanted SVZa cells by their BrdU immunoreactivity or their morphological appearance (see Results for a fuller explanation). The distance between either BrdU-labeled nuclear profiles or darkly stained SVZa cells and the midline of the transplant was measured, and average displacement distances were calculated. The percentage of BrdU-labeled cells in the transplant and migration

distances were calculated for two of the animals that survived 3 days after transplantation (Table 2). We determined the density of transplanted SVZa cells at all three intervals studied. Hematoxylin-stained coronal sections were viewed through a 403 objective containing a rectangular grid placed over the transplant at random sites. On average, 4 –14 squares of the grid per animal were analyzed to determine the relative packing density of cells within the graft. If the mean number of cells per square (1,640 mm2) was between 31– 40, the transplant was considered to be densely packed, and was represented as 1111 in Table 2. If the average number of cells ranged between 21–30 cells per square, it was considered to be moderately packed and represented by 111, an average density of 11–20 cells per square of the grid was represented as 11, and when the average density dropped below 11 cells per square, it was assigned a score of 1. Quantitative Estimation of SVZa Cell Survival. The number of surviving SVZa cells in the transplant 140 days posttransplantation was estimated by determining the number of SVZa cells per unit volume (numerical density, Nv), and then by multiplying the numerical density by the approximate volume of the graft (Vgraft). Within the selected sections, each cell profile was counted. To calculate the numerical density, the sections were captured on a Zeiss Axiophot microscope (403 ocular, 312.53 magnification) equipped with an Optronix camera connected to a Macintosh computer running Image 1 (National Institute of Health). The number of SVZa cell profiles and the area of the transplant were calculated from 11 coronal sections 170 6 30 mm apart. The real number of cells in these sections was then estimated using Method 1 of Abercrombie (1), which corrects for the likelihood that an individual cell will appear in adjacent sections. The approximate density of the cells in the graft was obtained by dividing the real number of cells by the volume of the transplant corre-

Grafting of neuronal progenitors into the striatum ● T. ZIGOVA

sponding to the measured graft areas (graft areas 3 the section thickness). The Abercrombie formula assumes that the cells are round and that their diameter is known; transplanted SVZa cells appear round or oval. To estimate the cellular diameter, 30 randomly selected cell profiles were chosen, using the same method as for the cell number and transplant area. The estimated mean profile diameter was 4.79 mm. This mean diameter was used in the Abercrombie formula. To calculate the entire volume of the graft we reconstructed the transplant, using 10 equally separated slices (see Fig. 8). The boundaries and the area of the graft in each section were determined using the same software as described above. To approximate a 3D representation, the surfaces of the sections were rotated 50° around the vertical axis of the transplant. The volume of each region between two adjacent selected sections was determined and summed to obtain the entire volume. RESULTS

In the present study 6-OHDA was injected into the nigrostriatal bundle to produce unilateral dopaminergic denervation of the striatum. The 6-OHDA–lesioned animals were maintained for 30 –34 days before receiving transplants of neuronal progenitor cells isolated from the anterior part of subventricular zone (SVZa). After short (3 days), intermediate (14 –21 days), and long (140 –152 days) periods of posttransplantation survival, animals were perfused and their brains processed and analyzed qualitatively and quantitatively to assess the distribution, survival, and phenotype of the transplanted SVZa cells. (Fig. 1). Appearance of the 6-OHDA Lesioned Site The brains of the experimental animals were immunostained with antibodies to tyrosine hydroxylase, to determine whether the dopaminergic neurons of the substanta nigra pars compacta had been eliminated by the 6-OHDA injection. Animals were included in the study if they exhibited a complete or nearly complete loss of tyrosine hydroxylase immunoreactivity in substantia nigra pars compacta neurons on the lesioned side. To reveal the morphology of cells at the lesioned site, some sections were counterstained with either hematoxylin or cresyl violet. Examination of the 6-OHDA lesion site in all three studied groups demonstrated a substantial loss of substantia nigra pars compacta neurons. The occasional presence of macrophages was evident along the needle track leading to the substantia nigra pars compacta and around the injection site. The degree of

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damage resulting from the 6-OHDA injections appeared to be very similar at all studied time points. Figure 2 illustrates the appearance of the injection site from the brain of an animal that survived 5 mo posttransplantation, corresponding to the longest period of survival. Neurons on the control side were tyrosine hydroxylase immunoreactive, while on the lesioned side, tyrosine hydroxylase positivity was not detected. We presume that there was a dopaminergic denervation of the striatum on the lesioned side of the brain secondary to the depletion of tyrosine hydroxylase-immunoreactive neurons in substantia nigra.

Comparison of the Morphological Characteristics of Striatal Neurons and Olfactory Bulb Interneurons To morphologically distinguish between the host striatal neurons and the grafted SVZa cells, we examined cresyl violet and hematoxylin-stained sections at the light microscopic level. Previous studies of the rat striatum revealed that it forms a large ‘‘striated’’ mass, penetrated by dispersed fiber bundles of the internal capsule (26). Most descriptions of striatal neurons in the rat are based on the soma size and the appearance of the processes (26). According to Chang et al. (12), the majority (.95%) of the striatal neurons have a medium size (10 –20 mm in diameter) with a smooth, spherical, or slightly ovoid nucleus (Fig. 3A and B); they represent the category of medium spiny neurons. Large (20 – 60 mm) and small (less than 10 mm) diameter neurons constitute only a small (;2–3%) contingent of the cells. The medium spiny neurons could be easily distinguished in the sections that were stained with cresyl violet by their lightly stained cytoplasm. Within the striatum, patches or ‘‘neuronal islands’’ were clearly separated by bridges of unstained fibers containing, small, elongated darkly stained glial cells irregularly spaced along the extent of the fiber bundles (Fig. 3A and B). Thus, the medium spiny neurons could be readily identified within the striatum and distinguished from the transplanted SVZa cells described below. Our previous studies demonstrated that SVZa-derived cells become the granule cell and periglomerular interneurons of the olfactory bulb (5,38,62). The granule cells are arranged in several parallel layers of uneven thickness (Fig. 3C and D) and the periglomerular cells surround the glomeruli. The light microscopic examination of cresyl violet-stained sections of the olfactory bulb revealed that the granule and periglomerular cells are smaller (5–10 mm in diameter) and more darkly stained than the medium spiny cells of the striatum. In addition, the packing density of the neurons in the granule cell layer is visibly much greater than that of the neurons in the striatum. The

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Fig. 2. A comparison of the morphology and phenotype of cells in the substantia nigra on the control and experimental side 152 days after a 6-OHDA lesion. (A,B) Photomicrographs showing cresyl violet-stained 18 mm coronal sections of the substantia nigra pars compacta on the experimental (A) and control (B) side of the brain. (C,D) Photomicrographs showing tyrosine hydroxylase immunostaining of the same regions shown in A and B. The arrows in B point to several large neurons of the intact substantia nigra. The same neurons expressing tyrosine hydroxylase are indicated by arrows in D. Note that the equivalent region of the substantia nigra on the experimental side does not reveal morphologically (A) or immunohistochemically (C) distinct neuronal cell bodies. Short arrows in A and C indicate the same cellular debris resulting from the lesion. The distances between the corresponding arrows in A vs. C, and B vs. D, are smaller due to the cresyl violet counterstaining of the immunohistochemically processed tissue. A comparison of the morphological and immunohistochemical differences between the experimental (A and C) and control side (B and D) of the brain demonstrates that the injection of 6-OHDA into the nigrostriatal pathway destroyed the neurons of substantia nigra responsible for the dopaminergic innervation of the striatum. Scale bars in A–D represent 100 mm.

SVZa and SVZa-derived cells can be distinguished from the small (2–3%) striatal neurons by the punctate appearance of their nuclei. These characteristics allowed us to distinguish the transplanted cells and to unequivocally delineate the borders of the implants of SVZa cells within the host striatum. BrdU Incorporation by SVZa Cells Donor pups were intraperitoneally injected with BrdU twice within 24 h prior to tissue harvesting (see Materials and Methods) to determine what proportion of SVZa cells incorporated the cell proliferation marker BrdU before transplantation. Single-cell suspensions of SVZa cells from two litters (13 pups per litter) were plated on poly-D-lysine-coated slides at 5.8 3 105 cells/cm2 (41). Twenty-four hours later, the cell cultures were fixed by 4% paraformaldehyde and stained with anti-BrdU antibody (Fig. 4). In both sets of cultures we determined the number of cells that incorporated BrdU and the total number of cells/field in 10 fields per experiment. We found that on average 22.5% of SVZa cells expressed cell proliferation marker BrdU 1 day after plating (Table

1). These findings suggest that approximately one-quarter of all SVZa cells injected into the host brain should be BrdU-labeled and immunohistochemically detectable in the host striatum. Short-Term Survival In one set of experiments we examined the appearance of transplanted SVZa cells within the striatum of three rats that survived 3 days posttransplantation. These animals were given unilateral 6-OHDA injections into the nigrostriatal bundle and 30 –34 days later two of them received three deposits (3 mL each) of BrdU-labeled SVZa cells (79,000 – 89,600 cells/mL). We did not find three separate implantation sites in the striatum of either of these animals. One plausible explanation for this finding is that, because the distance between the three injections was small, the injected cells might have formed one continuous mass of transplanted cells (see Discussion). The third animal received only one injection of the SVZa cell suspension (3 mL), which resulted in a considerably smaller area of SVZa cells in the host striatum (see Table 2).

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Fig. 3. The morphological appearance of striatal medium spiny neurons compared to granule cell interneurons within the olfactory bulb and following their implantation into the adult striatum. (A,B) Low- and high-magnification bright-field photomicrographs of an 18-mm sagittal section of the adult striatum stained with cresyl violet. Readily apparent in A and B are the two compartments of the adult striatum: the patch (*) portion containing mostly medium size striatal neurons and the striated portion, comprised of fiber bundles (f) of the internal capsule. Note that the striatal neurons are relatively lightly stained by cresyl violet (arrow) and distinct from the more darkly stained glia in the striated portion. Scale bars in A and B are 100 and 20 mm, respectively. (C,D) Low- and high-magnification photomicrographs of an 18 mm sagittal section of the adult granule cell layer of the olfactory bulb stained with cresyl violet. As shown in C, the small, darkly stained granule cells are arranged in layers (arrowheads) of irregular width. The regions of low cell density contain bundles of myelinated fibers, which are considerably thinner than the fiber bundles in the striatum. Note the difference in the cell size and intensity of staining between the granule cells of the olfactory bulb (D) and striatal neurons shown at the same magnification in B. Scale bars in C and D represent 100 and 20 mm, respectively. (E) Bright-field photomicrograph demonstrating the appearance of the transplanted SVZa neuronal progenitor cells within the striatum 152 days after implantation. Most notably, the morphology of the SVZa cells remained unchanged. They are dark, small (e.g., small arrow) and easily distinguished from the pale medium-sized cells of the striatum (large arrows). Note the occurrence of striatal cells within the region dominated by the presence of transplanted cells (arrowhead). Scale bar 5 20 mm.

Fig. 4. Dissection of SVZa cells for transplantation and assessment of BrdU incorporation. (A) SVZa cells from neonatal P0 –P2 rat forebrains were used as donors for transplantation into the adult striatum. The SVZa (hatched area) was microdissected from hand cut sagittal slices, trypsinized, and mechanically dissociated into a single cell suspension. For subsequent identification of the transplanted donor cells in the host brains, SVZa cells were prelabeled by two intraperitoneal injections of the cell proliferation marker BrdU prior to SVZa harvesting (see below and Materials and Methods). CC: corpus callosum; CTX: cerebral cortex; OB: olfactory bulb; V: lateral ventricle. (B,C) Phase (B) and fluorescent (C) photomicrographs of the same field of cultured SVZa cells labeled by administering BrdU to a P0 rat pup 24 h and 1 h before the region of the SVZa was harvested. Cells of the SVZa were cultured on poly-D-lysine coated dishes for 24 h before fixation. The arrowheads in B and C point to identical cells. On average, 20 –24% of the SVZa cells were BrdU positive. The 100 mm scale bar in B also applies to C.

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Fig. 5. The distribution of SVZa cells in the host striatum 3 days after transplantation. (A) Camera lucida drawing of an 18-mm hematoxylin-stained coronal section of the adult forebrain showing the region of the transplanted SVZa cells within the host striatum. Note that the transplant (black) is an elongated mass of cells situated lateral to the ventricle and considerably ventral to the overlying corpus callosum. Bar 5 200 mm. Magnification 5 312.53. Abbreviations: AC: anterior comissure; CC: corpus callosum; CTX: cerebral cortex; LV: lateral ventricle; ST: striatum. (B) Camera lucida drawing of a single coronal section of the same brain shown in A demonstrating the position of the SVZa cell nuclei (black dots). Note the relatively high packing density of the SVZa cells. Furthermore, some of the SVZa cells occur within the parenchyma at various distances from the core of the transplant. Bar 5 200 mm. Magnification 5 312.53. (C) Bright-field photomicrograph from the hematoxylin-stained section represented in B, which illustrates the central portion of SVZa transplant. Small, round, darkly stained SVZa cells resembling olfactory bulb interneurons (see Results) correspond to the transplanted cells. The host striatum is composed mainly of medium-sized striatal neurons that are distinct from the appearance of the transplanted SVZa cells. Bar 5 100 mm.

In all three animals hematoxylin and cresyl violetstained sections revealed grafts located within the striatum close to the lateral ventricle (Fig. 5A). The grafts were generally elongated in appearance (Fig. 5B), and their boundaries were easily distinguished from the surrounding striatal parenchyma. In two animals there was no indication of excessive cellular infiltration judging from the low number of macrophages. Darkly stained nuclei of transplanted SVZa cells, resembling darkly stained nuclei of olfactory bulb interneurons, were quite densely packed in the main mass of the graft (Fig. 5C).

Occasionally, some individual SVZa cells were found outside the main graft boundary. Our observations suggest that the short-term SVZa cells transplants were healthy and easily identifiable in the host striatum. The presence of SVZa cells within the parenchyma proper, beyond the graft boundary, indicated that some of the transplanted SVZa cells had migrated away in their new environment soon after their implantation. No difference was found in the distribution of BrdU-labeled cells compared to the SVZa cells morphologically identified (see below).

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Composition of the Graft. In addition to the previously described hematoxylin and cresyl violet-stained sections we also used BrdU immunohistochemistry to identify transplanted SVZa cells in the striatum. In two animals we counted the number of BrdU-labeled SVZa cells vs. all SVZa cells composing the transplant (see Materials and Methods). In one animal 31% of the grafted SVZa cells were BrdU positive, while 36% were BrdU positive in the other. When compared to our in vitro results, it seems that incorporation and expression of BrdU by SVZa cells in vivo was not affected by the transplantation procedure. These results indicate that there is not a preferential loss of BrdU-positive cells in the transplants. In summary, in the adult striatum, the grafts of SVZa cells were readily identified either morphologically or immunohistochemically. Density and Distribution of Transplanted SVZa cells. The hematoxylin and cresyl violet-stained sections were also used to determine the density of SVZa cells within the main body of the graft. Three days posttransplantation the implanted SVZa cells were very tightly packed within the graft (see Table 2). Some darkly stained SVZa cells were found in the striatal parenchyma (Fig. 6A, Fig. 4C), indicating that they had moved away from the main mass of the transplant. Frequently, we found SVZa cells around blood vessels (Fig. 6C) or in association with striatal fibers. The distance between the midline and the position of the cells displaced from the main mass of the transplant ranged from 47 to 250 mm (Table 2). Phenotype of SVZa Cells in Adult Striatum 3 Days Posttransplantation. To determine the phenotype of SVZa cells in the host striatum we combined BrdU immunohistochemistry with cell-type specific antibodies. The sections were double labeled either with antiBrdU and anti-TuJ1 to reveal the presence of transplanted cells with a neuronal phenotype (Fig. 6A and C) or with anti-BrdU and anti-GFAP to reveal transplanted GFAP-positive cells, as well as the distribution of glia in and around the graft (Fig. 6B). Three days posttransplantation we observed numerous BrdU-positive, TuJ1-positive, and double-labeled (BrdU-positive/TuJ1-positive) cells at the transplantation site in the striatum. TuJ1 neuronal staining was seen throughout the entire transplant, and the BrdU-positive cells were randomly scattered throughout the TuJ1-positive region. As expected, frequently, we observed BrdU-positive cells around blood vessels (Fig. 6C) in close proximity to the transplant. The presence of double-labeled cells (BrdU-positive/TuJ1-positive) indicates that some SVZa cells with a neuronal phenotype were able to migrate away from the main mass into the host parenchyma. We stained several sections, adjacent to sections

Fig. 6. Phenotype of SVZa cells 3 days after transplantation into the host striatum. (A) Fluorescent photomicrograph showing TuJ1-positive (green) and BrdU-positive (yellow) SVZa cells in the adult striatum shortly after transplantation. The full extent of the transplant corresponds to the region of intense TuJ1 fluorescence. In addition, many TuJ1-positive and double-labeled, TuJ1-positive/BrdU-positive, cells were found at varying distances from the site of implantation (arrows). Bar 5 100 mm. (B) Higher magnification, fluorescent photomicrograph from an adjacent section to the transplant shown in A. Note several brightly stained (green), TuJ1-positive SVZa cells leaving the interface between the graft and parenchyma. A collection of double-labeled (TuJ1-positive/BrdU-positive) cells were found close to the blood vessels. Bar 5 100 mm. (C) Fluorescent photomicrograph demonstrating GFAP immunoreactivity (green) and BrdU-positive SVZa cells (yellow) in the region of the transplanted SVZa cells. Note that the number of GFAP-positive cells around the transplant (*) was not significantly higher than in the other regions of the striatum, indicating that the glial response to the implantation of SVZa cells was negligible. In addition, note the low level of GFAP expression within the transplant. Bar 5 100 mm.

double labeled by anti-BrdU and anti-TuJ1, with antiGFAP and anti-BrdU. We found only a small number of double-labeled (BrdU-positive/GFAP-positive) cells within the transplant (Fig. 6B). We also noted that the GFAP immunoreactivity around the graft was not significantly higher than in the surrounding striatum, thus confirming that there was only a negligible glial response to the implantation of SVZa cells or to injury caused by

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Fig. 7. The distribution, morphology and phenotype of SVZa cells in the striatum 14 –21 days after transplantation. (A) A low-magnification bright-field photomicrograph of an 18-mm cresyl violet-stained sagittal section from a rat brain that received a suspension of SVZa cells (three injections, total volume 9 mL) into the previously denervated striatum. At 17 days posttransplantation the transplant (T) can be distinguished by the patches of darkly stained cells. The patches are separated by the fiber bundles running through the striatum. The higher magnification of the border between a patch of transplanted cells and the striatal parenchyma, demarcated by brackets, is shown in B. The arrow points to the same blood vessel in A and B. Bar 5 200 mm. (B) A high-magnification photomicrograph from the bottom left corner of the photomicrograph shown in A (brackets) demonstrating the spatial relationship between the patches of darkly stained SVZa cells (asterisks), host striatal fibers (f), and medium, lightly stained striatal neurons (arrowheads). Bar 5 100 mm. (C) A fluorescent photomicrograph showing the phenotype of the SVZa cells 14 days posttransplantation. The section was double-labeled with TuJ1 (green), to identify neurons and anti-BrdU (yellow). Numerous TuJ1-positive cells were found within and outside the core of the transplant (T). Note that several double-labeled TuJ1-positive/ BrdU-positive cells (e.g., arrows) migrated away from the transplant into the striatal parenchyma. Bar 5 50 mm.

the needle. Rarely did we find double-labeled (BrdUpositive/GFAP-positive) cells within the transplant (Fig. 6B). Intermediate Survival Because we were interested in knowing how the disposition of transplanted SVZa cells changed after various posttransplantation times, we examined another set of animals that received three injections (2.5– 4 mL each) of SVZa cells into the previously denervated striatum. These 19 animals constituted the intermediate survival cases and were perfused at 14 (n 5 4), 17 (n 5 8), 19 (n 5 2), 20 (n 5 2), and 21 (n 5 3) days posttransplantation. Similar transplant coordinates were chosen to those used in the short-term cases (Table 3). In 17 of the 19 animals the transplanted SVZa cells were confined to the striatum; in two of the cases the transplants were found in the striatum, as well as in the overlying cortex. In some cases we were able to follow the injection track running through the cortex and corpus

callosum into the striatum. In all cases, the transplant and the site of implantation could be discerned. Appearance and Density of the Graft. After intermediate survival periods the grafts of SVZa cells into the striatum were generally less compact and less cylindrical than in those brains studied 3 days posttransplantation. Although the density of grafted cells varied from one case to another, in the majority of transplants we could identify a darkly stained, densely packed core of cells. In most brains (14 of 19) the cells were very densely packed, while in others (2 of 19) they were moderately densely packed, or even loosely packed in one brain. Within the same graft, in several instances, we observed both regions of loosely packed SVZa cells as well as regions of tightly packed SVZa cells (Table 3, Fig. 7A and B). The borders of the transplant were less delineated and the graft was apparently better assimilated within the host parenchyma after the intermediate survival times compared to the short survival period (Fig. 7B). Numer-

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ous transplanted cells were observed in the vicinity of blood vessels (not shown) and striatal fibers (Fig. 7A and B) in the intermediate survival cases. Phenotype and Migration of SVZa Cells 14 –21 Days Posttransplantation. Representative sections from brains containing the grafts of SVZa cells were stained for neuronal (TuJ1) and glial (GFAP) markers as in the short-term cases. Double labeling with anti-BrdU and the neuronal marker TuJ1 revealed that the majority of cells within the transplant expressed a neuronal phenotype. Numerous double-labeled (BrdU-positive/TuJ1-positive) cells were found at various distances from the main mass of the transplant (Table 2, Fig. 7C). Sections stained for the presence of GFAP did not reveal a significantly different level of expression than that seen after short-term survival. In summary, the transplanted SVZa cells expressed a neuronal antigen and migrated considerable distances. Long-Term Survival To determine whether SVZa cells could survive and migrate for an extended period of time posttransplantation, the brains of three animals were examined approximately 5 mo after they received three injections per animal of an SVZa cell suspension into the striatum. Representative brain sections from each of the animal perfused 140 –152 days after transplantation were examined (Table 3). To approximate the number of transplanted SVZa cells that survived 140 days after grafting, we reconstructed the main mass of the transplant (Fig. 8A and B). The volume occupied by surviving SVZa cells was about 0.192 mm3, and there were about 97,700 cells in the transplant, representing 11.8% of the initially injected cells.

Fig. 8. Reconstruction of a graft (left) of SVZa cells 140 days after their implantation into the 6-OHDA lesioned striatum. (A) The position of the graft in a representative 18 mm coronal section. The drawing on the right illustrates the same section after it was rotated 50° around the vertical axis of the transplant. In both drawings the solid black area designates the graft in the striatum (St). The lateral ventricle is to the right, ventral to the corpus callosum (CC). (B) The overall configuration of the graft is illustrated by representative sections taken at intervals of 180 mm; the sections are rotated in the same way

Morphology and Density of Transplanted SVZa Cells. In hematoxylin and cresyl violet-stained sections the grafts were recognized easily in the host brains even after long-term survival (Fig. 9). Although we did not use the cell-type specific markers for this interval, most of the cells within the transplant had a distinctive SVZa cell morphology as observed after short-term and intermediate survival times (Fig. 3C and D). In some areas we observed lightly stained, medium-sized striatal neurons with processes that intermingled with smaller, roundoval SVZa cells (Fig. 9C). This intermingling of the two

as the section in A. The asterisk designates the section presented in the drawing in A. Note that in two of the sections the area of the graft extends further in the dorsal direction. These two regions may represent the positions of two different needle tracks. The grafted SVZa cells, however, appear to form a single transplant.

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Fig. 9. Distribution and morphology of SVZa cells 140 days after transplantation into the adult denervated striatum. (A) Bright-field photomicrograph of a cresyl violet-stained coronal section from an adult rat brain that received a unilateral injection of SVZa cells (;828,000 in 10 mL) into the striatum. Darkly stained SVZa cells, representing the transplant, are observed extending throughout the dorsoventral extent of striatum. The boxed area of the transplant is shown at higher magnification in B. Bar 5 100 mm. CC: corpus callosum; ST: striatum; V: lateral ventricle. (B) Higher magnification photomicrograph of the middle part of the transplant shown in A. Note the group of darkly stained SVZa cells (arrow) in the vicinity of the blood vessel. Note that the density of SVZa cells is lower than in the representative short-term survival case shown in Fig. 5. Bar 5 100 mm. (C) High-magnification bright-field photomicrograph of a portion of the SVZa transplant shown in A. The darkly stained SVZa cells, resembling interneurons of the olfactory bulb (arrowheads), are intermingled with pale, medium-sized neurons of the striatum (arrow). Bar 5 20 mm.

cell types was never observed in the short or intermediate survival cases. The packing density of the transplanted SVZa cells was less than in the granule cell layer of the olfactory bulb, but greater than in the typical ‘‘patch zones’’ of the striatum (Fig. 3A and B). When compared to the shortterm survival the packing density of cells within the main mass of the transplant was lower (Table 3). This could be accounted for by the migration of SVZa cells away from the graft and/or the death of the SVZa cells. Even though we could not rely on BrdU labeling to detect transplanted SVZa cells in the long-term survival cases, the main mass of the transplant as well as remote small groups of cells originating from the transplant were easily noticeable in the striatal parenchyma (Fig. 9B). The possible dilution of the BrdU marker together with the dispersion of transplanted cells and/or their intermingling with striatal cellular components made it difficult to assess how far and precisely how many of the SVZa cells migrated beyond the transplant boundary. Despite the possibility that our determination of the percentage of SVZa cells surviving 5 mo posttransplantation may underestimate the true value of surviving cells; nevertheless, there were large numbers of transplanted cells in each of the brains analyzed. DISCUSSION

The present study was undertaken to investigate the behavior of a novel population of neuronal progenitor cells following their implantation into the adult rat striatum previously lesioned by 6-OHDA. We used progenitor cells derived from the anterior part of the neonatal subventricular

zone, or SVZa, as donor tissue because their unique qualities may make them beneficial to the damaged or diseased host brain. First, because they comprise virtually a pure population of neuronal progenitor cells (41), they ought to yield only neurons following transplantation. Second, the SVZa cells maintain their capacity to proliferate in vivo (45) as well as in vitro, even though they posses a neuronal identity (41). This ability to divide suggests that they also retain features associated with undifferentiated cells, which have been shown to survive transplantation better than mature neurons (9). Third, in situ, the SVZaderived cells migrate long distances to reach their destination under normal (32,40,45) and various experimental (6,28,36,62) conditions. This migratory capacity, if retained after transplantation, should be advantageous in the dispersion of transplanted SVZa cells away from their site of implantation. Finally, after completing their migration, both unmanipulated and manipulated cells become incorporated into the circuitry of the olfactory bulb where they function either as GABAergic or dopaminergic interneurons (5). In this study we demonstrated that, indeed, as hypothesized, the SVZa progenitor cells transplanted into the denervated striatum of adult rats retained many of their seminal properties. Specifically, at all examined posttransplantation time points the SVZa cells survive in the denervated striatum. The transplanted SVZa cells display a neuronal phenotype and exhibit morphological characteristics of the olfactory bulb interneurons. Furthermore, SVZa cells disperse in the new environment and integrate well into the host parenchyma, particularly in the intermediate and longterm posttransplantation interval. Collectively, our results indicate that SVZa cells could possibly be a source of

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progenitor cells for transplantation into the brains damaged by Parkinson’s disease.

Technical Considerations Related to the Detection of Transplanted SVZa Cells In this study donor SVZa cells were labeled by the intraperitoneal administration of two injections of the cell proliferation marker BrdU, which can be detected immunohistochemically by the tagged cells (45,56) and their progeny following implantation into the host brain (6,62). Although at high concentrations BrdU can be deleterious to cells, there was no indication that it was toxic to the dividing SVZa cells at the concentration used. Unlike other markers (i.e., HRP, PHA-L, Fast blue, Hoechst 33258) BrdU is stably integrated into the cell nuclei without transfer to the host tissue. An additional advantage of using BrdU as a way to tag transplanted cells, that influenced our choice of markers, is that its nuclear localization allowed either concomitant or consecutive detection of cell-type specific markers expressed in the cytoplasm or on the cell surface. In our experiments we used anti-BrdU in conjunction with phenotypic markers to determine the identity of the newly generated SVZa cells. However, the shortcoming of BrdU labeling is that only a subset of cells incorporates BrdU during its administration. Our studies indicate that approximately one-fourth of SVZa cells are BrdU labeled at the time of transplantation. Overall, given the available markers that can be used to identify transplanted cells, BrdU was a suitable alternative. Even though not all grafted SVZa cells were BrdU labeled, we demonstrated another way to reliably identify the transplanted SVZa cells. Morphological markers, such as the size and staining intensity of SVZa cells relative to host striatal cells (Fig. 3), could be used to identify the transplanted SVZa cells and distinguish them from striatal cells. We determined that shortly after transplantation (3 days) the BrdU-labeled cells represented ;33% of the total number of grafted cells. One explanation to account for the greater percentage of BrdU-labeled cells in the transplants compared to the percentage of BrdU-labeled cells at the time of transplantation is that some of the transplanted cells may have escaped detection. Another issue about the labeling of transplanted cells that could not be fully resolved is the extent to which they undergo cell division posttransplantation and thereby dilute their label to the point that they cannot be detected unambiguously. To assess the ongoing division of SVZa cells it would be necessary to employ other methods, such as antibodies to the proliferating cell nuclear protein (PCNA), which is synthesized in early G1 and S phases of the cell cycle (59). We did not carry

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out definitive experiments to assess the continuous proliferation of SVZa cells in the host striatum, but we did notice a decline in the overall intensity of BrdU labeling in the 2–3 wk survival group compared to the shortsurvival animals, suggesting continued cell division by transplanted SVZa cells. The ongoing division of transplanted SVZa neuronal progenitor cells was also reported in our previous transplantation studies in neonates (6,62). A significant increase in the number of thymidine-labeled RN33B cells, a brainstem-derived cell line, transplanted into the neonatal and adult striatum, was also reported by Lundberg et al. (37) 2–3 wk posttransplantation, although PCNA immunostaining did not show detectable levels at any of the studied time points. Future studies using PCNA immunohistochemistry or some other more sensitive methods will be needed to reveal the level of SVZa cell proliferation in the host brain.

A Comparison of Properties of SVZa Cells With Other Transplanted Cell Types To evaluate the potential usefulness of SVZa neuronal progenitor cells for transplantation, and in particular in the therapeutic treatment of Parkinson’s disease, the properties of the SVZa cells must be compared with those of other types of cells that are being investigated for similar purposes. The ideal cell type for the replacement of degenerating neurons should be readily available in large numbers, survive for extended periods of time in the host brain and produce the required levels of biologically active substances (51). At the present time the cells isolated from the embryonic ventral mesencephalon, which generates the substantia nigra, seem to have the greatest potential for transplantation therapy in Parkinson’s disease. These cells can be harvested at the time they start to differentiate into dopaminergic neurons (11), and after transplantation they have been shown, in some cases, to form morphological and functional synapses (21,42). Unfortunately, only a small fraction of the transplanted cells survive, and only a fraction of those are tyrosine hydroxylase immunoreactive neurons (10, 46). Therefore, functional recovery depends on harvesting large amounts of tissue. One way to overcome the problem is to culture progenitor cells isolated from the embryonic ventral mesencephalon in the presence of the mitogenic growth factors, such as bFGF or EGF (44,55). Another approach that has been applied to circumvent the problem of limited tissue availability for transplantation is the immortalization of neural progenitors. The cell lines obtained usually produce an abundant number of cells, which can be homogenous and easily maintained for long periods of time. Following transplantation, the immortalized cells frequently migrate and integrate ap-

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propriately into the recipients brain in a nondisruptive, nontraumatic, and nontumorigenic manner (54). Although the immortalized cells can differentiate in the host brain, the vast majority are nonneuronal. Nevertheless, these immortalized cell lines have been used in targeted gene therapy for hemiparkinsonian rodents and primates with considerable success (2). Even so, the immortalized cell lines prepared by the transformation of progenitor cells with conditional oncogenes has met with some resistance for human use. Nonneuronal cell types (e.g., fibroblasts, glial cells, myoblasts) have also been considered as alternative sources for transplantation (13,16,20). As a group, these cells are easily obtainable, expandable in culture, and can be used for gene transfer combined with transplantation, or ex vivo gene therapy. For example, rat primary astrocytes were genetically modified to produce nervegrowth factor (15), brain-derived neurotrophic factor (60), and tyrosine hydroxylase using recombinant retroviruses (27). However, they were naturally unable to form reciprocal connections with the host brain. Nevertheless, genetically engineered nonneuronal cells have been reasonably effective in animal models of Parkinson’s disease (27), suggesting that the connectivity may not be the decisive factor for some functional recovery. By comparing the SVZa neuronal progenitor cells with the previously mentioned cell types we have demonstrated that SVZa cells possess a number of features prior to and following transplantation that make them candidates for cell replacement therapy. First, the SVZa cells are of CNS origin, which fosters their acceptance by the host brain. Furthermore we have previously shown that both the endogenous SVZa cells and cultured SVZa cells possess a neuronal phenotype, the preferred cell type for transplantation. However, although they have a neuronal identity, they have not lost the ability to proliferate, a property that should make the introduction of genes into SVZa cells attainable. Also of significance is our finding in this study, that the vast majority of the SVZa cells heterotopically transplanted into the denervated striatum express neuronal markers, and unlike some clonal neural cell lines (54), after grafting SVZa cells do not appear to revert to an earlier stage of differentiation. The majority of the grafted cells revealed a neuronal phenotype at all posttransplantation time points studied. In particular, at all posttransplantation intervals examined, the SVZa cells exhibited morphological features similar to those of olfactory bulb interneurons (Fig. 3). That is, unlike the behavior of some other cell types following transplantation into the brain, they did not show site-specific differentiation. One explanation for why the transplanted SVZa cells exhibit many of the properties they would exhibit ordinarily is because the SVZa progenitor cells are unusual precursors in that

they have made a commitment to become neurons, and possibly neurons with the properties of olfactory bulb interneurons even while they are still proliferating. Future studies will elucidate the neurotransmitter phenotype of the transplanted SVZa cells to determine whether it can be influenced by environmental cues operating in the host striatum. If the transplanted SVZa cells express the transmitter candidates that they did in the unmanipulated olfactory bulb, then some should be dopaminergic, as our preliminary experiments have indicated (7,64).

A Comparison of the Migratory Behavior of Homotopically and Heterotopically Transplanted SVZa Cells The mechanism of migration utilized by unmanipulated SVZa-derived cells does not appear to involve the radial glia that support most migrating neurons in the central nervous system (50), and yet SVZa-derived cells in the neonate and adult traverse relatively long distances from their site of generation to their final destination in the olfactory bulb (36,38). It is not known whether the ability of the SVZa-derived cells to migrate routinely several millimeters is a function of their intrinsic capacity to migrate or reflects a particularly favorable terrain. To begin to answer this question and to determine whether SVZa-derived cells could also migrate in a foreign environment, SVZa progenitor cells have been transplanted to homotopic and heterotopic locations (29, 62). It has now been well established that when SVZa cells are homotopically transplanted their behavior is essentially indistinguishable from that of unmanipulated SVZa-derived cells (62). When implanted into either the neonatal or adult SVZa the transplanted cells navigate to the olfactory bulb (at least in part) by following cues in their migratory pathway (36,62). Although the specific nature of the cues is not known, it is not a function of the astrocytic composition of the pathway (18); our preliminary experiments have shown that the network of astrocytes present in the adult migratory pathway are absent in the neonate (Smith, Gnani, and Luskin, unpublished observations). Given that the SVZa progenitor cells homotopically transplanted into the adult brain do not lose their migratory ability, we were encouraged to ask whether SVZa cells retain a capacity to migrate when placed in the 6-OHDA–lesioned striatum. In a related set of experiments we had previously shown that after SVZa cells are heterotopically transplanted into the neonatal striatum, they disperse from their site of implantation (6). However, there are many types of cells that can migrate when transplanted into the fetal or neonatal brain that display at best minimal migration when implanted in the adult brain. The inability of transplanted immature neurons to

Grafting of neuronal progenitors into the striatum ● T. ZIGOVA

migrate in the adult brain has been attributed primarily to the glial scar that forms around the implanted cells, or to a change in the extracellular matrix that acts to prevent migration (4). To analyze whether SVZa cells are similarly prevented from migrating in the adult brain, we transplanted them into the adult striatum. In contrast to the poor migratory behavior of some types of cells following transplantation into the adult striatum, the heterotopically transplanted SVZa cells dispersed from their site of implantation. This indicate that SVZa cells may possess some distinct properties that allow them to migrate not only in the immature brain, but also in the mature brain. The nature of these special properties have yet to be determined. We observed that the dispersion of the SVZa cells implanted in the adult striatum gradually occurred over a number of weeks. When examined within a couple of days of implantation, low numbers of SVZa cells migrated away from the site where they were deposited. However, when examined at 3 wk, or even more so, at several months posttransplantation, greater numbers of the transplanted cells were dispersed within the parenchyma surrounding the core of the transplant. This suggests that the cells had actively migrated away from their implantation site, which was notably devoid of a glial scar. The distribution of the transplanted cells after the longer survivals would also seem to indicate that the SVZa cells actively participate in their dispersion from their site of implantation. In particular, the transplanted cells were often found surrounding blood vessels at a considerable distance from their site of implantation, and further displaced from the graft than most of the transplanted cells. One interpretation of this finding is that the cells were attracted to some factor localized to or released from the walls nearby blood vessels. The other prominent feature of the disposition of the transplanted cells that supports the notion of active migration is that the SVZa cells were not evenly distributed within the location they were found. Rather, bands of transplanted cells separated by the fiber bundles composing the thalamic radiations were frequently observed, suggesting that the SVZa cells migrated towards their preferred destination in the striatal parenchyma. Alternatively, the SVZa cells could not penetrate into the fiber bundles. Despite the directed migration of SVZa cells away from their site of implantation, they did not migrate to the same extent as observed following transplantation to the SVZa, suggesting that SVZa cells are receptive to cues present in their endogenous environment that governs their migration. What remains to be elucidated are the signals that instruct an SVZa cell to terminate its migration and whether migration can be enhanced by the addition of growth promoting factors.

ET AL.

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CONCLUDING REMARKS

Our present studies provide further evidence, in an in vivo model of Parkinson’s disease, that SVZa neuronal progenitor cells may represent an additional cell type that, following transplantation, can serve to replace neurons lost to disease and damage of the central nervous system. The experiments reported here capitalize on the endogenous properties of SVZa cells, including a combination of immature and mature neuronal features that allow them to remain plastic but committed to a neuronal phenotype, and an intrinsic ability to migrate that fosters their dissemination and integration into an ectopic environment, such as the 6-OHDA–lesioned adult striatum. Although our findings support the premise that SVZa cells may be a promising cell type for transplantation, their functional utility and transmitter expression remains to be elucidated. Because the subset of unmanipulated SVZa-derived cells become dopaminergic (5), an outstanding goal would be to determine what signals are required to enhance the dopaminergic expression of SVZa cells in vitro and/or following transplantation into the compromised adult brain. Recent in vitro studies have shown that various growth factors can induce the expression of the rate limiting enzyme, tyrosine hydroxylase, in the expression of dopamine (58). Among the neurotrophins, brain-derived neurotrophic factor (BDNF) promotes the differentiation and survival of dopaminergic neurons (33), and GDNF can enhance their survival following transplantation (14). Furthermore, our recent in vivo studies have demonstrated that adult subventricular zone cells, including SVZa cells, are responsive to the administration of BDNF (61,63). Therefore, future studies will be designed to analyze the response of transplanted SVZa cells to the administration of one or more neurotrophic factors that may promote their neurotransmitter differentiation, proliferation, and/or survival, and set the stage for the exploitation of their clinical applications. Acknowledgment — We are grateful to Ms. Susannah Brock for critical and helpful comments on the manuscript, and to Dr. Anthony Frankfurter for his generous gift of TuJ1. We thank Mindy Minnen and Nojan Valadi for their technical assistance. This work was supported by Regeneron Pharmaceuticals, Inc., and grants from the Parkinson Foundation and from the National Institute of Deafness and Other Communicative Disorders (RO1 DC03190) awarded to M.B.L.

REFERENCES

1. Abercrombie, M. Estimation of nuclear population from microtome sections. Anat. Rec. 94:239 –247; 1946. 2. Anton, R.; Kordower, J.H.; Maidment, N.T.; Manaster, J.S.; Kane, D.J.; Rabizadeh, S.; Schueller, S.B.; Yang, J.; Rabizadeh, S.; Edwards, R.H. Neural-targeted gene therapy for rodent and primate hemiparkinsonism. Exp. Neurol. 127:207–218; 1994.

154

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3. Bankiewicz, K.S.; Leff, S.E.; Nagy, D.; Jungles, S.; Rokovich, J.; Spratt, K.; Cohen, L.; Libonati, M.; Snyder, R.O.; Mandel, R.J. Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson’s disease. Exp. Neurol. 144:147–156; 1997. 4. Barker, R.A.; Dunnett, S.B.; Faissner, A.; Fawcett, J.W. The time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesencephalon to the striatum. Exp. Neurol. 141:79 –93; 1996. 5. Betarbet, R.; Zigova, T.; Bakay, R.A.E.; Luskin, M.B. Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone. Int. J. Dev. Neurosci. 14:921–930; 1996. 6. Betarbet, R.; Zigova, T.; Bakay, R.A.E.; Luskin, M.B. Migration patterns of neonatal subventricular zone progenitor cells transplanted into the neonatal striatum. Cell Transplant. 5:165–178; 1996. 7. Betarbet, R.; Zigova, T.; Bakay, R.A.E.; Lindsay, R.M.; Wiegand, S.J.; Luskin, M.B. Short-term survival of neonatal subventricular zone progenitor cells transplanted into the striatum of adult rats. Soc. Neurosci. Abstr. 22:218; 1996. 8. Bignami, A.; Eng, L.F.; Uyeda, C.T. Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43:429 – 435; 1972. 9. Bjorklund, A.; Dunnett, S.B. Neural transplantation in adult rats. In: Dunnett, S.B.; Bjorklund, A., eds. Neural transplantation: A practical approach. New York: Oxford University Press; 1991:57–78. 10. Bjorklund, A. Dopaminergic transplants in experimental parkinsonism: Cellular mechanisms of graft induced functional recovery. Curr. Opin. Neurobiol. 2:683– 689; 1992. 11. Brundin, P.; Strecker, R.E.; Widner, H.; Clarke, D.J.; Nilsson, O.G.; Astedt, B.; Lindvall, O.; Bjorklund, A. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Immunological aspects, spontaneous and drug-induced behavior, and dopamine release. Exp. Brain Res. 70:192–208; 1988. 12. Chang, H.T.; Wilson, C.J.; Kitai, S.T. A Golgi study of rat neostriatal neurons: Light microscopic analysis. J. Comp. Neurol. 208:107–126; 1982. 13. Chang, P.L.; Capone, J.P.; Brown, G.M. Autologous fibroblast implantation. Feasibility and potential problems in gene replacement therapy. Mol. Biol. Med. 7:461– 470; 1990. 14. Choi-Lundberg, D.L.; Lin, Q.; Chang, Y.N.; Chiang, Y.L.; Hay, C.M.; Mohajeri, H.; Davidson, B.L.; Bohn, M.C. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275:838 – 841; 1997. 15. Cunningham, L.A.; Hansen, J.T.; Short, M.P.; Bohn, M.C. The use of genetically altered astrocytes to provide nerve growth factor to adrenal chromaffin cells grafted into the striatum. Brain Res. 561:192–202; 1991. 16. Dai, Y.; Roman, M.; Naviaux, R.K.; Verma, I.M. Gene therapy via primary myoblasts: Long-term expression of factor IX protein following transplantation in vivo. Proc. Natl. Acad. Sci. USA 89:10892–10895; 1992. 17. Date, I.; Yoshimoto, Y.; Imaoka, T.; Miyoshi, Y.; Furuta,

18.

19.

20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32.

T.; Asari, S.; Ohmoto, T. Effect of host age upon the degree of nigrostriatal dopaminergic system recovery following cografts of adrenal medulla and pretransected peripheral nerve. Brain Res. 637:50 –56; 1994. Doetsch, F.; Garcia-Verdugo, J.M.; Alvarez-Buylla, A. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17:5046 –5061; 1997. Emerich, D.F.; Winn, S.R.; Linder, M.D. Continued presence of intrastriatal but not intraventricular polymer-encapsulated PC12 cells is required for alleviation of behavioral deficits in parkinsonian rodents. Cell Transplant. 5:589 – 596; 1996. Fisher, L.J.; Jinnah, H.A.; Kale, L.C.; Higgins, G.A.; Gage, F.H. Survival and function of intrastriatally grafted primary fibroblasts genetically modified to produce L-Dopa. Neuron 6:371–380; 1991. Freund, T.F.; Bolam, J.P.; Bjorklund, A.; Stenevi, U.; Dunnett, S.B.; Powell, J.F.; Smith, A.D. Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochemical study. J. Neurosci. 5:603– 616; 1985. Gage, F.H.; Fisher, L.J.; Jinnah, J.H.A.; Rosenberg, M.B.; Tuszynski, M.; Friedmann, T. Grafting genetically modified cells to the brain: Conceptual and technical issues. Prog. Brain Res. 82:1–10; 1990. Goldman, J.E. Lineage, migration, and fate determination of postnatal subventricular zone cells in the mammalian CNS. J. Neurooncol. 24:61– 64; 1995. Halliday, A.L.; Cepko, C.L. Generation and migration of cells in the developing striatum. Neuron 9:15–26; 1992. Heim, R.C.; Willingham, G.; Freed, W.J. A comparison of solid intraventricular and dissociated intraparenchymal fetal substantia nigra grafts in a rat model of Parkinson’s disease: Impaired graft survival is associated with high baseline rotational behavior. Exp. Neurol. 122:5–15; 1993. Heimer, L.; Alheid, G.F.; Zaborsky, L. Basal ganglia. In: Paxinos, G., ed. The rat nervous system. Sydney: Academic Press; 1985:37– 86. Horellou, P.; Sabate, O.; Buc-Caron, M.; Mallet, J. Adenovirus-mediated gene transfer to the central nervous system for Parkinson’s disease. Exp. Neurol. 144:131–138; 1997. Hu, H.; Rutishauser, U. A septum-derived chemorepulsive factor for migrating olfactory interneuron precursors. Neuron 16:933–940; 1996. Hu, H.; Tomasiewicz, H.; Magnuson, T.; Rutishauser, U. The role of polysialic acid in migration of olfactory bulb interneuron precursors in the subventricular zone. Neuron 16:735–743; 1996. Johnson, G.V.W.; Jope, R.S. The role of microtubuleassociated protein 2 (MAP-2) in neuronal growth, plasticity and degeneration. J. Neurosci. Res. 33:505–512; 1992. Jones, K.H.; Senft, J.A. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J. Histochem. Cytochem. 33: 77– 80; 1985. Kishi, K. Golgi studies on the development of granule cells

Grafting of neuronal progenitors into the striatum ● T. ZIGOVA

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

of the rat olfactory bulb with reference to migration in the subependymal layer. J. Comp. Neurol. 258:112–124; 1987. Knusel, B.; Winslow, J.W.; Rosenthal, A.; Hefti, F. Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin-3. Proc. Natl. Acad. Sci. USA 88:961–965; 1991. Lee, M.K.; Rebhun, L.I.; Frankfurter, A. Posttranslational modification of class III b-tubulin. Proc. Natl. Acad. Sci. USA 87:7195–7199; 1990. Lee, C.S.; Sauer, H.; Bjorklund, A. Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by intrastriatal 6-hydroxydopamine in the rat. Neuroscience 72:641– 653; 1996. Lois, C.; Alvarez-Buylla, A. Long-distance neuronal migration in the adult mammalian brain. Science 264:1145– 1148; 1994. Lundberg, C.; Winkler, C.; Whittemore, S.R.; Bjorklund, A. Conditionally immortalized neural progenitor cells grafted to the striatum exhibit site-specific neuronal differentiation and establish connections with the host globus pallidus. Neurobiol. Dis. 3:33–50; 1996. Luskin, M.B. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189; 1993. Luskin, M.B.; McDermott, K. Divergent lineages for oligodendrocytes and astrocytes originating in the neonatal forebrain subventricular zone. Glia 11:211–226; 1994. Luskin, M.B.; Boone, M.S. Rate and pattern of migration of lineally-related olfactory bulb interneurons generated postnatally in the subventricular zone of rat. Chem. Senses 19:695–714; 1994. Luskin, M.B.; Zigova, T.; Soteres, B.J.; Stewart, R.R. Neuronal progenitor cells derived from the anterior subventricular zone of the neonatal rat forebrain continue to proliferate in vitro and express a neuronal phenotype. Mol. Cell. Neurosci. 8:351–366; 1997. Mahalik, T.J.; Finger, T.E.; Stromberg, I.; Olson, L. Substantia nigra transplants into denervated striatum of the rat: Ultrastructure of graft and host interconnections. J. Comp. Neurol. 240:60 –70; 1985. Martinez-Serrano, A.; Lundberg, C.; Bjorklund, A. Use of conditionally immortalized neural progenitors for transplantation and gene transfer to the CNS. In: Gage, F.H.; Christen, Y., eds. Isolation, characterization and utilization of CNS stem cells. New York: Springer Verlag; 1997:151– 168. Mayer, E.; Dunnett, S.B.; Fawcett, J.W. Mitogenic effect of basic fibroblast growth factor on embryonic ventral mesencephalic dopaminergic neuron precursors. Brain Res. Dev. Brain Res. 72:253–258; 1993. Menezes, J.R.L.; Smith, C.M.; Nelson, C.K.; Luskin, M.B. The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain. Mol. Cell. Neurosci. 6:496 –508; 1995. Nikkhah, G.; Cunningham, M.G.; Jodicke, A.; Knappe, U.; Bjorklund, A. Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspen-

47.

48.

49. 50. 51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

ET AL.

155

sions in the rat Parkinson model. Brain Res. 633:133–143; 1994. Okoye, G.S.; Freed, W.J.; Geller, H.M. Short-term immunosuppression enhances the survival of intracerebral grafts of A7-immortalized glial cells. Exp. Neurol. 128:191–201; 1994. O’Rourke, N.A. Neuronal chain gangs:homotypic contacts support migration into the olfactory bulb. Neuron 16: 1061–1064; 1996. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1982. Rakic, P. Principles of neural cell migration. Experientia 46:882– 891; 1990. Raymon, H.K.; Thode, S.; Gage, F.H. Application of ex vivo gene therapy in the treatment of Parkinson’s disease. Exp. Neurol. 144:82–91; 1997. Sabate, O.; Horellou, P.; Vigne, E.; Colin, P.; Perricaudet, M.; Buc-Caron, M.H.; Mallet, J. Transplantation to the rat brain of human neural progenitors which were genetically modified using adenoviruses. Nat. Genet. 9:256 –260; 1995. Smart, I.H.M.; Sturrock, R.R. Ontogeny of the neostriatum. In: Divac, I.; Oberg, R., eds. The neostriatum. Oxford: Pergamon Press; 1979:127–146. Snyder, E.Y.; Flax, J.D.; Yandava, B.D.; Park, K.I.; Liu, S.; Rosario, C.M.; Aurora, S. Transplantation and differentiation of neural ‘‘stem-like’’ cells: Possible insights into development and therapeutic potential. In: Gage, F.H.; Christen, Y., eds. Isolation, characterization and utilization of CNS stem cells. New York: Springer Verlag; 1997:173–196. Svendsen, C.N.; Fawcett, J.W.; Bentlage, C.; Dunnett, S.B. Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium. Exp. Brain Res. 102:407– 414; 1995. Takahashi, T.; Nowakowski, R.S.; Caviness, V.S., Jr. BUdR as an S-phase marker for quantitative studies of cytokinetic behavior in the murine cerebral ventricular zone. J. Neurocytol. 21:185–187; 1992. Ungerstedt, U.; Arbuthnott, G.W. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal system. Brain Res. 24:485– 493; 1970. Wang, M.Z.; Jin, P.; Bumcrot, D.A.; Marigo, V.; McMahon, A.P.; Wang, E.A.; Wolf, T.; Pang, K. Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein. Nat. Med. 1:1184 –1188; 1995. Woods, A.L.; Hall, P.A.; Shepherd, N.A.; Hanby, A.M.; Waseem, N.H.; Lane, D.P.; Levison, D.A. The assessment of proliferating cell nuclear antigen (PCNA) immunostaining in primary gastrointestinal lymphomas and its relationship to histological grade, S1G21m phase fraction (flow cytometric analysis) and prognosis. Histopathology 19:21– 27; 1991. Yoshimoto, Y.; Lin, Q.; Collier, T.J.; Frim, D.M.; Breakfield, X.O.; Bohn, M.C. Astrocytes retrovirally transduced with BDNF elicit behavioral improvement in rat model of Parkinson’s disease. Brain Res. 691:25–36; 1995. Zigova, T.; Wiegand, S.J.; Lindsay, R.M.; Luskin, M.B.

156

Cell Transplantation ● Volume 7, Number 2, 1998

Intraventricular infusion of BDNF in adult rats increases the proliferation of cells destined for the olfactory bulb. Soc. Neurosci. Abstr. 22:980; 1996. 62. Zigova, T.; Betarbet, R.; Soteres, B.J.; Brock, S.; Bakay, R.A.E.; Luskin, M.B. A comparison of the patterns of migration and the destinations of homotopically transplanted neonatal subventricular zone cells and heterotopically transplanted telencephalic ventricular zone cells. Dev. Biol. 173:459 – 474; 1996. 63. Zigova, T.; Valadi, N.; Cai, N.; Ge, P.; Lindsay, R.M.;

Wiegand, S.J.; Luskin, M.B. The continuous proliferation of cells in the subventricular zone of the adult rat brain: Effect of intraventricular infusion of BDNF. Soc. Neurosci. Abstr. 23:316; 1997. 64. Zigova, T.; Betarbet, R.; Minnen, M.; Bakay, R.A.E.; Lindsay, R.M.; Wiegand, S.J.; Luskin, M.B. Transplantation of neuronal progenitor cells from the neonatal subventricular zone into the neonatal and adult striatum of the rat. Abstracts of the 6th International Neural Transplantation Meeting. 42; 1997.