bFGF-dependent neurospheres derived from embryonic rat spinal cord

Brain Research 874 (2000) 87–106 www.elsevier.com / locate / bres

Research report

Characterization and intraspinal grafting of EGF / bFGF-dependent neurospheres derived from embryonic rat spinal cord a, a a b a Stella Y. Chow *, Jon Moul , Chris A. Tobias , B. Timothy Himes , Yi Liu , a a b a Maria Obrocka , Lisa Hodge , Alan Tessler , Itzhak Fischer a

Department of Neurobiology and Anatomy, MCP Hahnemann University, 3200 Henry Avenue, Philadelphia, PA 19129, USA b Department of Veteran Affairs Medical Center, Philadelphia, PA 19104, USA Accepted 25 April 2000

Abstract Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague–Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague–Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.  2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Transplantation Keywords: Precursor; Progenitor; Stem cell; Transplantation; BDNF; Retinoic acid

1. Introduction Intraspinal grafts of fetal tissue, peripheral nerve, primary cells or cell lines have been used in experiments to repair spinal cord injury. Grafts can act as a bridge for regenerating host axons, transplanted neurons can act as a relay between regenerating host axons and denervated host neurons, and molecules presented by transplanted tissue can be neuroprotective, rescuing host neurons that would otherwise die [60,61]. The trophic influences provided by transplanted cells may stimulate regenerative sprouting, diminish the immune response and reduce the glial scar. *Corresponding author. Tel.: 11-215-842-4635; fax: 11-215-8439082. E-mail address: [email protected] (S.Y. Chow).

Transplants of fetal CNS tissue or peripheral nerve, however, induce little regeneration into the host and permit only limited functional recovery. Optimal repair and recovery after CNS injury may require combinations of different factors to stimulate axonal growth and protect injured neurons. These factors may include neurotrophins to stimulate axonal sprouting and elongation and to protect injured neurons, molecules that will neutralize axonal growth inhibitors, provide a permissive extracellular environment, and ameliorate the toxic environment at the lesion site. Ex vivo gene therapy is a promising approach for improving spinal cord grafts since cells can be modified to supply factors needed for repair. With this strategy, cultured cells are genetically modified to express the necessary therapeutic gene products, such as neurotrophins, and

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02443-4

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then transplanted into the injury site where they can act both as a source of factors that support repair and as a bridge for regenerating host axons. Recently, intraspinal transplants of genetically modified primary fibroblasts have been shown to induce regeneration of host axons, as well as improving functional recovery after spinal cord injury [16,36]. Transplants of cells that can be genetically modified and also differentiate into neural cell types offer the same advantages that fibroblasts provide but in addition offer a potential for cellular replacement. CNS stem cells are defined by their ability to proliferate, self-renew, and retain the potential to generate progenitor cells that can differentiate into neurons and glia [30,38,47,65,66]. Progenitor cells have a more restricted lineage, differentiating into either neurons or glia. Precursor cells include both stem cells and progenitor cells [14,55]. Multipotent neural stem cells have been isolated from both the embryonic and adult brain [6,7,27,40,48,50,63] and spinal cord [46,64]. Treatment of these cells in vitro with specific growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or transforming growth factor-a (TGF-a) allows them to remain in the proliferative state [14]. During development, terminally differentiated neurons and glia are generated from multipotent stem cells [59]. The signals that regulate the fate and lineage commitment of these cells differ according to the specific region and developmental stage of the nervous system [4]. Such signals can have selective effects on the survival or proliferation of a subpopulation of progenitor cells, as well as instructive effects on phenotypic outcome [30,31,47,58]. For example, neural stem cells from adult mouse striatum survive and proliferate in response to EGF alone [49] or bFGF alone [17], but neural stem cells from adult mouse spinal cord only survive and proliferate when exposed to both EGF and bFGF [64]. EGF-responsive striatal stem cells do not proliferate in response to bFGF alone [49] but, when exposed to bFGF and fetal calf serum (FCS), they generate two populations of progenitor cells: a unipotent neuronal progenitor and a bipotent neuronal / astrocytic progenitor [62]. Extrinsic factors also affect phenotypic choice. Embryonic striatal cells expanded either with EGF or bFGF give rise to neurons, astrocytes, or oligodendrocytes but more astrocytes are generated when cells are expanded in the presence of EGF alone. Subsequent exposure to PDGF almost doubles the percent of bFGFexpanded cells that differentiate into neurons, while EGFexpanded cells show only a minimal increase in neuronal differentiation [24]. Optimal conditions may therefore need to be identified for each CNS region. Grafting experiments have, however, demonstrated that once these precursor cells are transplanted, many of them can respond to local environmental cues by differentiating into region appropriate cells [12]. Neural stem or progenitor cells that can be maintained in vitro in an actively proliferating state while maintaining

the capacity to differentiate into mature neurons and glia are attractive candidates for use as transplants to repair the damaged CNS. The potential to produce in vitro the desired proportions of glial or neuronal progenitors using defined extrinsic factors allows the design of cellular transplants that can fulfil specific needs in the repair of the damaged CNS. In this study we examined the proliferation and growth of embryonic rat spinal cord cells isolated in the presence of EGF and bFGF and the induction of glial and neuronal phenotypes by extrinsic factors in vitro. We then evaluated the potential of these cells as an intraspinal transplant by examining their survival and differentiation after grafting into the injured spinal cord. We found that cells isolated from the spinal cord of embryonic Sprague–Dawley and Fischer 344 rats proliferate in the presence of EGF and bFGF, can be expanded for multiple passages, and have the capacity to differentiate into neurons and glia in vitro. These cells show promise as intraspinal grafts because they survive well in injured spinal cord, differentiate into multiple cell types in vivo, are permissive for host axon growth, and are easily modified by adenoviral vectors.

2. Materials and methods

2.1. Isolation and expansion of embryonic spinal cord cells Rat embryonic day 14 (E14) spinal cord (Sprague– Dawley or Fischer 344 rats from Taconic Farms) was dissected in DMEM medium. The tissue was rinsed in Hank’s buffered saline solution (HBSS), cut into small pieces and transferred into full growth media composed of DMEM / F12 (1:1), HEPES buffer (5 mM), glucose (0.6%), sodium bicarbonate (3 mM), glutamine (2 mM), EGF (20 ng / ml, Collaborative Research), bFGF (20 ng / ml, Collaborative Research) and a defined hormone and salt mixture composed of insulin (25 mM / ml), transferrin (100 mM / ml), progesterone (20 nM), putrescine (60 mM), and sodium selenite (30 nM). The tissue was dissociated by trituration with a fire polished glass pipette and centrifuged to separate undissociated tissue. Cells were plated onto noncoated tissue culture flasks (Corning) in full growth media. Embryonic spinal cord cells initially attached to the plate. Floating neurospheres formed within 5–7 days in culture. The primary spheres were collected, mechanically dissociated, and replated to form secondary spheres. Cells were subsequently passaged every 7–10 days with a 4–5-fold expansion in the number of stem cells with each passage. The cells were cryopreserved in full media containing 10% DMSO. To examine the expansion and phenotype of single neurospheres, primary neurospheres were cultured for 13 days. Individual spheres were placed into a microcentrifuge tube with 200 ml of full growth media and dissociated

S.Y. Chow et al. / Brain Research 874 (2000) 87 – 106

into a single cell suspension. All cells from one sphere were plated together into one well of a 96-well plate [50]. The plate was examined 7 days later for the presence of secondary spheres. The secondary spheres were also isolated after 13 days in culture and the process repeated. Sphere samples at each stage were taken for immunocytochemical analysis.

2.2. Culturing EGF and bFGF-generated cells with extrinsic factors Embryonic spinal cord cells were treated with extrinsic factors to examine the ability of these factors to induce differentiation of these cells into neurons. The EGF and bFGF-generated spheres were collected, mechanically dissociated, and placed into full growth media without EGF or bFGF. One or a combination of the following factors were then added: PDGF (10 ng / ml), NT-3 (10 ng / ml), BDNF (10 ng / ml), NGF (10 ng / ml), EGF (10 ng / ml), bFGF (20 ng / ml), fetal calf serum (FCS: 0.1% or 10%), and 10 26 M all-trans retinoic acid. The cells were then plated onto poly-L-ornithine-coated coverslips in 12-well plates at a concentration of 20,000–50,000 cells per well. The cells were incubated with each factor or a combination of factors for 4 or 8 days. Media were changed every other day. At the conclusion of the experiments, cells were fixed with 4% paraformaldehyde and their phenotype analyzed immunocytochemically.

2.3. Recombinant adenovirus We used a recombinant adenovirus containing the lacZ gene under the control of the cytomegalovirus (CMV) promoter (Ad.CMV.lacZ) for genetic modification of the spinal cord cells. The virus contained deletions in the E1a and part of the E1b regions (mu 1–9) in order to make it replication defective [35].

2.4. Preparation of cells for transplantation For genetic modification of the cells with an adenovirus, the spinal cord spheres were dissociated into single cells 24–48 h before grafting and placed into a centrifuge tube at a concentration of 5–10310 6 cells in 3 ml of full growth media. Stock Ad.CMV.lacZ (2310 10 pfu / ml) was used to infect the cells with a final pfu / cell ratio of 50–100 pfu / cell. The cells were incubated at 378C for 1 h, gently shaken every 15 min, and then plated onto tissue flasks at an approximate concentration of 1310 6 cells / 4 ml in full growth media for 24–48 h. The viability of the cells was determined following the infection procedure by using Trypan blue exclusion. To label cells with the nuclear dye bisBenzimide (1 mg / ml, Sigma Aldrich, Irvine, UK), spinal cord spheres were treated 24 h before surgery as described [39].

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On the day of surgery, cells were centrifuged, washed with HBSS, centrifuged, dissociated into single cells, resuspended in full growth media, and counted. The concentration of viable cells ($90% viable cells), as determined by Trypan blue exclusion, was adjusted to 10 5 cells / ml. The cells were maintained on ice during surgery. After each surgery, some of the remaining cells were stained with Trypan blue (Sigma Aldrich) to verify viability, and the rest were replated and stained by X-gal histochemistry to verify transgene expression or for in vitro immunocytochemical analysis.

2.5. Immunosuppression of Sprague–Dawley rats with cyclosporin A ( CsA) CsA (Sandoz Pharmaceuticals, East Hanover, NJ) was injected subcutaneously at a dose of 1 mg / 100 g body weight into Sprague–Dawley rats. Daily CsA injections began 2–4 days before the transplantation procedures and continued for the next 2 weeks. Oral CsA solution (Sandoz) was then administered via the drinking water (50 mg / ml) and throughout the survival period. Fischer 344 rats did not receive CsA treatment since they are an inbred strain of rats and do not require immunosuppression.

2.6. Transplantation animal groups A total of 32 female Sprague–Dawley (SD) rats weighing 225–275 g and 19 (15 females and four males) Fischer 344 (F344) rats weighing 175–225 g (Taconic Farms) received cell transplants. One group of 25 rats (16 SD, nine F344) received a partial hemisection that completely disrupted the lateral funiculus, part of the ventral funiculus and gray matter and a transplant of cells alone. A second group of 26 rats (16 SD, 10 F344) received a partial hemisection and a transplant of cells in gelfoam soaked with recombinant human BDNF (1.5 mg / ml). BDNF was used because it has previously been shown to improve the permissiveness of a fibroblast cell graft for axonal regeneration [36]. The animals were sacrificed and spinal cords examined at 1-, 2-, and 4-month survival times (see Table 1).

2.7. Surgical procedures The surgical procedure for a subtotal hemisection has been described elsewhere [36]. Briefly, rats were anesthetized with an intraperitoneal (i.p.) injection of acepromazine maleate (0.5 mg / kg, Fermenta Animal Health, Kansas City, MO), ketamine (63 mg / kg, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (6.3 mg / kg, Bayer, Shawnee Mission, KS), and the C4 or T8 spinal segment was exposed by a laminectomy. The dura was cut and the dorsolateral portion of the right side of the C4 or T8 segment was removed by gentle aspiration. For T8 spinal transplants, a piece of gelfoam was placed in the

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90 Table 1 Animal surgery groups Prelabeling

Rat strain

C4 transplant

1 month survival

2 month survival

bisBenzamide

Sprague– Dawley

Spinal cord cells alone Spinal cord cells1BDNF Spinal cord cells alone Spinal cord cells1BDNF

n56

n510

n56

n510

Fischer 344

b-Galactosidase

Fischer 344

n55 n55

T8 transplant

4 month survival

Spinal cord cells alone

n54 males

n55

cavity and 10 ml of cells (50,000 cells / ml) were injected into the gelfoam. For C4 spinal transplants, a piece of gelfoam soaked with cells alone or cells with BDNF (10 5 cells / ml) was implanted into the cavity and immediately an additional 5 ml of cells suspended in media (50,000 cells / ml) were slowly injected into the gelfoam. The dura was closed with interrupted 10-O sutures (Ethicon), and the muscle and skin were closed in layers. Immediately following the completion of the surgery procedure, all rats received a single bolus intravenous injection of methylprednisolone (30 mg / kg, Pharmacia & Upjohn, Kalamazoo, MI) through the tail vein. After the surgery, animals were kept on heating pads, closely observed until fully awake and then returned to their home cages. All procedures were approved by MCP/ Hahnemann University’s institutional animal welfare committee in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals.

2.8. In vitro histology and immunocytochemistry Cell cultures were fixed with 4% paraformaldehyde in PBS (140 mM NaCl, 2.6 mM KCl, 8 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 0.2 mM thimerasol; pH 7.4) for 30 min and rinsed three times with PBS buffer, pH 7.2. For immunocytochemical staining using peroxidase conjugated secondary antibodies (see below), the coverslips were then placed in a culture dish with PBS, heated in a 608C water bath for 30 min, and treated with a 3% H 2 O 2 solution in methanol to inactivate endogenous peroxidase activity. This step was omitted for coverslips that were to be processed for fluorescence staining. All coverslips were then rinsed with PBS, permeabilized with 0.2% Triton X-100 (Thomas Scientific) for 5 min, rinsed with PBS, and incubated with 10% goat serum in PBS for 30 min. The coverslips were not treated with Triton X-100 when cells were immunolabeled with O1, an antibody against a surface membrane protein. Primary antibodies (Table 2) were added and incubated for 30 min, followed by three

rinses with PBS, and incubation with the appropriate secondary antibodies (30 min). Appropriate peroxidaseconjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgM and IgG, Jackson ImmunoResearch Laboratories) were used at 1:200 dilution. Coverslips were washed for 15 min with 0.05 M Tris buffered saline, incubated with diaminobenzidine (DAB, Sigma Fast) for 5–10 min, and dehydrated by increasing concentrations of ethanol and then Hemo-De (Fisher). Following dehydration, coverslips were attached to glass slides with DPX. For fluorescent reactions, sections were incubated with fluorescent secondary antibodies, including fluorescein (FITC)-conjugate donkey anti-rabbit IgG(H1L), Texas red-conjugate donkey anti-rabbit IgG(H1L), FITC-conjugate goat anti-mouse IgG1IgM, and Texas red-conjugate goat anti-mouse IgG (H1L) purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, diluted 1:100). Coverslips were placed on glass slides with vectashield containing the nuclear dye DAPI (Vector). To control for nonspecific background staining, some reactions were conducted omitting either the primary or secondary antibody.

2.9. X-gal histochemistry This procedure has been described in detail elsewhere [32]. Briefly, cells were fixed in PBS with 0.5% glutaraldehyde for 10 min, rinsed three times (10 min first wash, 5 min next two) with PBS followed by incubation in X-gal reagent (Molecular Probes, 1 mg / ml final concentration) with X-gal mixer (35 mM K 3 Fe(CN) 6 , 35 mM K 4 Fe(CN) 6 ?3H 2 O, 2 mM MgCl 2 in PBS) at 378C overnight.

2.10. In vivo immunocytochemistry Rats were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) solution, pH 7.4. Spinal cord segments containing the injury sites were dissected, rinsed in 0.1 M PB solution, and placed into 0.1 M PB solution containing 30% sucrose and thimerosal (0.2 g / l) for 48 h at 48C. The spinal cord tissue was then frozen in embedding media (O.C.T. compound, Tissue-Tek) and serially sectioned on a freezing microtome (20 mm sections). Immunocytochemical procedures were performed as previously described [32]. Tissue sections were washed three times in PBS, permeabilized with 0.2% Triton X-100 for 5 min, and incubated for 1 h with 10% normal goat serum (NGS) in PBS. The sections were then incubated with the appropriate primary antibody (Table 2) overnight in 5% NGSPBS at room temperature. The following day, sections were washed five times (5 min each) in PBS and incubated with appropriate secondary antibodies for 2 h. Tissue sections that were to be visualized with diaminobenzidine (DAB, using the Vectastain ABC Kit from Vector Labs,

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Table 2 Primary antibodies Primary antibodies

Antibody selectivity

Type

Dilution

Sources

Anti-b-gal Anti-RT-97

b-Galactosidase Phosphorylated neurofilament (neurons) Serotonin

pAb IgG mAb IgG1

1:1000 1:100

pAb IgG

1:10,000

Calcitonin gene related peptide Cholineacetyltransferase Glial fibrillary acidic protein (astrocytes) Glial fibrillary acidic protein (astrocytes) Immature phenotype

pAb IgG

1:10,000

pAb IgG

1:500

pAb IgG

1:500

mAb IgG

1:500

pAb IgG

1:1000

Immature phenotype

mAb IgG

1:1000

pAb IgG

1:1000

pAb IgG

1:1000

Black et al., 1994 [2]

Anti-NGF

Microtubule associated protein-2 (stains dendrites in mature neurons) Microtubule associated protein-1B (stains axons in neurons) Nerve growth factor

59→39 Inc., Boulder, CO Boehringer Mannheim, GmbH, Germany Eugene Tech., Ridgefield, NJ Peninsula Laboratories Inc., Belmont, CA Incstar, Stillwater, MN Biomedical Technologies Inc., Stoughton, MA Boehringer, Mannheim Drs. M. Marvin and R. McKay Developmental Studies Hybridoma Bank, Univ. of Iowa Fischer et al., 1991 [8]

mAb IgG

1:20

Anti-Substance P

Substance-P

pAb IgG

1:1000

Anti-RIP

Oligodendrocytes (in vivo) Oligodendrocytes (in vitro) g-Amino butyric acid Tyrosine hydroxylase

mAb IgG mAb IgM

hyb. Sup. 1:2 1:40

pAb IgG

1:5000

Boehringer Mannheim GmbH, Germany Peninsula Laboratories Developmental Studies Hybridoma Bank, Univ. of Iowa A gift from Dr. C. Dyer University of Pennsylvania Sigma, St. Louis, MO

pAb IgG

Undiluted

Anti-5-HT Anti-CGRP Anti-ChAT Anti-GFAP Anti-GFAP Anti-Nestin Anti-Nestin (Rat-401) Anti-MAP2 Anti-MAP1B

Anti-O1 Anti-GABA Anti-TH

Burlingame, CA) were then washed three times with PBS, incubated for 2 h in the ABC reagent, washed again for 5 min with PBS, washed for 15 min with 0.05 M Tris buffered saline (pH 7.6), and reacted with DAB. Sections were briefly rinsed with distilled water, dehydrated with increasing ethanol and Hemo-De (Fisher), and coverslipped with DPX. For fluorescent reactions, sections were washed three times (5 min each) with PBS following incubation with fluorescent secondary antibodies, including fluorescein (FITC)-conjugate donkey anti-rabbit IgG(H1 L), Texas red-conjugate donkey anti-rabbit IgG(H1L), FITC-conjugate goat anti-mouse IgG1IgM, and Texas red-conjugate goat anti-mouse IgG(H1L) (diluted 1:100, from Jackson ImmunoResearch Laboratories). Sections were coverslipped with vectashield without DAPI. To control for nonspecific background, some reactions were conducted omitting either the primary or secondary antibody. For X-gal histochemistry, slides containing tissue sections were rinsed three times (5 min each) with PBS and stained with X-gal reagent as described above for in vitro staining.

A gift from Dr. L. Iacovitti, Thomas Jefferson University

2.11. Primary antibodies See Table 2.

2.12. In vitro and in vivo cell counting In vitro: immunopositive cells were visualized with a Leica DMRBE microscope and counted using either brightfield (DAB staining) or fluorescence microscopy at a magnification of 1003 or 2003 within a field of standardized area (SA51.09310 6 mm 2 (1003); 2.7310 5 mm 2 (2003)) which was determined with a micrometer. For each experiment, a minimum of three coverslips were immunostained with one or more antibodies. Immunopositive cells were counted within several randomly selected fields on each coverslip (100–300 cells / coverslip). The number of immunopositive cells was divided by the total number of surviving cells, determined by Nomarski optics and DAPI staining, to calculate the percentage of immunopositive cells in the culture. The total number of cells on each coverslip was compared to the number of cells

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originally cultured to determine the percentage of cells that survived. In vivo: every fifth section of each spinal cord lesion site was stained with cresyl violet and the area occupied by the grafted cells was measured using NIH image software under 253 magnification. Average graft area was calculated from the measured sections and multiplied by the thickness of the graft (estimated from the total number of sections containing the graft) in order to approximate the total volume. To count the cells, images were taken at 2003 of 2–3 non-overlapping areas of the graft region on every fifth section (|3 images per section). A grid of 36 fields was generated using Adobe Photoshop and placed over each image, six random fields were chosen within the image, and bisBenzimide labeled cells were counted under fluorescent microscopy at 2003 magnification. The estimated number of cells for each slide was calculated by taking the number of cells counted in the sample fields of each section times the graft area divided by the area occupied by the sample fields: cells / slide 5 Sc 3 A g /A s . Then the total number of cells in the graft was determined according to Konigsmark [28].

2.13. Image analysis Digital images were captured using a Photometric Sensys KAF-1400 CCD camera (Photometric Inc.) attached to a Leica DMRBE microscope. Images were processed on a Macintosh Power PC 8500 with IP Lab Spectrum (Scanalytics) and NIH image analysis software packages.

2.14. Statistics In vitro: the total number of cells counted, percent of each cell phenotype, and percent survival were defined for each growth factor condition. Cells were counted from at

least three independent cultures for each treatment. The percentages for each experiment were averaged, the standard error of the mean was calculated, and all statistical analyses were performed using Microsoft Excel software (Microsoft, Redmond, WA).

3. Results

3.1. In vitro 3.1.1. Isolation, expansion, and storage of embryonic spinal cord cells E14 spinal cords were dissociated and plated in medium containing bFGF and EGF. The dissociated spinal cord cells rapidly proliferated and the dividing cells aggregated and formed free-floating spheres (Fig. 1A). These spheres resembled the neurospheres described previously for adult murine forebrain subependymal and spinal cord stem cells [49,64]. The majority of cells within the sphere expressed the intermediate filament protein, nestin, a marker for neural precursor cells [69,70] (Fig. 1B), but were not immunopositive for neuronal or glial markers. This nestin staining of the neurospheres was seen at all time points examined (5–13 days in culture) and in early and late passages. The cells were expanded by mechanically dissociating the neurospheres into single cell suspensions and cultured in flasks with full growth media. The single cells proliferated and formed new neurospheres within 5–7 days. Cells from Sprague–Dawley rats were expanded every 7–10 days for over 20 passages. During the first five passages, they expanded 4–5-fold. A crisis developed between passages 5 and 7, when the number of viable cells decreased dramatically. With careful harvesting of the surviving neurospheres and increasing the time between passages to 12–15 days, viable spheres continued to grow.

Fig. 1. E14 spinal cord cells. (A) Typical neurosphere generated from a single cell in medium containing EGF and bFGF for 10 days, under Nomarski optics. (B) Fluorescent image of a neurosphere expanded for 5 days showing that cells were immunopositive for nestin, an intermediate filament characteristic of undifferentiated cells, throughout the neurosphere. Scale bars: 25 mm.

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Once the cells passed this critical period, they again expanded an average of 4–5-fold every 7 days until passage 19–20. Their rate of expansion then progressively decreased. By passage 21, while the cells survived and proliferated, the total number of viable cells remained the same at each successive passage (Table 3). We also isolated spinal cord cells from E14 Fischer 344 rats. Fischer rats are an inbred strain and immunosuppression is not required when Fischer 344 cells are transplanted as allografts into a Fischer 344 host [5]. Fischer 344 cells were also expanded in the presence of EGF and bFGF, passaged every 7–10 days, and showed a 4–5-fold increase in viable cell numbers with every passage until passage 5. The rate of their expansion then progressively decreased and by passage 9, no neurospheres survived. Therefore unlike the Sprague–Dawley spinal cord cells, the Fischer 344 spinal cord cells did not survive beyond the critical period between passages 5 and 8 under our culture conditions (Table 3). Stocks of both Sprague–Dawley and Fischer 344 embryonic spinal cord cells were collected and cryopreserved. Neurospheres were pelleted and resuspended in full growth media containing 10% DMSO, frozen and stored in liquid nitrogen. The embryonic spinal cord cells remained viable for up to 2 years. Dissociated cells from thawed neurospheres proliferated and reformed neurospheres that were positive for nestin. Proliferating undifferentiated cells can therefore be isolated from embryonic rat spinal cord, expanded by multiple passages and remain viable after storage in liquid nitrogen. These are important criteria for selection of appropriate cells for grafting experiments.

3.1.2. Properties of embryonic spinal cord cells Single primary neurospheres were dissociated and all cells from a single sphere were plated into one well of a 96-well plate containing medium with EGF and bFGF. After 5–7 days the dissociated cells reformed individual secondary neurospheres which were positive for nestin, similar to primary neurospheres. Cells dissociated from secondary neurospheres divided and formed nestin-positive Table 3 Expansion index of spinal cord stem cell neurospheres a Passage number

P1–P4

P5–P8

P9–P19

$P20

Sprague–Dawley

4.760.7 n510 4.560.7 n55

2.260.3 n55–6 1.460.4 n55

4.660.6 n55–6 No survival

1.0 n55 No survival

Fischer 344 a

Stem cells were isolated from E14 Sprague–Dawley (n511) and Fischer 344 (n55) rat spinal cords, and passaged weekly by dissociation of neurospheres. Stem cells from both strains showed a 4–5-fold expansion during the first four passages. By P5–8, both strains reach a critical period at which cell survival dramatically decreased. Fischer 344 stem cells did not survive beyond this point. However, Sprague–Dawley stem cells recover from this critical period and once again expand 4–5-fold every passage until P20. From this point on, the cells continue to survive but no longer expand exponentially.

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tertiary neurospheres. As proliferating neurospheres, these cells remain nestin-positive and do not exhibit glial or neuronal phenotypes (Fig. 1). When single primary spheres were dissociated, plated onto poly-L-ornithine-coated glass slides and incubated for 8 days without EGF and bFGF, but with 10% FCS, the spheres generated neurons, astrocytes and oligodendrocytes. This was also true for secondary spheres derived from single primary sphere dissociations. We also examined spheres taken from passage 2–4 that were dissociated and cultured in medium containing 10% FCS without EGF or bFGF for 4 or 8 days to induce differentiation. We used this condition as our standard treatment, which provided the comparison group for other experiments. At both 4 and 8 days, the cells showed morphologies characteristic of neuronal or glial cells. Cell phenotypes were then examined by staining with antibodies specific for neurons (MAP1B, MAP2), astrocytes (GFAP), oligodendrocytes (O1), and immature cells (nestin). Neuronal cells exhibited axonal and dendritic polarity, with large axons intensely stained for MAP1B (Fig. 2A and D) and many short dendrites stained by MAP2 (Fig. 2B and E). GFAP immunostaining identified both commonly seen astrocyte morphologies, flat (Fig. 2C) and stellate (Fig. 2F). Very few cells were oligodendrocytes (Fig. 2G). The majority of the cells remained nestinpositive (Fig. 2H) and many of these also showed an astrocytic morphology. A subpopulation co-expressed GFAP and nestin (Fig. 3A and B). These results show that E14 spinal cord cells generated in the presence of EGF and bFGF are multipotent. However, while they can differentiate into neurons and glial cells, many of them remain nestin-positive, and are thus presumably immature. This pattern was maintained in both primary and secondary neurospheres, as well as later passaged cells, as described below.

3.1.3. Comparison of early and late passaged embryonic spinal cord cells in 10% FCS It is important to show that the embryonic spinal cord cells from different passages and different strains do not change their properties over time or following long-term storage. Cells from Sprague–Dawley and Fischer 344 passages 2–4 (early) (Fig. 4A and C) were compared to late passaged Sprague–Dawley cells (10–13 passages) (Fig. 4B). Because Fischer 344 cells did not survive beyond passage 8, only the early passages were examined. Cells were grown in the presence of 10% FCS, without EGF and bFGF, for 4 or 8 days. The percentage of cells positive for MAP1B, O1, and nestin was similar in early and late passages of Sprague–Dawley cells at 4 and 8 days (Fig. 4A and B). Less than 4% were MAP1B- or O1positive and 65 to 75% were nestin-positive. The percentage of GFAP-positive cells differed in the early and late passages; early passages had 17–30% (4 or 8 days) GFAP-positive cells, which increased to 50% in the late passages. Fischer 344 spinal cord cells differentiated in a

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Fig. 2. Representative photomicrographs illustrating cell phenotypes of E14 Sprague–Dawley spinal cord cells treated for 4 or 8 days with 10% FCS. Cells were fluorescently immunostained for (A, D) MAP1B, (B, E) MAP2, (C,F) GFAP, (G) O1, and (H) nestin, and demonstrated the morphology of neurons (A, B, D, E), astrocytes (C, F), oligodendrocytes (G) and immature cells (H). Although all these cell types were identified in each culture, most cells in this standard culture condition were nestin- and / or GFAP-positive (see Figs. 3 and 4). The GFAP-positive cells showed either a flat (C) or stellate (F) morphology. The few neurons identified exhibited axonal and dendritic polarity with large axons stained by MAP1B (A, D) and short dendrites stained by MAP2 (B, E). Scale bars: 50 mm.

Fig. 3. Fluorescent images of E14 Sprague–Dawley spinal cord cells treated for 8 days with 10% FCS. The cells were double-labeled for the expression of (A) GFAP and (B) nestin. Most cells were nestin-positive, but a subpopulation of cells co-expressed GFAP and nestin (arrows). Scale bars: 50 mm.

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Fig. 4. The percentage of different cell phenotypes of spinal cord precursor cells at early (2–4) and late (10–13) passages following 4- or 8-day treatment with 10% FCS. Sprague–Dawley precursor cells at early (A) and late (B) passages and (C) Fischer 344 precursor cells at early passages were immunostained for MAP1B (neurons), GFAP (astrocytes), O1 (oligodendrocytes), and nestin (immature cells). Cell phenotypes present in Sprague–Dawley and Fischer 344 precursor cells at early passages (A and C) did not differ significantly. The percentage of astrocytes increased at both 4 and 8 days in the late passaged Sprague–Dawley cells (P,0.05, asterisks), as compared to early passaged Sprague–Dawley or Fischer 344 cells. The majority of cells were nestin- and / or GFAP-positive for both strains of animals and at all passages examined.

fashion that closely resembled that of Sprague–Dawley cells from early passages (Fig. 4A and C). Examination of cells that had been cryopreserved showed no differences compared to cells that were continuously cultured (data not shown). These data demonstrate that embryonic spinal cord cells maintain their ability to differentiate into multiple cell types as they are passaged, but their propensity to differentiate into astrocytes increased, at least for the Sprague– Dawley cells, in the later stages.

3.1.4. The effect of extrinsic factors on embryonic spinal cord cells To study the influence of extrinsic factors on cell phenotype, E14 Sprague–Dawley spinal cord cells were dissociated between passages 9 and 13 and plated in

defined medium with one or a combination of several extrinsic factor(s). We used the late passaged cells since it was important for us to examine the effects of extrinsic factors on cells that have been expanded long-term. These later passaged cells are likely to be the cells that will be utilized for spinal cord transplants. Extrinsic factor(s) was added on the day the cells were plated (day 0) and replenished on alternate days. Embryonic spinal cord cells cultured with medium containing 10% FCS, which gives a low percentage of neurons, were used as a control (Fig. 4). Spinal cord cells were treated with a low concentration of FCS and / or several different extracellular factors, including PDGF, NGF, NT-3, BDNF, bFGF, EGF, and retinoic acid (RA). Treatment with PDGF, NGF, NT-3, BDNF, or bFGF did not appear to significantly affect

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differentiation of the spinal cord cells. Most cells remained nestin-positive, and there were only a few identified neuronal or glial cells (data not shown). These factors were not further investigated. The treatments that had the most significant effects on cell phenotype differentiation were 0.1% FCS, EGF and RA. When cells were grown in medium containing 0.1% FCS, 69.7% of the spinal cord cells remained nestin-positive at 4 days and very few cells expressed MAP1B, GFAP, or O1 (Fig. 5A). At 8 days there was a substantial increase in GFAP-positive cells (from 9 to 50.6%) but few cells expressed MAP1B or O1. These GFAP-positive cells exhibited both stellate and flat morphologies. Survival was relatively unaffected after 4 days compared to the control (cells treated with 10% FCS) but at 8 days in culture, the survival decreased by half (Fig. 7). When the spinal cord cells were grown in medium containing only EGF for 4 or 8 days, more than 90% of the cells remained nestin-positive (Fig. 5B). Less than 2% of the cells expressed either GFAP or O1 (Fig. 5B), and the few GFAP-positive cells were stellate in appearance. The percentage of MAP1B-positive cells did not differ from cells treated with 10% FCS at 4 days, but this number increased by threefold at 8 days and became significantly

higher than for cells treated with 10% FCS (P,0.03, compare Figs. 4B and 5B). Mature neurons were not present since MAP2 immunolabeling was not detected. There was approximately a 30% decrease in cell survival when treated with EGF for 4 or 8 days as compared to 10% FCS (Fig. 7). EGF-treated cultures observed prior to fixation revealed that some cells remained as free floating round cells. This may reflect the mitogenic properties of EGF. Since these cells did not adhere to the plate, they were lost when the medium was changed which probably contributed to their apparently poor survival. When cells were grown in medium containing RA alone for 4 days, more than 40% were neuronal, i.e. MAP1Bpositive (Fig. 6A). As with EGF treatment, we did not detect MAP2-positive cells. There was also a significant increase in oligodendrocytes, but the percentage of cells expressing nestin was similar to the 10% FCS control (compare Figs. 4B and 6A). The population of cells expressing GFAP was ,4% (Fig. 6A); as with cells treated with EGF, these GFAP-positive cells were stellate in shape. After 4 days in the presence of RA, almost 30% of the spinal cord cells were still viable, but the cells did not survive at 8 days (Fig. 7). Retinoic acid has been reported to induce apoptosis, which may account for the

Fig. 5. The percentage of different phenotypes of Sprague–Dawley spinal cord cells treated with 0.1% FCS or EGF. Cells were treated with (A) 0.1% FCS or (B) EGF (10 ng / ml) for 4 or 8 days. Most cells remain nestin-positive at both 4 and 8 days with either treatment. Treatment with 0.1% FCS increased the percentage of astrocytes at 8 days (P,0.001, asterisk). Although the percentage of neurons remained low, there was a significant increase in neurons at 8 days with the EGF treatment as compared to 10% FCS (P,0.03, see Fig. 4). The percentage of oligodendrocytes was low under all conditions.

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Fig. 6. The effect of retinoic acid on the percentage of different phenotypes of Sprague–Dawley spinal cord cells. The cells were treated with (A) retinoic acid (RA), (B) RA and EGF (10 ng / ml), and (C) RA and 0.1% FCS for 4 or 8 days. Over 40% of the cells expressed MAP1B (neurons) and 20% were positive for O1 (oligodendrocytes) when treated with RA alone at 4 days. Most of these cells were nestin-positive (immature cells). Very few cells were GFAP-positive (astrocytes). Cells did not survive at 8 days with this treatment. (B, C) Combining RA treatment with EGF or 0.1% FCS improved the survival of these cells at 8 days. Both combined treatments yielded an even greater percentage of neurons, 65–85% (B, C) with small numbers of astrocytes and oligodendrocytes at both 4 and 8 days. A subpopulation of MAP1B immunopositive cells also double-labeled with nestin. All three RA treatments significantly increased the percentage of neurons as compared to 10% FCS (P,0.001 for each treatment, see Fig. 4).

reduced survival in these cultures [20,21]. This suggests that a careful balance is needed to promote differentiation and survival. Because both RA and EGF treatment promoted a marked increase in the percentage of neurons compared to the 10% FCS control, the cells were treated with a combination of the two factors in an attempt to further increase the population of neurons. This had a dramatic effect. When treated with the combination of EGF and RA, the spinal cord cells gave rise to 67.7% MAP1B-positive cells at 4 days and 83.9% MAP1B-positive cells at 8 days (Fig. 6B). There were no MAP2-positive cells. The percentage of both astrocytes and oligodendrocytes was

,8% at each time point. Over 90% of the cells still expressed nestin, similar to cells treated with only EGF (compare Figs. 5B and 6B). Again, as with RA treatment alone, the survival was very low (from 10% to 17%) (Fig. 7). Fetal calf serum was then combined with RA in an attempt to increase survival of the cells. When the cells were grown in 0.1% FCS and RA, 60% of the cells at 4 days and 72% at 8 days were neuronal (MAP1B and MAP2 immunopositive) (Fig. 6C). More than 65% of the cells were nestin-positive (Fig. 6C) but only a few cells expressed GFAP or O1. Those cells grown with RA and either EGF or 0.1% FCS that differentiated and expressed MAP1B did not have a mature neuronal morphology. A

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Fig. 7. The percent survival of embryonic spinal cord cells at 4 or 8 days following treatment with extrinsic factors. The initial number of cells plated was between 2310 4 and 5310 4 cells / well. The number of viable cells after 4 or 8 days was counted and divided by the initial plating density to determine survival. Cells were counted from at least 12 coverslips and averaged to determine survival.

subpopulation of these MAP1B-positive cells was also nestin-positive. No MAP2-positive cell double-labeled with nestin. Cell survival after 4 days with RA and 0.1% FCS was almost twice that for cells grown only in RA, but by the eighth day survival was reduced to 15% of the original cell population (Fig. 7). Together, these results show that E14 spinal cord cells grown in the presence of EGF and bFGF differentiate primarily into astrocytes when exposed to FCS and into neurons when exposed to RA.

3.1.5. Neuronal phenotypes of retinoic-acid-treated embryonic spinal cord cells Immunofluorescent staining was used to identify the phenotypes of neurons in the cultures treated with RA and either EGF or 0.1% FCS. The spinal cord cells were immunolabeled with several antigen markers, including those for GABA, Substance P, TH, CGRP, low affinity NGF receptor (p75), ChAT, and 5-HT (Table 2). Many of the cells with a neuronal morphology were positive for GABA expression (Fig. 8A, B, E and F). A few cells were positive for ChAT (Fig. 8C and D), but only after treatment with both RA and EGF. None of the cells were immunopositive for Substance P, TH, CGRP, low affinity NGF receptor, or 5-HT in any treatment condition. 3.2. In vivo 3.2.1. Survival and differentiation of embryonic spinal cord cells transplanted into the injured spinal cord To examine the potential usefulness of the embryonic spinal cord cells as an intraspinal transplant, these cells alone or with addition of exogenous BDNF were transplanted into a partial hemisection cavity aspirated in the C4 or T8 spinal cord. The cells were labeled either by the nuclear dye bisBenzimide or by a recombinant adenovirus containing the lacZ reporter gene (Ad.CMV.lacZ). The adenovirus readily infected the cells. In initial experiments,

the cells were infected at a ratio of 200 pfu / cell. This concentration of the virus caused 66% cell loss 48 h following the infection procedure. When the infection ratio was reduced to 50–100 pfu / cell, cell loss was reduced to 15% 48 h following the infection procedure. With an infection ratio of 50–100 pfu / cell, 90–100% of the cells expressed b-gal transgene product (Fig. 9). We used an infection ratio of 50–100 pfu / cell in all subsequent infections. We have also infected the spinal cord cells with the recombinant adenovirus Ad.CMV.green fluorescent protein (Ad.CMV.GFP) with similar efficiency (data not shown). In our initial experiments, Sprague–Dawley rats received only oral CsA for immunosuppression and did not receive methylprednisolone. Some cells survived at 1 week, but at 2 weeks and 1 month post-transplantation, survival was poor (data not shown). Several strategies improved cell survival in subsequent experiments. (1) Delaying transplantation to 1 week after injury and transplantation of cells in combination with other cells that may provide neurotrophic support, such as primary embryonic spinal cord cells (data not shown). (2) Using Fischer 344 rats as a source of spinal cord cells and as hosts. Fischer 344 embryonic spinal cord cells infected with Ad.CMV.lacZ survived for over 4 months and continued to express the lacZ transgene when transplanted into hemisected Fischer 344 rats that were not immunosuppressed (Fig. 10A). (3) Including parenteral CsA in our immunosuppression protocol. It has been reported that the serum level of CsA is decreased 24 h post spinal cord injury in rats when CsA is given orally (by gavage) [22]. This decrease in serum CsA levels combined with decreased intake of water during the recovery period may provide insufficient levels of immunosuppression immediately following our spinal cord surgery. We found the best survival and integration of transplanted spinal cord cells into Sprague–Dawley rats that received a bolus injection

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Fig. 8. Fluorescent images of neuronal cell types found in cultures of RA and EGF or 0.1% FCS treated cells. Spinal cord cells were grown in RA with either EGF (A, C) or 0.1% FCS (B) for 4 days or 8 days. Cells were immunolabeled for GABA (A, 8 days; B, 4 days) or ChAT (C, 4 days). Both GABAand ChAT-positive cells were found when cells were treated with RA and EGF, but only GABA-positive cells were found in cells treated with RA and 0.1% FCS. Scale bars: A, 100 mm; B and C, 50 mm.

of methylprednisolone immediately following grafting, injections of CsA 2–3 days before grafting and continuing for 2 weeks, followed by oral CsA administration for the remaining survival period (Fig. 10B). This protocol produced survival of about 50% of the transplanted cells, identified by bisBenzimide labeling, which was comparable to that observed in non-immunosuppressed Fischer 344 rats even up to 2 months following grafting (Fig. 10B). Cells injected into gelfoam soaked with BDNF also survived well and filled the lesion cavity. Most of the cells remained within the grafts, but some migrated out both rostrally and caudally (up to |1–2 mm).

3.2.2. Spinal cord cell phenotypes Immunocytochemical analysis of the transplants revealed that E14 spinal cord cells differentiated into neurons, astrocytes, and oligodendrocytes in vivo, but neurons (cells double-labeled for MAP1B and bisBenzimide or MAP2 and bisBenzimide) were only present in animals supplied with exogenous BDNF at the time of the transplant (Fig. 10). Some of the bisBenzimide-labeled cells differentiated into astrocytes, based on immunoreactivity for GFAP, and oligodendrocytes, based on immuno-

reactivity for RIP (Fig. 10). Both GFAP and RIP-positive cells were present in animals with and without exogenous BDNF. The GFAP-positive cells were located within the graft, whereas RIP-positive cells were located around the periphery of the graft. In animals given exogenous BDNF, we also identified RIP and bisBenzimide double-labeled cells within the graft. In all groups, most of the cells appeared undifferentiated.

3.2.3. Axon growth in the grafts Spinal cord precursor cell transplants with exogenous BDNF in both Sprague–Dawley and Fischer 344 rats contained numerous host axons that were immunoreactive for the neurofilament antibody RT-97 (Fig. 11C). Grafts contained MAP1B-positive axons regardless of whether BDNF was administered. The axons within the grafts could have originated from either host or transplanted neurons. Some axons were immunopositive for 5-HT, indicating they were derived from the raphe nuclei of the host, and some were immunopositive for CGRP indicating they were of dorsal root origin. The 5-HT-positive axons were distributed throughout the grafts (Fig. 11A), whereas most of the CGRP-positive axons were located in the

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Fig. 9. Expression of b-gal in spinal cord cells genetically modified with a recombinant adenoviral vector. Spinal cord cells were dissociated and infected with an adenoviral vector containing the lacZ reporter gene (Ad.CMV.lacZ). The cells were transferred onto poly-L-lysine-coated glass slides, incubated for 48 h and processed for X-gal histochemistry. The cells were efficiently infected by the recombinant adenovirus and in (A), all of the cells expressed the b-gal transgene product although some are more intensely stained than others. (B) The recombinant adenovirus vector used to genetically modify the spinal cord cells. The reporter gene (lacZ) is driven by a CMV promoter. Scale bar: 50 mm.

dorsal regions of the grafts (Fig. 11D) and appeared medially near the host / graft interface in only a few animals. Transplants of spinal cord cells without supplementary BDNF also contained RT-97 and 5-HT-positive axons in both Sprague–Dawley and Fischer 344 rats, but these were much fewer than in grafts with exogenous BDNF. Immunostaining of CGRP axons did not differ with or without BDNF. These results suggest that E14 spinal cord cells are permissive for host axonal growth, but that the number and types of axons that grow into the graft can be greatly increased by the addition of BDNF.

4. Discussion We show that spinal cord cells isolated from embryonic day 14 rats and grown in the presence of EGF and bFGF, proliferate as undifferentiated cells and can be expanded over long periods of time. When induced to differentiate by extrinsic factors, they can become neurons, astrocytes, or oligodendrocytes. These cells survive when grafted into the injured spinal cord and therefore can be used as an intraspinal transplant in models of spinal cord injury. They are easily modified by adenoviral vectors, differentiate into multiple cell types in vivo, and are permissive for host axonal growth, especially when grafted in the presence of an exogenous growth factor.

4.1. In vitro 4.1.1. Isolation, and expansion of spinal cord cells Cells isolated from the embryonic spinal cord of both

Sprague–Dawley and Fischer 344 rats proliferate in the presence of EGF and bFGF. Cells from both strains can be expanded as free-floating neurospheres similar to those described for murine EGF-responsive striatal stem cells [48,50] and for EGF- and bFGF-responsive adult spinal cord stem cells [64]. The neurospheres are composed of undifferentiated cells, as indicated by nestin-positive immunostaining, and remain undifferentiated with successive passages. Isolation of a single primary neurosphere and evaluation of its progeny demonstrate that cells that compose the primary neurospheres, as well as those in the secondary neurospheres are predominantly nestin-positive, and serve as a source of tertiary neurospheres that also grow as undifferentiated cells. Following withdrawal of EGF and bFGF and addition of 10% FCS to the media, the dissociated cells of the neurospheres differentiated into phenotypes that included neurons, astrocytes, and oligodendrocytes. Because our analysis of these cells did not include clonal analysis of a single isolated cell induced to proliferate and examination of its progeny, we do not have direct evidence that the neurospheres contain multipotent stem cells. It is, however, very likely that the neurospheres are composed of a heterogeneous population of multipotent progenitor and stem cells. Rao and collaborators reported that neuroepithelial (NEP) stem cells from the embryonic rat spinal cord undergo a multi-stage process of cell differentiation [25,37,46]. Their results indicate that E10.5 NEP stem cells, which were cultured as adherent cells in the presence of bFGF and chick embryo extract, are multipotent and give rise to more restricted neuronal and glial precursors by E13.5. The neuronal precursors generate several neuronal but no glial

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Fig. 10. Spinal cord cells grafted into the injured spinal cord. (A) Fischer 344 spinal cord cells infected with the recombinant adenovirus, AD.CMV.lacZ, were grafted into the partially hemisected T8 spinal cord of a non-immunosuppressed Fischer 344 rat host. A montage of sagittal section photographs shows fluorescent immunostaining for b-galactosidase 4 months following surgery. Abundant immunoreactive product is visible. (B) Sprague–Dawley cells pre-labeled with bisBenzimide, a nuclear dye, were grafted with exogenous BDNF into the partially hemisected C3 / 4 spinal cord of an immunosuppressed Sprague–Dawley rat host. Two months following surgery, many blue stained cells remain. (C–F) Analysis of cell phenotypes expressed by Fischer 344 cells grafted with exogenous BDNF into the partially hemisected C3 / 4 spinal cord of non-immunosuppressed Fischer 344 rats. Double immunofluorescent staining shows the nuclear dye staining of the cells (blue) combined with MAP2 (C) and MAP1B (D) staining (green) or GFAP (E) and RIP (F) staining (red). Scale bars: A and B, 100 mm; C–F, 50 mm.

phenotypes in culture [26,37]. Glial precursors generate oligodendrocytes and two populations of astrocytes [46], but no neurons. By E14, cells isolated from the spinal cord are composed of more restricted progenitors or a mixed population of progenitors and multipotent stem cells. Weiss et al. [64] have shown that spinal cord stem cells can be isolated from adult mice as neurospheres in the presence of EGF and bFGF indicating that a population of multipotent stem cells remain in the spinal cord even in late stages of development.

It has been shown that culture conditions may differentially affect cells isolated from the same region in mice and rats. Embryonic day 16 Sprague–Dawley rat striatal cells grown in the presence of EGF or EGF and bFGF can only be expanded for 21–28 days after which a crisis ensues and the cells eventually die [56]. In contrast, embryonic mouse striatal cells can be maintained long-term [56], and for over 40 passages [50]. Our Fischer 344 spinal cord cells could only be maintained up to passage 9. Our isolation, growth, and passage protocols may have selected

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Fig. 11. Sagittal sections showing host axonal growth into grafts of spinal cord cells pre-labeled with bisBenzimide and transplanted into a C3 / 4 injury site. (A–C) Show the same field from a Sprague Dawley rat showing bisBenzimide labeled cells transplanted with exogenous BDNF 2 months following surgery (B), fluorescently labeled 5HT axons (A) and RT97 axons (C). (D) From a Fischer 344 rat that did not receive BDNF and shows bisBenzimide labeled cells and fluorescently labeled CGRP host axons 2 months following surgery. Numerous 5HT-positive (A) and RT97-positive (C) axons are present throughout the grafts, whereas most of the CGRP-positive axons are in the dorsal regions (D). Scale bars: 50 mm.

a more restricted progenitor cell population with limited mitogenic potential in Fischer 344 rats. Sprague–Dawley spinal cord cells can overcome the crisis period, expand for over 20 passages and then continue to be maintained at a steady state. Furthermore, late passaged Sprague–Dawley spinal cord cells continue to grow as undifferentiated nestin-positive neurospheres and to respond to different extrinsic factors by differentiating into multiple phenotypes.

4.1.2. The effect of extrinsic factors on embryonic SC precursor cells We found that 10% FCS generated more cells with astrocytic than neuronal or oligodendrocytic phenotypes and that late passaged Sprague–Dawley cells showed an

even higher proportion of GFAP immunopositive cells. It is possible that harvesting the surviving Sprague–Dawley neurospheres during the crisis period at passage 5–7, when many cells cease proliferating and adhere, selects for a subpopulation of precursor cells that are committed to an astrocyte phenotype when exposed to FCS. Indeed, recent studies with fetal human spinal cord precursor cells have shown that continued passage in EGF and bFGF can select for precursors that are restricted for the astrocytic lineage [44]. Palmer et al. [42] have shown that clones of adult hippocampal stem cells can generate both neurons and glia but differentiate primarily into astrocytes when exposed to 10% FCS. Although exposure to 10% FCS increases the proportion of GFAP-positive cells, most of the cells remain nestin-

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positive, many of them show an astrocytic morphology but only a subpopulation co-expresses GFAP and nestin. Double-labeling of GFAP and nestin has been noted in FGF-expanded E14 neuroepithelial stem cells that were induced to differentiate into astrocytes with ciliary neurotrophic factor (CNTF) [45] and in E16 hippocampal cells grown in the presence of bFGF [63]. Co-expression of GFAP and nestin suggests that the cells are not yet fully committed to a mature astrocyte phenotype. Consistent with this idea is the report that some CNTF-induced astrocytes double-label with nestin, but those that are intensely immunostained with GFAP are nestin negative [45]. Serum may therefore direct some of the spinal cord cells towards an astrocytic pathway, but the cells may require longer exposure to the serum than our 4- and 8-day treatments or full commitment to a mature astrocyte phenotype may require additional factors. Alternatively, the cells may be committed to a lineage but have not become fully differentiated, have not ceased proliferating and thus are still in an immature state. Treatment of the spinal cord cells with RA greatly increased the percentage of cells differentiating into neurons and combined treatment with EGF and RA or 0.1% FCS and RA further increased the percentage of cells differentiating into neurons, without affecting glial differentiation. Retinoic acid also promotes neuronal differentiation of embryonic carcinoma cells [20], primary cultures of embryonic spinal cord cells [67] as well as stem / progenitor cells [37,57]. The spinal cord precursor cells become immunopositive for the neuronal marker MAP1B, and a sub-population is also nestin-positive. MAP 2, a marker for more mature neurons, was only detected in the RA and 0.1% FCS treatment, and these cells did not double-label with nestin. Similar to the GFAP and nestin double-stained cells, this suggests that in the period examined, the cells are directed towards becoming neurons, but are not yet fully committed to a mature neuronal phenotype. Perhaps longer exposure of the cells to the treatment would have resulted in a decrease in the nestin immunostaining and more cells becoming MAP2-positive. RA greatly decreased the survival of precursor cells, suggesting that RA may promote differentiation of only a selective subpopulation of neuronal progenitors or may even have adverse effects on cell survival [20,21]. Since our measurements of survival are based on the number of cells surviving at the end of the culture period compared to the initial plating concentration, we cannot address possible selective effects on proliferation and survival, or the possibility that RA may affect differentiation instructively. Future experiments are needed to examine the effects of RA treatment in combination with factors that promote both survival and proliferation of the spinal cord cells. Taken together, the results of these in vitro experiments show that spinal cord precursor cells treated with EGF and bFGF generated cells that can be expanded, maintained, cryopreserved, and can differentiate into multiple cell

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types. These cells can also be manipulated towards an enriched astrocytic population by treatment with FCS and towards an enriched neuronal population with RA.

4.2. In vivo 4.2.1. Survival and differentiation of embryonic spinal cord precursor cells transplanted into the injured spinal cord The results of the in vivo studies confirm that embryonic spinal cord cells are promising as intraspinal transplants. About 90–100% of the precursor cells exposed to the Ad.CMV.lacZ recombinant adenovirus expressed the reporter gene, demonstrating that these cells are amenable to genetic modification with adenoviral vectors. We also found robust expression of the transgene at 4 months following transplantation in non-immunosuppressed Fischer 344 rats, the longest time examined. Liu et al. [35] have shown that the host’s immune response may contribute to diminished transgene expression in chronic transplants that have been transfected by adenovirus. Therefore immunosuppression with CsA, especially our revised protocol of using injectable CsA, is likely to be important for long-term expression of transgene in outbred Sprague– Dawley rats. The ability to genetically modify these precursor cells should allow additional molecules to be supplied that would increase the potential of the transplant to promote regeneration. Indeed our results show that the precursor cells alone allow or enhance growth by host dorsal root and supraspinal serotonergic axons, but that the amount of growth of the 5-HT axons is increased when transplants of precursor cells are supplemented with exogenous BDNF. Our observation that transplanted spinal cord precursor cells differentiate into multiple cell types makes them potentially more attractive than other cells that have been used previously for transplants such as fibroblasts. Although transplants of genetically modified fibroblasts promote regeneration and recovery of motor function [16,36], they can only act as a permissive bridge for regenerating axons and as a biological pump for neurotrophic factors. Precursor cells have the additional potential to differentiate into neurons that can replace those lost during injury and into oligodendrocytes that can remyelinate regenerated and surviving but demyelinated axons. Precursor cells respond to local signals by acquiring region-specific phenotypic fates when grafted into the CNS of developing animals, and in some cases when grafted into the intact adult CNS [9,10,13,52–54]. For example, neural progenitor cells isolated from the embryonic human forebrain [10] or from the dentate gyrus of adult rat hippocampus [13] differentiated into regionally appropriate neurons when grafted into neurogenic areas of adult rat brain. Neural stem cells may also be able to distinguish among cues present in damaged regions of the CNS. For example, the immortalized neuronal precursor C-17 cell

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line appeared to replace pyramidal neurons that had degenerated following photolytic injury but differentiated into glia when grafted into the intact adult neocortex or into adult neocortex injured by kainic acid injection [52]. Reports that endogenous neural stem cells differentiate into glia after brain or spinal cord injury [11,19,23,41] suggest the presence of cues for glial specification, as did our immunocytochemical results indicating that precursor cells transplanted into damaged spinal cord differentiated into astrocytes and oligodendrocytes. Because most of the transplanted cells are immature based on nestin staining, the opportunity to influence their differentiation remains, and we observed increased differentiation into neurons following the exogenous administration of BDNF. Neurotrophic factors are known to promote neuronal differentiation and maturation of stem / progenitor cells [1,15,29,42,63]. Stem-like cells (C-17 cells) that have been genetically modified to express a neurotrophic factor (NT3) can differentiate into neurons after transplantation into the spinal cord [34,43]. This indicates that the strategy is also effective in directing transplanted cells toward a desired phenotype. Neurotrophic factors have also been shown to increase the survival of bulbospinal [33] and intraspinal [3,18] neurons after axotomy and to encourage sprouting [51] and regeneration [36,68] of supraspinal axons after spinal cord injury. Future experiments will investigate the most effective strategies for providing precursor cell transplants with a combination of factors that will promote further neuronal differentiation to replace damaged cells and encourage the survival and regeneration of injured CNS neurons. In summary, cells derived from embryonic spinal cord are amenable to genetic modification and therefore can be used to deliver therapeutic gene products into the injured spinal cord. Their robust survival and integration with the host tissue may provide the necessary bridge or relay for regenerating or sprouting axons. Their ability to differentiate into multiple cell types and to respond to exogenous factors indicates the potential to replace damaged neurons and to remyelinate regenerating axons.

Acknowledgements This work was supported by National Institutes of Health Grant NS24707 and Training Grants NS10090 and HD07467, The Spinal Cord Research Foundation of the Paralyzed Veterans Association, The Eastern Paralyzed Veterans Association, the International Spinal Cord Research Trust, a Center of Excellence Grant from Medical College of Pennsylvania / Hahnemann University, and the Research Service of the Department of Veteran Affairs. We thank Dr. Marion Murray for her suggestions and critical comments on this manuscript. We thank Theresa Connors and Kathy Bozek for their technical assistance. We thank the following people for their generous gifts: Drs. M.

Marvin and R. McKay for nestin antibody, Dr. L. Iacovitti for TH antibody, and Dr. C. Dyer for O1 antibody. The Rat-401 (nestin) antibody and RIP hybridoma cells developed by Dr. Susan Hockfield were obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-73263 from the NICHD.

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