www.elsevier.com/locate/devbrainres

Developmental Brain Resea

Research report

Mouse neural progenitor cells differentiate into oligodendrocytes in the

brain of a knockout mouse model of Canavan disease

Sankar Surendrana, Lamya S. Shihabuddinb, Jennifer Clarkeb, Tatyana V. Taksirb,

Gregory R. Stewartb, Geoffrey Parsonsb, Wendy Yangb, Stephen K. Tyringc,

Kimberlee Michals-Matalona, Reuben Matalona,*

aPediatrics Rm # 3.350, Department of Pediatrics, Childrens Hospital, The University of Texas Medical Branch, Galveston, TX 77555-0359, United StatesbGenzyme Corporation, Framingham, MA 01701, United States

cDepartment of Dermatology, University of Texas Health Center, Houston, TX, United States

Accepted 7 July 2004

Available online 19 August 2004

Abstract

Canavan disease (CD) is an autosomal recessive disorder that leads to spongy degeneration in the white matter of the brain.

Aspartoacylase (ASPA) synthesizing cells, oligodendrocytes, are lost in CD. Transplantation of neural progenitor cells (NPCs) offers an

interesting therapeutic approach for treating neurodegenerative diseases by replacing the lost cells. Therefore, the NPCs transplantation to the

brain of the CD mouse was studied. Injection of mouse NPCs to the striatum and cerebellum of juvenile CD mouse showed numerous BrdU

positive cells at 1 month after injection. The same result was also observed in the adult CD mouse brain after 5 weeks of post-transplantation

period. The implanted cells differentiated into oligodendrocytes and fibrous astrocytes, as observed using glial cell marker. This is the first

report to describe the survival, distribution and differentiation of NPCs within the brain of CD mouse and a first step toward the potential

clinical use of cell therapy to treat CD.

D 2004 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Transplantation

Keywords: Neural progenitor cell; Cell therapy; Canavan disease; Retrovirus; Knockout mouse; Oligodendrocyte; Astrocyte

1. Introduction

Canavan disease (CD) is an autosomal recessive leuko-

dystrophy, caused by defect in the aspartoacylase (ASPA).

The enzyme is synthesized by oligodendrocytes and these

cells are lost in the white matter of the brain in CD. The

ASPA deficiency leads to accumulation of N-acetylaspartate

(NAA) in the brain [11]. The clinical symptoms of CD

include megalencephaly, hypotonia, mental retardation and

early death [37]. The knockout mouse model for CD showed

0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2004.07.003

* Corresponding author. Tel.: +1 409 772 3466; fax: +1 409 772 9595.

E-mail address: rmatalon@utmb.edu (R. Matalon).

similar abnormalities to those in patients with CD including

aspartoacylase deficiency, accumulation of NAA and spongy

degeneration of the brain [13,32,33]. The animal model gave

information to understand molecular and pathophysiological

mechanisms involved in CD, and to evaluate potential

therapies for the treatment of this disease [14,32–34].

At present, treatment for CD is symptomatic although

various studies have been carried out to correct the pathology

of CD [12]. One approach to restore ASPA enzyme in CD is

through the use of gene therapy to replace the missing gene.

In the mouse model for CD, injection of an adeno-associated

virus (AAV) carrying the ASPA gene into the striatum of the

brain increased local levels of enzyme activity normalizing

NAA levels and improved spongiform pathology near the

rch 153 (2004) 19–27

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–2720

injection site [14]. Ongoing clinical trials have used plasmid

or AAV based delivery of the ASPA gene into a limited series

of CD patients with varying results [10].

Stem cell therapy is another promising approach for the

treatment of neurodegenerative diseases [28]. To replace the

lost cells to recover the lost function in the disease process,

different types of cells have been used, obtained from

various sources including bone marrow, peripheral blood,

amniotic epithelial cells, embryonic layers and brain tissue

[2,15,27,29–31]. Of particular interest are neural Stem Cells

and neural progenitor cells (NPCs) that can be readily

isolated from the central nervous system of fetal or postnatal

animals [9,26,39].

The neurosphere cultures contain both multipotent cells

with stem cell-like characteristics and more restricted NPC

populations at different states of differentiation [35,38,43].

Neurosphere cultures are able to differentiate into neurons,

astrocytes and oligodendrocytes in vitro [25,26]. Neuro-

spheres derived from fetal brain have been shown to retain

at least some of their regionally specific markers [8,17,19].

Transplantation of neurospheres into the rat brain differ-

entiated predominantly into glial cells [43]. Since loss of

ASPA synthesizing cells, oligodendrocytes, is an important

event in CD, it is interesting to understand whether NPCs

transplantation have capable to survive, distribute and

differentiate into oligodendrocytes to replace the lost cells

as well as to improve the ASPA activity in CD. Therefore, in

the present study ASPA overexpressed NPCs were

implanted into the brain in the mouse model of CD to

understand their role as a source for cell replacement as well

as enzyme replacement was evaluated.

2. Materials and methods

2.1. Isolation and culture of mouse neural progenitor cells

Isolation of adult mouse neural progenitors was

described previously [24]. Briefly, brains minus cerebellum

from C57bl/6 mice were cut into 1–2 mm pieces, then

mechanically and enzymatically dissociated with papain–

protease–DNase solution. The dissociated cells were parti-

ally purified from contaminating debris by using Percoll

density gradients [17]. The dissociated cells were collected

and thoroughly washed. Isolated cells were grown on

uncoated plates in N2 medium containing EGF (20 ng/

ml), FGF (20 ng/ml) and heparin (5 ng/ml) as free-floating

dneurospheresT. Cells were passaged and could be frozen,

thawed and recultured.

2.2. Construction of retrovirus vector carrying ASPA gene

The ASPA retroviral vector was constructed by insert-

ing the full-length human ASPA cDNA (hASPA) into the

pLXIN retroviral vector [16]. The ASPA gene was inserted

into the HpaI and XhoI sites of the retroviral vector

pLXIN (Clontech, CA). The construction was performed

as a three-part ligation to enable the correct orientation of

the ASPA gene. 1.5�107 293 cells were triple transfected

with plasmids encoding the MMLV gag/pol genes,

pVPack-GP (Stratagene, CA), vesicular stomatitis virus G

protein (VSV-G) as an envelope protein, pVPack-VSV-G

(Stratagene), and pLXIN-ASPA. Equal amounts of vector

were transfected using calcium phosphate. Ten micrograms

of DNA (total) was used to transfect 2.5�106 cells in a 6-

cm plate. Fifteen hours post-transfection media was

removed and replaced with 3 ml media/6-cm plate.

Forty-eight hours post-media change supernatants were

pooled, filtered through a 0.45-AM filter, and spun in an

SW28 rotor at 20,000 rpm for 2 h at 4 8C. Supernatantswere removed and virus was resuspended in 800 Al stem-

cell media. Polybrene was added to a final concentration of

8 Ag/ml.

2.3. Transduction of NPCs

Adult mouse progenitor cells were treated with a

replication-defective retroviral vector expressing the

hASPA gene and neomycin resistance gene from long

terminal repeat (LTR) prepared using standard methods

[21]. To achieve retroviral transduction, NPCs were made

into a cell suspension (2�106 cells/ml) in N2 medium

containing the concentrated vector and the cells were

exposed to the vector for 3–4 h. The cell suspension was

then centrifuged, resuspended and plated in N2 medium

containing EGF, FGF and heparin. The transduced

cultures were expanded and grown under selection in

the presence of 100 Ag/ml G418 starting at 5 days post-

transduction.

2.4. Labeling methods and preparing cells for

transplantation

To enable the detection of cells in vivo, expanded cultures

were labeled with bromodeoxyuridine (BrdU), which was

added to the culture medium at 0.5 AM concentration 72 h

before the preparation of cells for transplantation. Cultures

were trypsinized, and detached cells were washed with 0.1 M

PBS, collected by centrifugation at 2500 rpm for 3 min, and

resuspended in 1 ml of PBS. An aliquot of the cell

suspension was removed and mixed with trypan blue to test

viability. The cell suspension was centrifuged a second time

and resuspended in a smaller volume of 0.1 M PBS to give

the equivalent of 100,000 viable cells/Al.

2.5. Transplantation and tissue processing

Adult and juvenile mice were anesthetized by ketamine

60 mg/kg and xylacine 10 mg/kg and placed in a stereotaxic

instrument (Kopf, CA). All animal handling procedures were

followed by the rules and regulations of Institutional Animal

Care committee. The coordinates were derived according to

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–27 21

Paxinos and Franklin [20]. In adult and juvenile mice, the

transplantation site was 1.2 mmML, 1.7 mm AP and 1.9 and

3.5 mm DV for striatum transplants. For cerebellum, the

coordinates used were 2.3 mm ML, 10.0 mm AP and 1.8

mm DV. The BrdU treated NPCs were stereotaxically

injected unilaterally into the right side of the brain of CD

mice and wild type (1 Al/injection site) at a concentration of

100,000 cells/Al at different ages (juvenile and adults).

Same volume of the buffer without cells was injected into

the left side of the same regions of the brain and this was

used as sham control. A total of 9 juvenile (7 CD mice and

2 wild type) and 18 adults (14 CD and 4 wild type) mice

were implanted. Juvenile mice were sacrificed at 4 weeks

and adult mice at 5 weeks post-transplantation. Injected

animals (12 adults, 5 juveniles) were perfused with 4%

paraformaldehyde and the brains were sectioned (50 Amcoronal sections) using a vibrating blade microtome. These

sections were used to stain different markers as mentioned

below. Remaining animals (6 adults, 4 juveniles) were used

for biochemical analysis.

2.6. Immunostaining of the transplanted cells for

oligodendrocyte progenitor, oligodendrocytes and fibrous

astrocytes

For immunofluorescence staining, sections were pre-

treated for BrdU detection as described previously [4,5] and

stained with rat anti-BrdU (1:100, Accurate, NY), and with

the anti-oligodendroglial progenitor marker rabbit anti-NG2

(1:500; Chemicon, CA). The secondary antibody used was

donkey anti-species FITC or Texas red (Accurate Chemical,

NY). Sections were visualized and photographed under

Olympus microscope.

Cells were stained using oligodendrocytes or fibrous

astrocytes marker as described earlier [2,42]. To determine

oligodendrocytes, brain sections were incubated for 1 h in

blocking solution containing Tris buffered saline (TBS),

5% donkey serum and 0.1% Triton X-100. Subsequently,

rat anti-BrdU (1:100 dilution) (Accurate Chemical) and

mouse anti-CNPase (1:200 dilution) (Sternberger, MD)

were added and incubated overnight at 4 8C. Sections werewashed with TBS and incubated for 1 h at room temper-

ature in the presence of a 1:250 dilution of biotin–

streptavidin-conjugated donkey anti-mouse IgG (Jackson

Labs, Maine) in TBS containing 1% donkey serum and

0.1% Triton X-100. Then, sections were incubated for 1 h

at room temperature with a 1:250 dilution of Texas Red

labeled donkey anti-rat IgG (Jackson Labs), and a 1:250

dilution of fluorescein (DTAF)-conjugated streptavidin

(Jackson Labs) in TBS containing 0.1% Triton X-100.

Tissues were visualized and photographed under confocal

microscopy.

To determine fibrous astrocytes, tissue sections were

incubated for 1 h in blocking solution containing TBS, 5%

goat serum and 0.1% Triton X-100. Then astrocyte marker,

glial fibrillary acidic protein (GFAP) (diluted 1:2500,

Sigma, MI) and rat anti-BrdU antibodies were incubated

overnight at 4 8C. Sections were washed with TBS and

incubated for 1 h at room temperature in the presence of

1:250 dilution of FITC labeled rabbit anti-goat IgG (Jackson

Labs) and a 1:250 dilution of Texas Red labeled donkey

anti-rat IgG (Jackson Labs) in TBS containing 0.1% Triton

X-100. Tissues were visualized and photographed under

microscopy.

2.7. Aspartoacylase activity assay

ASPA activity in the transduced and non-transduced cell

cultures was measured as followed earlier [33]. The NPCs

injected parts of the cerebrum and cerebellum from the right

half of the brain was separated and pooled to prepare

homogenate as we followed earlier [14]. Same parts from

left side of the brain was collected and used as untreated

control. ASPA assay was carried out in a total volume of

600 Al with 50 mM Tris–HCl (PH 8.0), 0.5% (w/v) NP-40,

50 mM sodium chloride, 1 mM calcium chloride, 2.8 mM

N-acetylaspartic acid and the enzyme at 37 8C for 3 h. The

reaction was coupled with malic dehydrogenase, glutamic

oxalacetic transaminase and h-nicotinamide adenine dinu-

cleotide reduced form and the amount of l-aspartate

released in the aspartoacylase reaction was quantified by

spectrophotometry at 340 nm. One milliunit aspartoacylase

activity is equivalent to 1 nano mole of aspartate released in

1 min.

3. Results

NPCs retrovirally transfected with the human ASPA gene

expressed greater than fourfold higher levels of enzyme

activity than non-transduced cells (0.995 mU/mg protein

compared to 0.234 mU/mg protein). Therefore, NPCS

retrovirally modified to overexpress ASPA were used in

all subsequent transplantation experiments.

Injection of NPCs to the brain of juvenile CD mouse

striatum showed numerous cells (Fig. 1B) as observed in the

wild type (Fig. 1A) at 4 weeks after transplantation.

Transplanted cells identified as BrdU positive cells spread

away from the injection site, as observed in some brain

sections (Fig. 1B). In the striatum, the cells spread about 3

mm in the rostrocaudal axis. Injection of NPCs to the

cerebellum of juvenile CD mouse showed high NPCs

survival rate with little migration (Fig. 1D) as also observed

in the wild type (Fig. 1C).

In some juvenile animals the BrdU positive cells seemed

to favor the white matter tracts (Fig. 2A), and to migrate

about 3 mm in the rostrocaudal axis. Some of the

transplanted cells migrated from the white matter tracts into

the neighboring cortical matter (Fig. 2B). Some of these

BrdU-labeled cells co-expressed the glial progenitor marker,

the proteoglycan NG2 (Fig. 3A,B). Cells expressing NG2

had unipolar, bipolar or multipolar morphologies.

Fig. 1. Neural progenitor cells transplantation to the brain of juvenile CD mouse BrdU positive neural progenitor cells (arrow) in the forebrain of (A) wild type

and (B) CD mouse. Neural progenitor cells in the cerebellum of (C) wild type and (D) CD mouse. Interestingly, implanted cells migrated from the injected site

in the CD mouse both in wild type and CD mice (bar=50 Am).

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–2722

Staining of the transplanted cells with CNPase showed

positive staining for oligodendrocytes. NPCs differentia-

tion into oligodendrocytes was seen both in the striatum

and cerebellum areas of the brain. CNPase positive

implanted cells in the striatum of the CD mouse are shown

in Fig. 4A,B.

In wild type mice, ASPA activity in the injected side

of the brain slightly increased (Table 1). While no ASPA

Fig. 2. Neural progenitor cells lining up towards the white matter of the CD mo

(B) The transplanted cells migrated from the white matter tracts into the neighbo

activity was observed in the uninjected site of the CD

mouse brain, the NPCs injected side was 16% of wild

type ASPA activity after 4 weeks of transplantation

(Table 1).

The transplanted NPCs have differentiated into fibrous

astrocytes, as evident from GFAP staining. Differentiation

into fibrous astrocytes was seen in the striatum and in the

cerebellum areas of the transplanted brain. The GFAP

use brain. (A) BrdU positive cells seemed to favor the white matter tracts.

ring cortical matter.

Table 1

Aspartoacylase activity in the neural progenitor cells (NPCs) transplanted

wild type and knockout mice brains

ASPA activity (mU/mg protein; n=FS.E)

Juvenile mice (4 weeks post-transplantation)

Wild type (n=1)a Knockout mouse (n=3)

Cells injected

side

Cells uninjected

side

Cells injected

side

Cells uninjected

side

0.189F0.004 0.141F0.006 0.030F0.008 0.000F0.000

Adult mice (3 weeks post-transplantation)

Wild type Knockout mouse (n=2)

ND Cells injected Cells uninjected

0.026F0.003 0.007F0.001

Adult mice (5 weeks post-transplantation)

Wild type (n=1)a Knockout mouse (n=3)

Cells injected Cells uninjected Cells injected Cells uninjected

0.280F0.010 0.248F0.009 0.007F0.004 0.001F0.001

The NPCs implanted at the side of the brain (right side) showed increased

activity of ASPA in the juvenile knockout mouse brain compared to the left

uninjected side, even after 4 weeks of implantation. The juvenile as well as

adult wild type mice showed mild increase in ASPA activity in the injected

side compared to the uninjected side of the brain. NPCs transplantation to

the adult knockout mouse brain improved ASPA activity during 3 weeks of

post-transplantation period, but the level was reduced after 5 weeks of

transplantation.

ND=not determined.a Same brain homogenate was assayed two times.

Fig. 3. Transplanted neural progenitor cells differentiate into oligoden-

drocyte progenitor cells in the CD mouse brain. Neural progenitor cells

stained with oligodendrocyte progenitor marker, NG2, showed positively

stained cells. A and B show different magnifications.

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–27 23

positive transplanted cells in the striatum of the CD mouse

are shown in Fig. 5A,B.

Injection of NPCs to the adult CD mouse striatum

showed numerous BrdU positive cells near the injection site

Fig. 4. Image indicates CNPase (green) and BrdU (red) immunoreactive transplan

bars, (A) 100 Am; (B) 10 Am.

at 3 and 5 weeks of transplantation (Fig. 6B) as also

observed in the wild type (Fig. 6A). Transplantation of these

cells to the adult CD mouse cerebellum also showed a

similar result (Fig. 6C,D). The average spread of BrdU

positive cells was about 1.8 mm along the rostrocaudal axis.

The NPCs implanted side of the CD mouse brain showed

increased ASPA activity compared to the other side of the

brain (Sham control) (Table 1) after 3 weeks of trans-

ted cells in the striatum of the CD mouse at different magnifications. Scale

Fig. 5. Implanted neural progenitor cells differentiate into astrocytes in the striaum of the CD mouse. (A) Staining with GFAP showed astrocyte positive neural

progenitor cells. Image indicates GFAP (green) and BrdU (red) immunoreactive transplanted cells in the striatum. The rectangle enclosed area is shown at

higher magnification in B.

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–2724

plantation. However, the increased activity was declined

during 5 weeks of post-transplantation period (Table 1).

4. Discussion

Transplantation of NPCs represents an alternative route

to replace lost or damaged cells in the central nervous

Fig. 6. Neural progenitor cell transplantation to the brain of adult CD mouse BrdU

CD mouse. Neural progenitor cells (arrow) in the cerebellum of (C) wild type an

system. Transplantation of NPCs derived from the CNS has

shown the unique ability to integrate in the brain with

terminal differentiation into mature neurons and glia to

replace the lost function [9,26,43,44]. Thus transplantation

of NPCs represents a viable therapeutic opportunity for

replacing lost or damaged cells in the CNS. Oligodendro-

cytes are one of the major glial cells in the CNS, play an

important role in maintaining healthy myelin [7]. In CD,

positive neural progenitor cells (arrow) in the forebrain of (A) wild type (B)

d (D) CD mouse (bar=50 Am).

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–27 25

oligodendrocytes are lost and therefore replacing oligoden-

drocytes to produce ASPA activity is one of the important

strategies to correct CD.

The CD mouse displays a neuropathology analogous to

the human disease [32–34]. It was therefore of therapeutic

interest in this initial study to characterize the survival,

migration and oligodendrocytes differentiation potential of

NPCs following implantation into the CD mouse brain. The

CD mouse shows clear neurophenotype by 1 month of age

[32,33] and therefore it was of interest to implant juvenile

animals in the early stages of degeneration as well as adult

animals with advanced disease.

The cultured NPCs derived from mouse neurospheres in

the present study had normal ASPA activity, which was

similar to the ASPA activity reported in the wild type mouse

brain [33]. Since the NPCs enzyme activity seen in vitro was

presumably not sufficient to increase ASPA activity in the

CD mouse brain, we transfected the NPCs with ASPA gene

carrying retroviral vector. After transduction, the transfected

NPCs showed higher enzyme activity in vitro and therefore

these cells were used for transplantation in the CD mouse

brain to replace the lost cells as well as the enzyme.

The NG2 stained cells in the central nervous system has

been used as a marker to identify oligodendrocyte progen-

itor cells (OPCs) [3]. Since there is little evidence that

mature oligodendrocytes can divide, it is probable that

OPCs generate oligodendrocytes and OPCs can be labeled

by antibodies to NG2, a chondroitin sulfate proteoglycan

[22]. Certainly, NG2 positive cells in the spinal cord

differentiated into mature oligodendrocytes [41]. In addi-

tion, OPCs are likely to replenish oligodendrocytes, result-

ing in the remyelination in the peri-infarct area after

ischemic insult [36]. These studies suggest that NG2 marker

can be used not only to identify OPCs but also to interpret

the generation of mature oligodendrocytes from OPCs for

remyelination in the disease process.

Following intracranial implantation, we demonstrated

that the transfected NPCs survived, migrated and differ-

entiated into OPCs in the juvenile knockout mouse brain as

observed in the wild type brain. Since previous studies

suggest that NG2 positive cells differentiated into mature

oligodendrocytes [41] and likely to replenish oligodendro-

cytes to result to remyelination [36], the NG2 positive

implanted cells observed in the CD mouse brain are likely to

differentiate into mature oligodendrocytes to result to

remyelination in the CD mouse brain.

The myelin specific enzyme, 2V, 3V-cyclic nucleotide 3V-phosphodiesterase (CNPase) is an oligodendroglial marker,

being expressed in cell bodies before the onset of

myelination [1,18]. Loss of ASPA synthesizing cells,

oligodendrocytes, is one of the events in CD. In order to

replace the lost cells in CD, whether NPCs differentiate into

oligodendrocytes was examined. CNPase positive implanted

cells in the knockout mouse brain observed in our study

suggests that NPCs differentiate into oligodendrocytes

likely to result to remyelination.

The transplanted NPCs yielded measurable ASPA

activity in the mouse brain up to 1 month post-trans-

plantation period, the maximum time we studied. Differ-

entiation of NPCs into oligodendrocytes and the resulting

increased ASPA activity after implantation in the CD mouse

suggest that NPCs can be used as a potential source of cell

replacement therapy in CD.

Astrocytes play a major role in neuron protection and

gliosis [6,40]. Fibrous astrocytes are localized within the

white matter and protoplasmic astrocytes are present in the

gray matter [23]. Fibrous astrocytes can be identified by a

nonsoluble acidic cytoskeletal protein, glial fibrillary acid

protein (GFAP) [4,23]. Since spongiform degeneration was

seen in the white matter of the brain in CD, fibrous

astrocytes are also affected in addition to oligodendrocytes

[32–34]. Therefore we investigated whether the trans-

planted NPCs differentiate into fibrous astrocytes to replace

the lost cells in CD. We observed GFAP positive trans-

planted cells in the CD mouse brain. This observation

suggests that NPCs differentiate into fibrous astrocytes and

therefore NPCs can be used to replace the lost cells in the

CD brain.

We observed good survival of NPCs following trans-

plantation into adult mice. Although increased ASPA

activity was seen at 3 weeks after implantation, the activity

at 5 weeks post-transplantation declined below the thre-

shold of detection. This is likely due to the short-term

expression of the retroviral vector in vivo. Adeno-asso-

ciated virus (AAV) injection in the CD mouse brain showed

long-term expression with sustained enzyme activity [14].

Therefore, NPCs transduced with the parvoviral vector,

AAV, may be used for implantation in CD for long-term

efficacy.

Acknowledgments

The authors thank Sylvia Szucs for technical assistance.

References

[1] P.C. Barradas, S.S. Gomes, L.A. Cavalcante, CNPase expression in

the developing opossum brainstem and cerebellum, NeuroReport 6

(1995) 289–292.

[2] O. Brqstle, U. Maskos, R.D.G. McKay, Host-guided migration allows

targeted introduction of neurons into the embryonic brain, Neuron 15

(1995) 1275–1285.

[3] A.M. Butt, A. Duncan, M.F. Hornby, S.L. Kirvell, A. Hunter, J.M.

Levine, M. Berry, Cells expressing the NG2 antigen contact nodes of

Ranvier in adult CNS white matter, Glia 26 (1999) 84–91.

[4] L. Eng, J. Vanderhaeghen, A. Bignami, B. Gerstel, An acidic

protein isolated from fibrous astrocytes, Brain Res. 28 (1971)

351–354.

[5] F.H. Gage, P.W. Coates, T.D. Palmer, H.G. Kuhn, L.J. Fisher, J.O.

Suhonen, D.A. Peterson, S.T. Suhr, J. Ray, Survival and differ-

entiation of adult neuronal progenitor cells transplanted to the adult

brain, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 11978–11883.

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–2726

[6] J.H. Garcia, Y. Yoshida, H. Chen, Y. Li, Z.G. Zhang, J. Lian, S. Chen,

M. Chopp, Progression from ischemic injury to infarct following

middle cerebral artery occlusion in the rat, Am. J. Pathol. 142 (1993)

623–645.

[7] J.B. Grinspan, M. Coulalaglou, J.S. Beesley, D.F. Carpio, S.S.

Scherer, Maturation-dependent apoptotic cell death of oligoden-

drocytes in myelin-deficient rats, J. Neurosci. Res. 54 (1998)

623–634.

[8] S. Hitoshi, V. Tropepe, M. Ekker, D. van der Kooy, Neural stem cell

lineages are regionally specified, but not committed, within distinct

compartments of the developing brain, Development 129 (2002)

233–244.

[9] H. Jeltsch, J. Yee, E. Aloy, P. Marques Pereira, S. Schmchowitsh,

L. Grandbarb, S. Caillard, E. Mohier, J.C. Cassel, J. Helene, Y.

Jason, A. Elisabeth, M.P. Patricia, S. Sarah, G. Luc, C. Sophie, M.

Eliane, C. Jean-Christophe, Transplantation of neurospheres after

granule cell lesions in rat cognitive improvements despite no long

term immunodetection grafted cells, Behav. Brain Res. 143 (2003)

177–191.

[10] P. Leone, C.G. Janson, L. Bilianuk, Z. Wang, F. Sorgi, L. Huang, R.

Matalon, R. Kaul, Z. Zeng, A. Freese, S.W. McPhee, E. Mee, M.J.

During, Aspartoacylase gene transfer to the central nervous system

with therapeutic implications for Canavan disease, Ann. Neurol. 48

(2000) 27–38.

[11] R. Matalon, K. Michals, D. Sebasta, M. Deanching, P. Gashkoff, J.

Casanova, Aspartoacylase deficiency and N-acetyl-aspartic aciduria

in patients with Canavan disease, Am. J. Med. Genet. 29 (1988)

463–471.

[12] R. Matalon, R. Kaul, K. Michals, Canavan disease: biochemical and

molecular studies, J. Inherit. Metab. Dis. 16 (1993) 744–752.

[13] R. Matalon, P.L. Rady, K.A. Platt, H.B. Skinner, M.J. Quast, G.A.

Campbell, K. Matalon, J.D. Ceci, S.K. Tyring, M. Nehls, S. Surendran,

J. Wei, E.L. Ezell, S. Szucs, Knockout mouse for Canavan disease: a

model for gene transfer to the central nervous system, J. Gene Med. 2

(2000) 165–175.

[14] R. Matalon, S. Surendran, P.L. Rady, M.J. Quast, G.A. Campbell,

K.M. Matalon, S.K. Tyring, J. Wei, C.S. Peden, E.L. Ezell, N.

Muzyczka, R.J. Mandel, Adeno-associated virus-mediated aspartoa-

cylase gene transfer to the brain of knockout mouse for Canavan

disease, Mol. Ther. 7 (2003) 580–587.

[15] J.W. McDonald, X.Z. Liu, Y. Qu, S. Liu, S.K. Mickey, D. Turetsky,

D.I. Gottlieb, D.W. Choi, Transplanted embryonic stem cells survive,

differentiate and promote recovery in injured rat spinal cord, Nat.

Med. 5 (1999) 1410–1412.

[16] A.D. Miller, G.J. Rosman, Improved retroviral vectors for gene

transfer and expression, BioTechniques 7 (1989) 980–990.

[17] J. Ourednik, V. Ourednik, W.P. Lynch, M. Schachner, E.Y.

Snyder, Neural stem cells display an inherent mechanism for

rescuing dysfunctional neurons, Nat. Biotechnol. 20 (2002)

1103–1110.

[18] K. Ozawa, G. Suchanek, H. Breitschopf, W. Brqck, H. Budka, K.Jellinger, H. Lassmann, Patterns of oligodendroglia pathology in

multiple sclerosis, Brain 117 (1994) 1311–1322.

[19] M. Parmar, C. Skogh, A. Bjfrklund, K. Campbell, Regional

specification of neurosphere cultures derived from subregions of the

embryonic telencephalon, Mol. Cell. Neurosci. 21 (2002) 645–656.

[20] G. Paxinos, K. Franklin, The Mouse Brain in Stereotaxic Coordinates,

Academic Press, San Diego, 2001.

[21] W.S. Pear, G.P. Nolan, M.L. Scott, D. Baltimore, Production of high-

titer helper-free retroviruses by transient transfection, Proc. Natl.

Acad. Sci. U. S. A. 90 (1993) 8392–8396.

[22] A. Peters, C. Sethares, Oligodendrocytes, their progenitors and other

neuroglial cells in the aging primate cerebral cortex, Cereb. Cortex

(2004) 995–1007.

[23] A. Privat, P. Rataboul, Fibrous and protoplasmic astrocytes, in: S.

Fedoroff, A. Vernadakis (Eds.), Astrocytes, vol. 1, Academic Press,

San Diego, 1986, pp. 105–130.

[24] J. Ray, F.H. Gage, Neural stem cell isolation, characterization and

transplantation, in: U. Windhorst, H. Johansson (Eds.), Modern

Techniques in Neuroscience Research, Springer Verlag, New York,

1999, pp. 339–360.

[25] B.A. Reynolds, S. Weiss, Clonal and population analyses demonstrate

that an EGF-responsive mammalian embryonic CNS precursor is a

stem cell, Dev. Biol. 175 (1996) 1–13.

[26] C.M. Rosario, B.D. Yandava, B. Kosaras, R. Zurakowski, L. Sidman,

E.Y. Snyder, Differentiation of engrafted multipotent neural progen-

itors towards replacement of missing granule neurons in meander tail

cerebellum may help determine the locus of mutant gene action,

Development 124 (1997) 4213–4224.

[27] R. Seggewiss, E.C. Buss, D. Hermann, H. Goldschmidt, A.D. Ho, S.

Fruehauf, Kinetics of pheripheral blood stem cell mobilization

following G-CSF-supported chemotherapy, Stem Cells 21 (2003)

568–574.

[28] L.S. Shihabuddin, J. Ray, F.H. Gage, Stem cell technology for

basic science and clinical applications, Arch. Neurol. 56 (1999)

29–32.

[29] L.S. Shihabuddin, P.J. Horner, J. Ray, F.H. Gage, Adult spinal cord

stem cells generate neurons after transplantation in the adult dentate

gyrus, J. Neurosci. 20 (2000) 8727–8735.

[30] S. Surendran, Possible role of prostaglandin E2 in human amniotic

epithelial cell death: an in vitro study, Inflamm. Res. 50 (2001)

483–485.

[31] S. Surendran, Possible role of fas antigen (CD 95) in human

amniotic epithelial cell death in vitro study, Cell Biol. Int. 25 (2001)

485–488.

[32] S. Surendran and R. Matalon, Progress in Canavan disease. The

Science advisory board, 2004 at: http://www.scienceboard.net/

community/perspectives.108.html.

[33] S. Surendran, P.L. Rady, K. Michals-Matalon, M.J. Quast, D.K.

Rassin, G.A. Campbell, E.L. Ezell, J. Wei, S.K. Tyring, S. Szucs, R.

Matalon, Expression of glutamate transporter, GABRA6, serine

proteinase inhibitor 2 and low levels of glutamate and GABA in the

brain of knockout mouse Canavan disease, Brain Res. Bull. 61 (2003)

427–435.

[34] S. Surendran, K. Michals-Matalon, M.J. Quast, S.K. Tyring, J.

Wei, E.L. Ezell, R. Matalon, Canavan disease: a monogenic trait

with complex genomic interaction, Mol. Genet. Metab. 80 (2003)

74–80.

[35] O.N. Suslov, V.G. Kukekov, T.N. Ignatova, D.A. Steindler, Neural

stem cell heterogeneity demonstrated by molecular phenotyping of

clonal neurospheres, Proc. Natl. Acad. Sci. U. S. A. 99 (2002)

14506–14511.

[36] K. Tanaka, S. Nogawa, S. Suzuki, T. Dembo, A. Kosakai,

Upregulation of oligodendrocyte progenitor cells associated with

restoration of mature oligodendrocytes and myelination in peri-infarct

area in the rat brain, Brain Res. 989 (2003) 172–179.

[37] L. Van Bogaert, I. Bertrand, Su rune idiotie familiale avec

degerescence sponglieuse de neuraxe (note preliminaire), Acta

Neurol. Belg. 49 (1949) 572–587.

[38] A.L. Vescovi, E.Y. Snyder, Establishment and properties of neural

stem cell clone: plasticity in vitro and in vivo, Brain Pathol. 9 (1999)

569–598.

[39] C. Vicario-Abejoin, M.G. Cunningham, R.D.G. McKay, Cerebellar

precursors transplanted to the neonatal dentate gyrus express

features characteristic of hippocampal neurons, J. Neurosci. 15

(1995) 6351–6363.

[40] W. Walz, Cerebral ischemia, in: C. Iadecola (Ed.), Mechanisms

of Cerebral Ischemic Damage, Humana Press, Totowa, NJ, 1999,

pp. 3–32.

[41] M. Watanabe, Y. Toyama, A. Nishiyama, Differentiation of prolif-

erated NG2-positive glial progenitor cells in a remyelinating lesion,

J. Neurosci. Res. 69 (2002) 826–836.

[42] S.R. Whittemore, D.J. Morassutti, W.M. Walters, R.-H. Liu, D.S.K.

Magnuson, Mitogen and substrate differentially affect the lineage

S. Surendran et al. / Developmental Brain Research 153 (2004) 19–27 27

restriction of adult rat subventricular zone neural precursor cell

populations, Exp. Cell Res. 252 (1999) 75–95.

[43] C. Winkler, R.A. Fricker, M.A. Gates, M. Olsson, J.P. Hammang,

M.K. Carpenter, A. Bjfrklund, Incorporation and glial differentiation

of mouse EGF-responsive neural progenitor cells after transplanta-

tion into the embryonic rat brain, Mol. Cell. Neurosci. 11 (1998)

99–116.

[44] Z.G. Zhang, Q. Jiang, R. Zhang, L. Zhang, L. Wang, L. Zhang, P.

Arniego, M. Chopp, magnetic resonance imaging and neurosphere

therapy of stroke in rat, Ann. Neurol. 53 (2003) 259–263.