Brain Research 974 (2003) 17–24

www.elsevier.com / locate / brainres

Research report

Transplanted neuronal progenitor cells in a peripheral nerve gap

promote nerve repair
Takeshi Murakami*, Yoshinori Fujimoto, Yuji Yasunaga, Osamu Ishida, Nobuhiro Tanaka,
Yoshikazu Ikuta, Mitsuo Ochi
Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, 1 -2 -3 Kasumi, Minami-Ku,
Hiroshima-city 734 -8551, Japan

Accepted 20 February 2003

Abstract

A basic experiment of peripheral nerve regeneration using neuronal progenitor cells embedded in collagen gel was performed in a rat
sciatic nerve defect. First, when neuronal progenitor cells derived from the fetal rat hippocampus were cultured in atelocollagen-
containing medium, neurospheres positive for anti-nestin antibody were confirmed after 8 days. These cells differentiated into astrocytes
positive for anti-glial fibrillary acidic protein (GFAP) antibody, oligodendrocytes positive for anti-galactocerebroside (GalC) antibody and
neurons positive for anti-neurofilament 200 (NF200) antibody, and they were capable of extending axons. They also differentiated into
Schwann-like supportive cells positive for anti-s100 and anti-p75 antibody. Next, a 15-mm defect was prepared in the sciatic nerve of
mature rats, and the nerve was bridged with a silicone tube filled with neuronal progenitor cells (1310 5 ) embedded in collagen gel. The
transplanted neuronal progenitor cells were labeled in advance with 5-bromo-2-deoxyuridine (BrdU). When the regenerated tissue was
examined 6 weeks and 10 weeks after grafting, the number and diameter of myelinated fibers were significantly increased compared with
a control tube without neuronal progenitor cells. Action potentials were detected in the regenerated nerve. Also, cells positive for both
anti-BrdU antibody and anti-S100 or anti-p75 antibody were observed in the regenerated tissue, and part of the grafted neural stem cells
were considered to have differentiated into Schwann cell-like supportive cells. From these results neuronal progenitor cells derived from
the fetal rat hippocampus are considered to retain their proliferative and differentiating abilities in collagen gel, and when transplanted to a
site of peripheral nerve defect, part of them differentiate into supportive cells and they contributed to promotion of axonal regeneration.
 2003 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Regeneration

Keywords: Neuronal progenitor cell; Peripheral nerve; Regeneration; Artificial nerve

1. Introduction trophic factors [3,5,10] with non-biological materials is

The development of artificial nerves is needed to treat
patients with a peripheral nerve defects. Many attempts
have been made to induce nerve regeneration at nerve
defects using non-biological materials such as a silicone
tube [17,23,35]. However, the regeneration-inducing abili-
ty of non-biological materials has been concluded to be
inadequate, and the development of artificial nerves by
combining cells such as Schwann cells [4,15] and neuro-
*Corresponding author. Tel.: 181-82-257-5233; fax: 181-82-257-
5234.
E-mail address: muraken@hiroshima-u.ac.jp (T. Murakami).
attempted today.
The presence of cells with multiple differentiation
potentials in the central nervous system of the fetal rat was
demonstrated in 1989 [31], and the self-replicating po-
tential of the stem cells and their presence in the central
nervous system were confirmed in 1994 [6]. Thus, neural
stem cells are defined as cells that have both the multiple
differentiation potential, or the potential to differentiate
into different cell types such as neurons, astrocytes, and
oligodendrocytes, and the self-proliferation potential, or
the potential to proliferate in an undifferentiated state
before differentiation into these cells. These cells have
been reported to be present around the cerebral ventricles

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0006-8993(03)02539-3
18 T. Murakami et al. / Brain Research 974 (2003) 17–24
and in the hippocampus and spinal cord of the rat or
mouse, and to be responsive to basic fibroblast growth
factor (bFGF) or epidermal growth factor (EGF)
[13,28].Recently, neural progenitor cells were experimen-
tally used for the source of transplantation to the disease
models such as Parkinson’s disease [29], spinal cord injury
[19], stroke [26], and multiple sclerosis [11].
In this study, we prepared an artificial nerve by filling a
silicone tube with neuronal progenitor cells three-dimen-
sionally embedded in atelocollagen gel, and examined its
effectiveness for repairing a 15-mm defect in the rat sciatic
nerve.
2. Materials and methods
2.1. Cell culture condition
Neuronal progenitor cells were cultured by a modi-
fication of the method of previous report [8]. After Fischer
344 rats on day 17 of pregnancy were anesthetized
intraperitoneally with sodium pentobarbital (30 mg / kg)
and killed with 0.9% NaCl, hippocampal tissue was
resected from the fetuses using a surgical microscope. The
tissue was mechanically minced, 2 ml of Dulbecco’s
modified Eagle’s medium (DMEM) / F12 medium (Gibco,
Grand Island, NY) with a 1% N2 supplement (Gibco) was
added, and this mixture was flushed about 20 times using a
Pasteur pipette. It was centrifuged at 1000 rpm for 3 min,
the supernatant was discarded, and 2 ml of the same
medium was added. Flushing was performed about 20
times similarly using a reduced-bore pipette. Neuronal
progenitor cells redifferentiated by this procedure were
cultured in a medium supplemented with bFGF (20 ng / ml,
Sigma, St. Louis, MO) and 1% atelocollagen gel (Koken,
Tokyo, Japan).
The medium was renewed every 4 days, and the cells
cultured for 12 days were used for immunological staining.
Differentiation of the adsorbed neuronal progenitor cells
into neurons and gliacytes was induced by removing bFGF
from the medium and adding 0.5% of fetal bovine serum.
Also, part of the cells were induced to differentiate and
stained immunologically to demonstrate neurons and
gliacytes.
2.2. Immunocytochemistry
After neuronal progenitor cells were embedded in
collagen gel, we examined whether they would remain
undifferentiated in a medium containing atelocollagen gel.
First, the cells were adsorbed on covering glass coated
with poly-o, and were fixed with 4% paraformaldehyde 10
days after differentiation. They were treated with 0.5%
Triton X-100 for 10 min, and reacted with mouse anti-
nestin monoclonal antibody (1:200; Chemicon Internation-
al, Temecula, CA), a primary antibody specific to neural
stem cells or undifferentiated neuronal progenitor cells
[16], at 37 8C for 60 min. They were further reacted with
the secondary antibody anti-mouse IgG Cy3 (1:200;
Chemicon) at 37 8C for 45 min. After nuclear staining
using Hoechst 33342, the cells were mounted on glass
slides and observed under the fluorescent microscope.
The differentiated cells were fixed with 4% paraformal-
dehyde, treated with 0.5% Triton X-100 for 10 min, and
reacted with mouse anti-NF200 monoclonal antibody
(1:200; Chemicon) to observe axons or with rabbit anti-
S100 antibody (1:50; Dako, Glostrup, Denmark) or anti-
p75 antibody (1:100; Santa Cruz Biotechnology, Santa
Cruz, CA) to observe Schwann-like supportive cells at
37 8C for 60 min. They also reacted anti-GFAP (1:200;
Chemicon) to observe astrocytes or anti-GalC antibody
(1:200; Chemicon) to observe oligodendrocytes. They
were further reacted with the secondary antibody anti-
mouse IgG FITC (Cy3) or anti-rabbit IgG FITC or anti-
goat Cy3 at 37 8C for 45 min. After nuclear staining using
Hoechst 33342, the cells were mounted on glass slides, and
examined under the fluorescent microscope.
2.3. Grafting procedure
Twenty days after starting culture, neuronal progenitor
cells were used as the source of grafting. These cells were
labeled with BrdU (10 mM Roche, Mannheim, Germany)
added to the medium 48 h before grafting, and 1310 5 cells
were placed in 0.2 ml of collagen gel medium not
containing bFGF. The cells were suspended evenly by
repeatedly flushing using a micropipette. Thirty-six 8-
week-old female Fischer 344 rats were anesthetized by
intraperitoneal administration of sodium pentobarbital (30
mg / kg), and a 15-mm defect was prepared in the left
sciatic nerve. This defect was bridged with a silicone tube
1.5 mm in internal diameter and 20 mm long. Both the
proximal and distal stump of the nerve was inserted 2.5
mm into the tube and connected with three stitches each
using a nylon thread. In 18 animals, 0.2 ml of neuronal
progenitor cells (1310 5 ) embedded in collagen gel were
infused into the tubes using a micropipette. In the remain-
ing 18 animals, 0.2 ml of the collagen gel medium alone
was infused into the silicone tubes as a control group.
2.4. Examination of regenerated nerve
Nine rats each were killed in the group grafted with
neuronal progenitor cells (NPC-grafted group) and the
control group 6 and 10 weeks after surgery. The tissue that
formed in the tube was removed and fixed with 2.5%
glutaraldehyde. The regenerated tissues were embedded in
Epon by standard method and cut cross-sectionally by 0.5
mm thick, stained with toluidine blue or hematoxylin–
eosin (H&E). The number and diameter of regenerated
axons were measured using the image analysis software
Mac Scope (Mitani Corp., Tokyo, Japan). We estimated the
 T. Murakami et al. / Brain Research 974 (2003) 17–24
regenerated axons by calculating two sections (the mid
point of the tube and 10 mm from the distal stump of the
nerve). The number of regenerated axons was calculated
from the number of axons per unit area in a microscopic
field at 3400. Four fields of each section were examined.
The diameter of regenerated axons was evaluated by
measuring the diameter of 40 axons each in five micro-
scopic fields at 31000 [12].
Part of the resected tissue of regenerated nerve was
embedded in paraffin, and microscopic sections were
prepared. These sections were reacted with anti-mouse
anti-BrdU antibody (1:10; Roche) and anti-rabbit anti-S100
antibody (1:200; Dako) or anti-p75 antibody (1:100; Santa
Cruz Biotechnology), and then with the secondary anti-
bodies anti-mouse IgG FITC and anti-rabbit (anti-goat)
IgG Cy3.
Electric stimulation was applied to the proximal side of
the silicone tube, and nerve action potentials (NAP) were
measured on the distal side, 10 weeks after surgery. The
rats were anesthetized with intraperitoneal pentobarbital
injection (40 mg / kg). The proximal side of the tube was
stimulated at a supermaximum intensity, a duration of 0.01
ms, and a frequency of 4.9 Hz using a clip electrode of an
electromyograph (Viking 4 System, Nicolet, Madison, WI).
Records were obtained at the distal end of the tube through
a microneedle electrode as means of 50 stimulations. The
distance between two electrodes was 20 mm. The filtration
range was 20–2000 Hz. Both types of electrodes and nerve
were covered with mineral oil and petroleum jelly to
prevent drying. When NAPs were recorded, their delay
velocity and amplitude were measured and compared with
those of the normal nerve
This study was performed with the permission of the
Fig. 1. (A) Six days after starting culture, neurospheres were recognized. Scale bar540 mm. (B) Four days after washing out bFGF, neuronal progenitor
cells differentiated into neurons. Their axons elongated in the collagen medium. Scale bar55 mm.
19
ethics committee of Hiroshima University’s Faculty of
Medicine.
2.5. Statistical analysis
To determine the independent and combined effects of
cell transplantation and feeding period, comparisons be-
tween groups were performed by two-way analysis of
variance followed by a multiple comparison test using the
Fisher LSD post hoc test.
3. Results
3.1. Proliferation and differentiation of cells in collagen
medium
Cell proliferation was confirmed from 2 days after the
beginning of culturing. Cells began to aggregate from the
4th day of culturing, and neurospheres were noted on the
8th day. Neuron formation was observed 4 days after
removal of bFGF and the addition of 0.5% of fetal bovine
serum. Further increases in the diameter and length of
neurons were observed continuously until the 14th day
after the beginning of differentiation (Fig. 1). When cells
before differentiation were stained immunologically using
anti-nestin antibody, expression of nestin was demon-
strated in the cytoplasm. Part of the differentiated cells
were positive for anti-NF200 antibody, and a small number
of cells positive for anti-S100, anti-p75, anti-GFAP and
anti-GalC antibody were also noted (Fig. 2). The propor-
tion of S100-positive cells was 4.360.9% and the propor-
tion of p75 positive cells was 4.260.6%. Thus, the
20 T. Murakami et al. / Brain Research 974 (2003) 17–24

Fig. 2. (A) Immunological staining of neuronal progenitor cells using anti-nestin antibody 12 days after starting culture. Nestin (red) was expressed
diffusely in the cytoplasm. The blue is nuclear stain using Hoechst 33342. Scale bar55 mm. (B–F) Immunological staining of axons using anti NF200
antibody (B), anti-GFAP antibody (C), anti-GalC antibody (D), anti-s100 antibody (E) and anti-p75 antibody (F) 10 days after the beginning of
differentiation. Scale bar55 mm.

neuronal progenitor cells that we cultured either prolifer-
ated in an undifferentiated state or differentiated into
neurons having axons, Schwann-like supportive cells,
astrocytes and oligodendrocytes also in a collagen-con-
taining medium.
3.2. Examination of nerve regeneration
Nerve regeneration was confirmed in only four of the
nine tubes 6 weeks after surgery but in seven tubes after 10
weeks in the NPC-grafted group (Fig. 3). In the control
group, scar-like tissue was noted in some tubes, but clear
nerve tissue could be confirmed in only two tubes ex-
amined after 10 weeks. Although many myelinated fibers
were observed in the NPC-grafted group, only a few
myelinated fibers were noted in scar tissue in the control
group. The angiogenesis of the regenerative nerve was
observed at HE staining specimen (Fig. 4). Table 1 shows
the number and diameter of regenerated myelinated fibers
in each group. Axon formation was clearly richer in the
NPC-grafted group after both 6 and 10 weeks.
When regenerated tissue was stained immunologically
with anti-BrdU antibody, many positive cells were ob-
served around the nerve fibers, and survival and differen-
tiation of neuronal progenitor cells were confirmed. More-
over, some cells were positive also for anti-S100 or anti-
p75 antibody on double staining, indicating that part of the
transplanted neuronal progenitor cells differentiated into
Schwann cell-like supportive cells (Fig. 5).
NAPs were observed in all regenerated nerves in the
NPC-grafted group 10 weeks after surgery, but no NAP
was noted in the control group (Fig. 6). The delay time of
NAP was 7.9261.22 ms, and the amplitude was
1.3860.14 mV, in the regenerated nerves after neuronal
progenitor cell grafting. They were 7.8060.24 ms and
3.6260.17 mV, respectively, in normal nerves. The delay
time was similar, but the amplitude was about one-third, in
the regenerated nerves compared with normal nerves.
 T. Murakami et al. / Brain Research 974 (2003) 17–24 21

Fig. 3. This tube was harvested 10 weeks after neural stem cells transplantation. A fine nerve was regenerated.

Fig. 4. (A) Regenerated tissue 10 weeks after grafting of collagen gel not containing neuronal progenitor cells. Although myelinated fibers were noted, they
were few and small in diameter. (B,C) Regenerated nerve 10 weeks after grafting of neuronal progenitor cells embedded in collagen gel. Myelinated cells
with a large diameter were observed in a sufficient number. Many fibers had a thick myelin sheath. Angiogenesis was remarkable around the nerve fibers.
A,B: toluidine blue stain, 31000; C: H&E stain, magnification 3400.

Table 1
Numbers and diameters (mm) of myelinated fibers
Without NPCs With NPCs
6 weeks 10 weeks 6 weeks 10 weeks
Regenerated nerve 0/9 2/9 4/9 7/9
At the middle – 247654 12766546 64536680**
of regenerative nerve 2.2260.3 3.4561.91 5.7860.94*

10 mm from distal – 39624 8366352 356661430*

stump of the nerve 0.1560.11 1.0560.28 3.0260.64*
Number of rats that showed nerve regeneration and the number and diameter of myelinated fibers in the regenerated nerves 6 and 10 weeks after grafting.
Significant promotion of regeneration of myelinated fibers was observed 10 weeks after neuronal progenitor cell grafting compared with grafting of
collagen gel alone (control). *P,0.05; **P,0.01.
22 T. Murakami et al. / Brain Research 974 (2003) 17–24
Fig. 5. Double immunological staining of regenerated tissue using anti-BrdU antibody and anti-s100 antibody (A) and anti-p75 antibody (B). Both
BrdU-antibody-positive (green) and s100 or p75-antibody-positive (red) cells were noted, and the grafted neural stem cells are considered to have
differentiated into Schwann cell-like supportive cells. Scale bar52 mm.
4. Discussion
4.1. Damage and regeneration of peripheral nerves
Autologous nerve grafting is performed for peripheral
nerve defects, but the length of available nerve grafts is
limited. Nerve repair using non-biological materials such
as a silicone tube has been attempted experimentally for
large nerve defects that cannot be treated by autologous
nerve grafting. However, it has been shown that the length
of the nerve that can be regenerated using a non-biological
material not containing a neurogenic protein is limited, and
methods using a polyglycolic acid (PGA) tube [18] or a
Fig. 6. In the tubes filled with collagen gel alone, no NAP could be
recorded on the distal side on electric stimulation of the proximal side
(A). However, clear NAPs could be recorded in the tubes filled with
neuronal progenitor cell graft (B). Arrows show recorded NAPs.
tube coated with laminin, which is a neurotrophic factor
[22], have been devised. Moreover, several studies of
artificial nerves prepared from cultured Schwann cells have
also been carried out [7,9,20,30]. As for cells derived from
the central nervous system, axonal regeneration was noted
using olfactory bulb ensheathing cells [34]. Direct nerve
regenerating activity is considered to be the largest in
Schwann cell grafting for regeneration of peripheral
nerves. However, from the viewpoint of securing donor
cells for grafting, it is difficult to maintain a large quantity
of Schwann cells in an adequate condition. Neuronal
progenitor cells that we used had high proliferative activity
and can be preserved for a long period. They may also be
collected from embryonic stem cells (ES cells) [2,24,33] so
that cells with controlled antigenicity may be obtained in a
larger quantity in the future. Pemela et al. reported that
transplanted fetal spinal cord tissue supported the repair of
injured spinal cord [25]. In fact, tissues or cells derived
from embryos have the capacity to repair injured tissues,
especially neuronal tissues. There has been no definite
conclusion about optimal embryonic cells or tissues to
enhance nerve regeneration. However, according to previ-
ous reports that transplanted neural progenitor cells into
neuronal injury or disease models, these cells were sup-
posed to have an excellent ability to make neuronal tissues.
Therefore neural progenitor cells are considered appro-
priate as donor cells for the regeneration of nervous
system. In the case of embryo, neural stem cells were
located in the cerebral cortex, hippocampus, cerebellum
basal forebrain and spinal cord in Temple’s report [32].
However, there was no previous report that described
differences among cells from these sites. Gage et al.
reported that neural progenitor cells derived from the
hippocampus exhibit site specific differentiation [8], and
these cells were stable for long-term culture [27]. Thus, we
consider that these cells were suitable for the donor of
transplantation.
 T. Murakami et al. / Brain Research 974 (2003) 17–24
4.2. Tissue engineering and regeneration of peripheral
nerves using neural progenitor cells
The recent discovery of various stem cell systems has
made tissue engineering of many organs possible. Applica-
tion of neural stem cells or progenitor cells to various
disease and trauma models has been proposed with gradual
clarification of the mechanisms of their differentiation and
proliferation. In this study, also, neuronal progenitor cells
proliferated while maintaining an undifferentiated state
also in a medium containing atelocollagen and retained
their characteristic that they have the potential to differen-
tiate into neurons or gliacyte. Also, neural stem cells that
differentiated into neurons could extend axons even in this
viscous medium. This suggests that neuronal progenitor
cells embedded in a collagen medium can be grafted
securely to the recipient site by retaining their multipoten-
tiality and that they can thus be applied to various
experiments. To our knowledge, there has not been a
report of application of progenitor cells derived from the
central nervous system to regeneration of peripheral
nerves. Recently, however, there have been reports on
precursor cells of central and peripheral nerves. Mujtaba et
al. reported that common precursor cells of central and
peripheral nerves are present in the fetal rat spinal cord
[21], and Keirstead et al. reported that precursor cells of
gliacytes collected from the central nervous system of rat
neonates formed myelin sheath similar to that of peripheral
nerves in the central nervous system [14]. Furthermore,
Akiyama et al. demonstrated by the electrophysiologic
technique that myelinated fibers formed in the spinal cord
by nerve precursor cells collected from the adult brain
functioned effectively [1]. All these reports were about
differentiation of nerve precursor cells in the central
nervous system. In our experiment, cells derived from the
central nervous system were shown to supply myelinated
fibers also in peripheral nerves. In rat models of sciatic
nerve defect, defects 10 mm or smaller are generally
considered to be repaired by bridging with a non-biological
material such as a silicone tube, but we succeeded in
repairing 15-mm nerve defects by using cultured neural
stem cells. A large number of myelinated axons were
formed in the tubes packed with the neuronal progenitor
cell graft, and the regenerated nerve fibers were shown to
be active as a conduction route by confirming descending
NAPs. S100 or p75 antibody-positive cells derived from
the grafted neuronal progenitor cells were noted in the
regenerated nerves. These cells are considered to be
supportive cells resembling Schwann cells and to play an
important part of the regeneration of sciatic nerve. How-
ever, the number of Schwann-like supportive cells was too
small to conclude that regeneration was led only by these
cells. In addition to the effects of Schwann like-supportive
cells, an unknown mechanism, for example some kind of
cytokine or inflammation, might be involved enhancing the
nerve regeneration. Further evaluation is needed to analyze
23
the fates of grafted cells and clarify the mechanism of
nerve regeneration.
Acknowledgements
We thank Dr. Satoshi Tashiro of the Department of
Biochemistry at the Biomedical Science Hiroshima Uni-
versity for his technical support and helpful comments.
References
[1] Y. Akiyama, O. Honmou, U. Kato, T. Uede, K. Hashi, J.D. Kocsis,
Transplantation of clonal neural precursor cells derived from adult
human brain establishes functional peripheral myelin in rat spinal
cord, Exp. Neurol. 167 (2001) 27–39.
[2] G.I. Bain, J.D. Bennett, R.S. Richards, G.P. Slethug, J.H. Roth,
Embryonic stem cells express neuronal properties in vitro, Dev.
Biol. 168 (1995) 342–357.
[3] D.J. Bates, J.A. Ransford, D.C. Mangelsdorf, Blot and culture
analysis of neuronotrophic factors in nerve regeneration chamber
fluids, Neurochem. Res. 16 (1991) 621–628.
[4] D.J. Brian, K.K. Wang, D.P. Chakalis-Haley, Effect of Schwann cells
in the enhancement of peripheral regeneration, J. Reconstr. Mi-
crosurg. 12 (1996) 439–446.
[5] N. Danielsen, S. Varon, Characterization of neurotrophic activity in
the silicone-chamber model for nerve regeneration, J. Reconstr.
Microsurg. 26 (1995) 231–235.
[6] A.A. Davis, S. Temple, A self-renewing multipotential stem cells in
embryonic rat cerebral cortex, Nature 372 (1994) 263–266.
[7] H. Fansa, G. Keilhoff, G. Wolf, W. Schneider, B.G. Gold, Tissue
engineering of peripheral nerves: A comparison of venous and
acellular muscle grafts with cultured Schwann cells, Plast. Reconstr.
Surg. 107 (2001) 485–494.
[8] F.H. Gage, P.W. Coates, P.D. Palmer, H.G. Kuhn, L.J. Fisher,
Survival and differentiation of adult neuronal progenitor cells
transplanted to the adult brain, Proc. Natl. Acad. Sci. USA 92
(1995) 11879–11883.
[9] T.A. Hadlock, C.A. Sundback, D.A. Hanter, J.P. Vacanti, M.L.
Cheney, A new artificial nerve graft containing rolled Schwann cell
monolayers, Microsurgery 21 (2001) 96–101.
[10] K. Harman, J. Katnick, R. Lim, A. Raheer, J.C. de la Torre, Glia
maturation factor beta stimulates axon regeneration in transected rat
sciatic nerve, Brain Res. 564 (1991) 332–335.
[11] R.Q. Hintzen, Stem cell transplantation in multiple sclerosis;
multiple choices and multiple challenges, Mult. Scler. 8 (2002)
155–160.
[12] O. Ishida, J. Davis, T. Tsai, W.C. Breidenbach, J. Firrell, Regenera-
tion following rejection of peripheral nerve allograft of rats on
withdrawal of cyclosporine, Plas. Reconstr. Surg. 52 (1993) 916–
926.
[13] K.K. Johe, T.G. Hazel, T. Muller, M.M. Dugich-Djordjevic, R.D.
Mckey, Single factors direct the differentiation of stem cells from
fetal and adult central nervous system, Genes Dev. 10 (1996)
3129–3140.
[14] H.S. Keirstead, S.V. Morgan, M.J. Wilby, J.W. Fawcett, Poly-
sialylated neural cell adhesion molecule-positive CNS precursors
generate both oligodendrocyte and Schwann cells to remyelinate the
CNS after transplantation, J. Neurosci. 19 (1999) 7529–7536.
[15] D.H. Kim, S.E. Connolly, D.G. Kline, R.M. Voorhies, A. Smith, M.
Powell, T. Yoes, J.K. Daniloss, Labeled Schwann cell transplants
versus sural nerve grafts in the nerve repair, J. Neurosurg. 80 (1994)
254–260.
24 T. Murakami et al. / Brain Research 974 (2003) 17–24
[16] U. Lendahl, L.B. Zimmerman, R.D. McKay, CNS stem cells express
a new class of intermediate filament protein, Cell 60 (1990) 585–
595.
[17] G. Lundborg, L.B. Dahlin, N. Danielsen, Nerve regeneration in
silicone model chambers: influence of gap length and distal stump
components, Exp. Neurol. 76 (1982) 361–375.
[18] S.E. Mackinnon, A.L. Dellon, Clinical nerve reconstruction with a
bioabsorbable polyglycolic acid tube, Plast. Reconstr. Surg. 85
(1990) 419–424.
[19] J.W. McDonald, X.Z. Liu, Transplanted embryonic stem cells
survive differentiate and promote recovery in injured rat spinal cord,
Nat. Med. 5 (1999) 1410–1412.
[20] A. Mosahebi, M. Butterworth, M. Wiberg, R. Martin, G. Terenghi,
Retroviral labeling of Schwann cells: in vitro characterization and in
vivo transplantation to improve peripheral nerve regeneration, Glia
34 (2001) 8–17.
[21] T. Mujtaba, M. Mayer-Proschel, M.S. Rao, A common neural
progenitor for the CNS and PNS, Dev. Biol. 200 (1998) 1–15.
[22] Y. Nakao, An experimental study on the effect of laminin in vivo on
promoting regeneration of axons, Nippon Seikeigeka Gakkai Zasshi
66 (1992) 334–349.
[23] M. Ochi, Experimental study on orientation of regenerating fibers in
the served peripheral nerve, Hiroshima J. Med. Sci. 31 (1983)
389–406.
[24] S. Okabe, K. Forsberg-Nielsen, A.C. Spiro, M. Segal, R.D. McKay,
Development on neuronal precursor cells and functional postmitotic
neurons from embryonic stem cells in vitro, Mech. Dev. 59 (1996)
89–102.
[25] S. Pamela, D.S. Bregman, B.S. Bregman, Fetal spinal cord trans-
plants supported the development of target reaching and coordinated
postural adjustment after neonatal cervical spinal cord injury, J.
Neurosci. 18 (1998) 763–778.
[26] K.I. Park, Transplanted of neural stem cells: cellular and gene
therapy for hypoxic ischemic brain injury, Yonsei Med. J. 41 (2000)
825–835.
[27] J. Ray, D.A. Peterson, M. Schinstine, F.H. Gage, Proliferation,
differentiation, and long term culture of primary hippocampal
neurons, Proc. Natl. Acad. Sci. USA 90 (1993) 3602–3606.
[28] B.A. Reynolds, W. Tetzlaff, S. Weiss, A multipotent EGF-responsive
striatal embryonic progenitor cell produce neuron and astrocytes, J.
Neurosci. 12 (1992) 4565–4574.
[29] C.N. Svendsen, M.A. Caldwell, J. Shen, Long-term survival of
human central nervous system progenitor cells transplanted into rat
model of Parkinson’s disease, Exp. Neurol. 148 (1997) 135–146.
[30] O.A. Sulaiman, T. Gordon, Effects of short- and long-term Schwann
cell denervation on peripheral nerve regeneration, myelination, and
size, Glia 32 (2000) 234–246.
[31] S. Temple, Division and differentiation of isolated CNS blast cells
in microculture, Nature 340 (1989) 471–473.
[32] S. Temple, The development of neural stem cells, Nature 414
(2001) 112–117.
[33] V. Tropepe, S. Hitoshi, C. Sirard, T.W. Mak, J. Rossant, D. van der
Kooy, Direct neural fate specification from embryo stem cells: a
primitive mammalian neural stem cells stage acquired through a
default mechanism, Neuron 30 (2000) 65–78.
[34] E. Verdu, X. Narvarro, G. Gudino-Cabrera, F.J. Rodriguez, D.
Ceballos, A. Valero, M. Nieto-Sampedro, Olfactory bulb ensheathing
cells enhance peripheral nerve regeneration, Neuroreport 10 (1999)
1097–1101.
[35] L.R. Williams, F. Longo, H.C. Powell, G. Lundborg, S. Varon,
Competence of nerve tissue as distal insert promoting nerve
regeneration in silicone chamber, Brain Res. 293 (1984) 201–211.