factor, was directly injected into the injured spinal cord parenchyma to manipulate proliferative cells in situ. We found that cells
formed neurospheres and differentiated into neurons, astrocytes, and oligodendrocytes in vitro, demonstrating that GFP retroviruses
factor into lesioned tissue could induce a significant fraction of GFP-labeled cells to express immature neuronal markers. Moreover,
retrovirus-mediated overexpression of the basic helix-loop-helix transcription factors Neurogenin2 and Mash1, together with growth
factor treatment, enhanced the production and maturation of new neurons and oligodendrocytes, respectively. These results demon-
strate that endogenous neural progenitors can be manipulated to replace neurons and oligodendrocytes lost to insults in the injured
The adult mammalian CNS is highly vulnerable to various in-
sults. It has long been thought that such vulnerability is attribut-
cells (Horner and Gage, 2000). Many lines of previous studies,
however, have revealed that neural stem and other progenitor
cells [herein collectively called neural progenitor cells (NPCs)]
persist in the adult CNS (Q. Cao et al., 2002). In fact, neurogen-
various species, including humans (Goldman, 2004).
Such continuous cell genesis, however, is confined to only a
few areas under physiological conditions, and moreover, regen-
eration of new cells appears to be very limited even after damage
in most regions of the CNS (Goldman, 2004). In particular, the
adult spinal cord has been considered to be one of the most
restrictive regions in which NPCs can contribute to cell replace-
Previous cell culture studies have demonstrated that the adult
spinal cord contains an abundant source of endogenous NPCs
(Weiss et al., 1996; Johansson et al., 1999; Shihabuddin et al.,
Correspondence should be addressed to Dr. Masato Nakafuku, Division of Developmental Biology, Cincinnati
Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail:
11948 • TheJournalofNeuroscience,November15,2006 • 26(46):11948–11960
(McTigue et al., 1998, 2001; Johansson et al., 1999; Yamamoto et
al., 2001a,b; Kojima and Tator, 2002; Zai and Wrathall, 2005;
tation studies have demonstrated that exogenous NPCs, which
retain strong neurogenic and/or oligodendrogenic activities in
vitro, differentiate only very poorly when grafted into the spinal
2001, 2002; Han et al., 2002, 2004; Hill et al., 2004; Enzmann et
tal restriction can be relieved by certain manipulations, endoge-
egies to manipulate endogenous NPCs remain unexplored to
In this study, we tested two strategies to manipulate neuronal
and glial differentiation of endogenous NPCs in vivo. The first
was direct administration of a mixture of growth factors (GFs),
fibroblast growth factor 2 (FGF2) and epidermal growth factor
(EGF), into injured tissue and the second was virus-mediated
overexpression of the transcription factors Neurogenin2 (Ngn2)
and Mash1. We show that the combination of these manipula-
drocytes by endogenous NPCs in the injured spinal cord.
Spinal cord injury. Young adult Sprague Dawley rats (7–9 weeks of age
and weighing 250–330 g) were used in all experiments. All experimental
procedures were performed according to the guidelines of the Institu-
tional Animal Care and Use Committee and National Institutes of
Health. Rats were anesthetized with 50 mg of ketamine HCl and 5 mg of
xylazine (100 and 20 mg/ml, respectively; Phoenix Pharmaceuticals, St.
Joseph, MO) per kilogram of body weight. Laminectomy and complete
transection of the spinal cord at the tenth thoracic (T10) level were per-
formed as described previously (Yamamoto et al., 2001a,b).
Growth factor treatment and retrovirus infection in vivo. Recombinant
retroviruses pMXIG and pMXIG-Ngn2, which are designed to express
green fluorescent protein (GFP) as a marker for infected cells, were de-
scribed previously (Morita et al., 2000; Yamamoto et al., 2001b).
Mash1 (Torii et al., 1999) into the pMXIG vector. For virus infection in
vivo, a 30 ?l solution of artificial CSF (aCSF) containing high-titer ret-
roviruses (2 ? 108colony-forming unit/ml), 0.1 mg/ml rat serum albu-
manually into three different locations (10 ?l each) of the transected
In some experiments, recombinant human FGF2 (1 ?g; Peprotech,
and coinjected with retroviruses. An equivalent amount of rat serum
albumin was used as control. To label proliferating cells, 5-bromo-
2?deoxyuridine (BrdU) (150 mg/kg of body weight; Sigma) dissolved in
0.9% sterile saline was injected intraperitoneally twice a day for 3 d be-
tween day after injury 0 (DAI0) and DAI2. The first administration of
repeated every 12 h.
In vitro culture. Spinal cord stumps ?4 mm-long both rostral and
caudal from the lesion epicenter were subjected to in vitro culture as
described previously (Yamamoto et al., 2001a,b) with some modifica-
tions. In brief, the harvested tissue was cut into small pieces in ice-cold
aCSF containing the following (in mM): 124 NaCl, 5 KCl, 1.3 MgCl2, 2
CaCl2, 26 NaHCO3, and 10 D-glucose. Subsequently, the tissue was dis-
sociated by incubation with 0.1% (w/v) trypsin (Sigma), 0.67 mg/ml
hyaluronidase (Sigma), and 0.1 mg/ml deoxyribonuclease I (Roche) in
aCSF at 37°C for 30 min, with aeration with 95% O2/5% CO2. Trypsin
was neutralized with 0.7 mg/ml ovamucoid (Sigma) and the resultant
tissue suspension was triturated mechanically to yield a single cell sus-
pension. In some experiments, the resultant cells were immediately
seeded onto poly-D-lysine (PDL; 100 ?g/ml; Sigma)-coated eight-well
chambers (Nalge Nunc International, Rochester, NY) and subjected to
immunostaining 2 h after plating.
To initiate neurosphere culture, fragmented neuropiles and other de-
bris were removed from the above-described dissociated single cell sus-
?m pore diameter; Becton, Dickinson and Company, Franklin Lakes,
NJ) (Yamamoto et al., 2001b). The resultant single cells were seeded at
the density of 2 ? 104cells/ml in a growth medium [1:1 mixture of
DMEM and F-12 medium supplemented with B-27 and N2 culture sup-
plements (Invitrogen, Carlsbad, CA), 20 ng/ml bovine FGF2, 20 ng/ml
mouse EGF, 20 ng/ml human platelet-derived growth factor (R & D
Systems, Minneapolis, MN), 2 ?g/ml heparin (molecular mass 3000;
Sigma), 1 mg/ml bovine serum albumin (Sigma), and 100 ?M
2-mercaptoethanol (Sigma)]. Culture dishes were coated with poly
[2-hydroxyethyl methacrylate] (Sigma) to prevent cell attachment
(Yamamoto et al., 2001b). At day 14 in vitro (DIV14), forming floating
the same condition or differentiation culture. Under these conditions,
0.9 ? 0.1% (n ? 6 independent experiments) of initially seeded viable
cells formed neurospheres, and this frequency was maintained in subse-
quent four passages.
To induce differentiation into neurons and glia, neurospheres grown
in the presence of GFs were seeded onto PDL-coated eight-well cham-
104cells per well, and subsequently cultured in the above medium with-
out GFs or heparin for 6 d. In some experiments, the following peptide
factors were added to the culture medium: human bone morphogenetic
& D Systems), human ciliary neurotrophic factor (CNTF; 50 ng/ml;
Sigma), and human BDNF (50 ng/ml; Sigma). To count cell numbers,
cell nuclei were stained with 1 ?g/ml 4?,6-diamidino-2-phenylindole
Retrovirus infection in vitro. The full-length cDNAs for mouse Smad6
et al., 2004) were kind gift from Drs. K Miyazono and Y. Gotoh (The
University of Tokyo, Tokyo, Japan), respectively, and cloned into
to virus infection as described previously (Yamamoto et al., 2001b). In-
fected cells were maintained in floating culture for a week, during which
?10% of the cells expressed GFP. The resultant secondary neurospheres
were dissociated, seeded onto PDL-coated chambers, and incubated for
additional 2 d without GFs to induce differentiation.
Immunostaining. Affinity-purified rabbit polyclonal antibodies
(pAbs) against nestin (diluted 1:1000), Olig2 (1:2000), Ngn2 (1:5000),
Rabbit antibody for microtubule-associated protein 2 (MAP2) (react
sity of Tokyo) (Yamamoto et al., 2001b). Mouse monoclonal antibodies
(mAbs) against nestin (Rat401, 1:500), Nkx2.2 (74.5A5, 1:1000), HB9
(81.5C10, 1:50), Islet1 (39.4D5, 1:50), Lim1 (4F2, 1:50), Lim3 (67.4E12,
ies Hybridoma Bank of the University of Iowa. Other antibodies were
purchased from commercial sources: GFP [mouse mAb, 1:500; rabbit
pAb, 1:5000 (Invitrogen); and rat mAb, 1:5000 (Nacalai Tesque, Kyoto,
and rat mAb (Oxford Biotechnology, Oxford, UK)], HuC/D (mouse
mAb, 1:1000; Invitrogen), MAP2 (mouse mAb clone AP20 detecting a
and b subunits, 1:100; Roche), ?-tubulin type III (TuJ1) (mouse mAb,
1:5000; Babco, Richmond, CA), NeuN (mouse mAb, 1:200; Millipore,
Temecula, CA), glial fibrillary acidic protein (GFAP) [mouse mAb,
1:1000 (Millipore) and rabbit pAb, 1:1000 (Sigma)], NG2 (mouse mAb,
Ohorietal.•RegenerationoftheInjuredSpinalCordJ.Neurosci.,November15,2006 • 26(46):11948–11960 • 11949
1:1000, and rabbit pAb, 1:1000; Millipore), myelin basic protein (MBP)
(mouse mAb, 1:1000; Millipore), proteolipid protein (PLP) (mouse
mAb, 1:100; Millipore), O4 (mouse IgM mAb, 1:400; Millipore), galac-
tocerebroside (GalC) (mouse mAb, 1:200; Millipore), glutathione-S-
transferase ? (GST-?) (mouse mAb, 1:50; Becton Dickinson), OX42
Millipore), ?-aminobutyric acid (GABA) (rabbit pAb, 1:500; Sigma),
synaptophysin (mouse mAb, 1:100; Roche), and Mash1 (mouse mAb,
1:200; BD Biosciences).
For immunohistochemistry of tissue sections, rats were killed and
fixed by intracardial perfusion of 4% (w/v) paraformaldehyde (Acros,
Geel, Belgium) in phosphate-buffered saline. Isolated spinal cord tissues
were cryoprotected with 10–30% (w/v) sucrose (Fisher Scientific, Pitts-
burgh, PA), and embedded into OCT compound (Sakura Finetek USA,
Torrance, CA). Staining was visualized with appropriate sets of second-
ary antibodies conjugated with Alexa Fluor 350, 488, 568, 594, and 633
(1:200; Invitrogen) as described previously (Yamamoto et al., 2001b;
Nakatomi et al., 2002).
To examine the total number of virus-infected cells in injured spinal
epicenter). Among these serial sections, representative 12 sections, at
least 280 ?m apart from each other, were subjected to immunostaining
with GFP antibody. The number of GFP?cells in the entire area of each
section was counted manually under Zeiss (Oberkochen, Germany) flu-
tiplied with the number of total sections obtained from each samples
(?360 sections), and then divided by 12 to yield the total number of
GFP?cells per spinal cord.
To examine the coexpression of various cell type-specific markers in
GFP?cells, six representative sections from the above serial transverse
microscope. To further validate the costaining of multiple makers in
single cells, 1–2 representative sections from each animal was further
examined by confocal Z-sectioning at an interval of 1.0 ?m under Zeiss
microscope LSM-501 as described previously (Nakatomi et al., 2002).
Only cells that appeared to retain the intact soma and nuclei within a
long spinal cord stumps (4 mm each for rostral and caudal to the lesion
at least 280 ?m apart from each other, were subjected to immunostain-
was examined as described above by scanning the entire area of individ-
staining for BrdU were included for counting.
Statistical analysis. The quantitative results were expressed as mean ?
SD, and the numbers of replicated experiments are shown in text or
figure legends. Statistical analyses were performed with two-tailed un-
paired t test or one-way ANOVA.
Previous studies have demonstrated that endogenous NPCs pro-
liferate in response to spinal cord injury (Johansson et al., 1999;
Yamamoto et al., 2001a,b; Kojima and Tator, 2002; Horky et al.,
genitors in situ, we used replication-incompetent, recombinant
retroviruses. Retroviruses almost exclusively infect dividing cells
(Leber and Sanes, 1991; Horky et al., 2006). Thus, when directly
administered to injured spinal cords, they are expected to infect
proliferating NPCs together with other cell types. The retrovirus
vector pMXIG used in this study was designed to express GFP so
that virus-infected cells were detected as GFP-positive (GFP?)
cells (Morita et al., 2000; Yamamoto et al., 2001a,b).
Immediately after transection at the thoracic level, a small
damaged parenchyma. At DAI3, virus-infected, GFP?cells were
GFP?cells spread out to broader areas, reaching at a distance of
?2.5 mm from the lesion epicenter both rostrally and caudally
from the lesion. In the areas proximal (?1 mm) to the lesion,
(Fig. 1B). At locations distal (?2 mm) to the lesion, however,
the gray matter where NeuN?neurons were densely populated
(Fig. 1C). Given such widespread distribution of virus-infected
cells, we included 8-mm-long spinal cord stumps encompassing
the T8 to T12 columns for quantitative analyses. As a whole,
2.87 ? 1.28 ? 104and 1.50 ? 0.67 ? 104GFP?cells were
detected at DAI3 and DAI7, respectively, per spinal cord (n ? 3)
after infection with control viruses.
Both FGF2 and EGF are required for proliferation of adult
spinal cord NPCs in vitro and in vivo (Weiss et al., 1996; Johans-
son et al., 1999; Yamamoto et al., 2001a,b; Kojima and Tator,
2002; Martens et al., 2002). Thus, to stimulate their proliferation
retroviruses (1 ?g each per animal). This GF treatment resulted
in 1.6- and 2.7-fold increases in the number of GFP?cells at
4.00 ? 1.80 ? 104cells at DAI7 per spinal cord, n ? 3). More-
significantly higher in GF-treated animals (85.6%) than that in
untreated animals (52.3%) ( p ? 0.01 in two-tailed unpaired t
and survival of virus-infected cells in vivo. Treatment with either
FGF2 or EGF alone, or their combination at a lower dose (0.1 ?g
each) resulted in a much smaller increase (?1.2-fold) in the
number of GFP?cells at DAI7 (data not shown), suggesting a
dose-dependent, combinatorial effect of FGF2 and EGF. We did
not observe, however, any significant difference in the overall
distribution pattern of GFP?cells within injured tissue between
GF-treated and untreated animals. The extent of tissue damage
and overall staining patterns of NeuN, MBP, GFAP, and OX42
also appeared to be similar between the two groups (data not
shown). Thus, although GFs have been shown to exert pleiotro-
pic effects in the injured spinal cord, including modulation of
inflammatory responses, glial scar formation, and survival of
neurons and glia (Cheng et al., 1996; Lee et al., 1999; Teng et al.,
1999; Rabchevsky et al., 2000; Kojima and Tator, 2002; Meijs et
al., 2004), we focused our analyses on their effects on the differ-
entiation of GFP-labeled cells in this study.
PropertiesofGFPvirus-labeledcells in vivo
We next examined the early phenotypes of GFP?cells in injured
short period of time (Leber and Sanes, 1991; Horky et al., 2006).
Therefore, when pMXIG viruses were administered immediately
marked by the BrdU labeling method. Intraperitoneal adminis-
tration of BrdU was initiated right after virus injection and sub-
sequently repeated twice a day for 3 d. In these animals, 28 ?
11950 • J.Neurosci.,November15,2006 • 26(46):11948–11960Ohorietal.•RegenerationoftheInjuredSpinalCord
that GFP viruses indeed infected a population of proliferative
cells in vivo. However, GFP?/BrdU?cells comprised only 6% of
total BrdU?cells, suggesting that the majority of BrdU-labeled
cells proliferated after the period of virus infection. Consistent
with our previous study (Yamamoto et al., 2001a), the major
fractions of these BrdU?cells were OX42?microglia and other
inflammatory cells (44.7%), RECA-1?vascular endothelial cells
cells, as a whole, comprised 66.0% of total BrdU?cells. In con-
trast, these cell types were rather minor among GFP?cells
cell types were preferentially infected with viruses.
It has been shown that cells expressing the proteoglycan NG2
are one of the predominant proliferative cell types in both the
intact and injured spinal cord (Horner et al., 2000; Ishii et al.,
2001; McTigue et al., 2001; Dawson et al., 2003; Horky et al.,
ing the transcription factors Olig2 and Nkx2.2 comprise sub-
populations of proliferative cells in injured tissue (Yamamoto et
al., 2001b; Han et al., 2004; Watanabe et al., 2004; Talbott et al.,
2005). We found that the vast majority of GFP?cells detected at
NG2 (80.7 ? 4.2%; n ? 3 animals) (Fig. 1D,I). These cells did
not overlap with OX42?, RECA-1?, or GFAP?cells (data not
shown) (Yamamoto et al., 2001b; Watanabe et al., 2002, 2004;
these three markers among BrdU?cells were significantly lower
than those among GFP?cells. Given the difference in the period
In line with this idea, when BrdU was administered only once at
DAI0, the fractions of Olig2?and NG2?cells among total
(E) cells (green) with various cell type-specific markers (red) at DAI3. Arrows and arrowheads indicate GFP?cells positive and negative, respectively, for markers shown in each panel. F–H,
Ohorietal.•RegenerationoftheInjuredSpinalCordJ.Neurosci.,November15,2006 • 26(46):11948–11960 • 11951
(59.6 ? 3.2 and 53.3 ? 4.7%, respectively;
n ? 3 animals), whereas the percentage of
OX42?/BrdU?cells became much lower
(23.4 ? 1.1%) compared with those after
repetitive injections for 3 d. Conversely,
when GFP viruses were administered at
both DAI0 and DAI2, the percentage of
than that detected after single administra-
results are in agreement with the recent
cells proliferate early after injury, which is
followed by expansion of OX42?and
GFAP?cells at later stages. Given these
results, we chose the condition of single
virus injection in subsequent studies.
The above results suggested that the
majority of GFP?cells coexpressed all
three markers. We further addressed this
issue using dissociated single cell prepara-
tions (Fig. 1F-H,J). To avoid possible re-
gional variability, cells were recovered
from 8 mm spinal cord stumps where the entire population of
GFP?cells distributed. In such preparations, GFP?cells com-
prised only 1.3 ? 0.6% (n ? 6 animals) of total cells at DAI3.
Among these GFP?cells, 93.3 ? 2.1 and 82.0 ? 7.0% were
Olig2?and Nkx2.2?, respectively (Fig. 1F–H,J). Likewise,
NG2?cells were highly enriched in the GFP?population
(90.7 ? 0.6%). Furthermore, a series of triple staining demon-
strated that the majority (?80%) of GFP?cells were positive for
all three markers (Fig. 1F–H). Most of these cells also expressed
nestin and Sox2, commonly used markers for undifferentiated
NPCs (Fig. 1J) (data not shown). These properties of GFP?cells
were essentially identical between GF-treated and untreated ani-
that represented the total cell population in injured tissue (Fig.
We next sought to examine the frequency of NG2?/Olig2?/
Nkx2.2?cells, which comprised the major fraction of virus-
ers could not be performed because of technical reasons, we
conducted a series of double staining. NG2?cells comprised
NG2?cells, NG2?/Olig2?and NG2?/Nkx2.2?cells were 43
and 60%, respectively (Fig. 2A). Likewise, only a fraction of
Olig2?cells expressed NG2 and Nkx2.2 [20% (2.8 ? 14.2) and
35% (5.0 ? 14.2), respectively], and only 74% (5.0 ? 6.8) and
57% (3.9 ? 6.8) of Nkx2.2?cells coexpressed Olig2 and NG2,
heterogeneous cell types coexisted in the spinal cord, consistent
with the results of previous studies (Yamamoto et al., 2001b;
Watanabe et al., 2004; Talbott et al., 2005; Kitada and Rowitch,
2006). Using the Venn diagram based on these results, we esti-
the total cells in the intact spinal cord (Fig. 2B), which corre-
sponded to 30–40% of total NG2?cells. The fact that the vast
majority of GFP?virus-labeled cells coexpressed three markers
indicates that such triple positive cells indeed exist in vivo. After
transection injury, this cell population increased to 3.9–6.0%
mainly because of a net increase (2.3-fold) in the number of
NG2?cells as observed under other injury conditions such as
cells significantly increased
contusion and demyelination (Watanabe et al., 2004; Talbott et
al., 2005; Horky et al., 2006; Kitada and Rowitch, 2006).
We next asked whether cells infected with GFP viruses in vivo
contained NPCs. Here, we operationally define NPCs as the cells
that can grow as neurospheres in the presence of GFs and differ-
entiate into neurons and glia after removal of GFs in vitro (Weiss
et al., 1996; Johansson et al., 1999; Yamamoto et al., 2001b; Mar-
tens et al., 2002). Injured spinal cords treated with GFP viruses
subsequently expanded as floating neurospheres. Although the
frequency of GFP?cells among initial viable cells was very low
(1.3 ? 0.6% at DIV0; n ? 6), they were significantly enriched
ered as neurospheres were GFP?at DAI14 (n ? 4; p ? 0.01
compared with DAI0 in two-tailed unpaired t test) (Fig. 3A–C).
About one-third of GFP?neurospheres were entirely composed
of GFP?cells (Fig. 3B,B’), and they repeatedly formed GFP?
of GFP?cells in the original samples subjected to culture, such
purely GFP?neurospheres were likely to have derived from sin-
gle GFP?cells. The majority of these GFP?cells in primary
and 81.7 ? 4.2% for Nkx2.2; n ? 3) (Fig. 3D,E,H). About one-
third of GFP?cells were also NG2?(32.0 ? 6.6%; n ? 3)
these markers were also the predominant cell type in virus unin-
rather minor among GFP?cells before neurosphere formation
After removal of GFs, both GFP?and GFP?neurospheres
gave rise to TuJ1?neurons, GFAP?astrocytes, and O4?oligo-
dendrocytes (Fig. 3I,I’). By a series of triple staining, we con-
firmed that most (?95%) of the GFP?spheres composed en-
tirely of GFP?cells contained all three neural lineages (data not
ronal and glial markers were quantified using preparations of
Occurrence of cells expressing NG2, Olig2, and Nkx2.2 in the spinal cord. A, Percentages of NG2?, Olig2?, and
11952 • J.Neurosci.,November15,2006 • 26(46):11948–11960 Ohorietal.•RegenerationoftheInjuredSpinalCord
dissociated single cells. We found that GFP?cells contained all
ulations (Fig. 3J). Altogether, these results demonstrate that a
fraction of GFP-labeled, virus-infected cells indeed exhibited the
properties of NPCs.
We next examined differentiation of
retrovirus-infected cells in vivo. Without
administration of GFs, no GFP?cells ex-
at any time point examined (Fig. 4I) (data
contrast, we found that in GF-treated spi-
nal cords, a significant fraction of GFP?
cells expressed the immature neuronal
markers HuC/D, TuJ1, and c subunit of
MAP2 at DAI3 and DAI7 (Fig. 4A,B,I).
The costaining of GFP and these markers
in the same cells was confirmed under
confocal microscope (Fig. 4A,B, bottom
tion pattern varied among sections exam-
ined. The size (9–14 ?m in diameter) and
shape (round, oval, or spindle) of their
soma were also variable at different loca-
tions. Yet, they commonly harbored mul-
tiple thin processes, typical of differentiat-
ing immature neurons. None of these
however, coexpressed NeuN, a marker
commonly used to identify mature neu-
rons (see below). Given the fact that the
cord are NeuN?, these results reinforce
the idea that GFP viruses did not infect
To further validate the coexpression of
neuronal markers and GFP in single cells,
GF-treated tissue was dissociated into sin-
gle cells and seeded on poly-D-lysine-
coated dishes. GFP?/neuronal marker-
positive cells immediately attached to the
culture surface and actively extended pro-
cesses within 2 h after plating (Fig. 4C–F).
Thus, they were indeed live neurons, not
bored multiple or abnormally enlarged
nuclei; hence, it is unlikely that fusion be-
neurons, which is known to occur at an
jured adult tissue (Alvarez-Dolado et al.,
2003), accounted for the emergence of
Moreover, when BrdU was coadminis-
tered with GFs between DAI0 and DAI2, a
cells among total 1090 BrdU?cells exam-
ined; 0.37%) were detected at DAI7, al-
though such cells were never detected in GF-untreated animals
(data not shown) (Yamamoto et al., 2001a,b). Thus, the results
using both BrdU and GFP viruses supported the idea that new
neurons were generated from endogenous cells in GF-treated
chamber. J, Differentiation of GFP?and GFP?neurosphere cells into neurons and glia. Primary neurospheres at DIV14 were
markers HuC/D (A) and MAP2 (B) (red) in GFP?cells (arrows) at DAI7. The bottom-right panel in each set shows a three-
Ohorietal.•RegenerationoftheInjuredSpinalCordJ.Neurosci.,November15,2006 • 26(46):11948–11960 • 11953
Nakamura and Bregman, 2001; Velardo et
al., 2004). Given the observed effect of ex-
ogenously administered GFs, however, it
appears that their endogenous levels are
not sufficient to support neurogenesis in
the injured spinal cord. This is in sharp
contrast to the situation in other parts of
the CNS, where detectable neurogenesis
occurs after injury without treatment with
katomi et al., 2002; Teramoto et al., 2003).
We next quantitatively assessed the in-
duction of new neurons by GFs. At DAI3,
3.0 ? 0.7% of GFP?cells (19 positive
cells/652 cells examined; n ? 3 animals)
to 22.8 ? 1.9% at DAI7 (224 cells/995
GFP?cells were also HuC/D?and
MAP2?, respectively. The percentage of
GFP?/HuC/D?cells in animals treated
(?5%), suggesting a dose-dependent ef-
fect of GFs on neuronal differentiation.
Given that 4.67 ? 104and 4.00 ? 104
GFP?cells were detected at DAI3 and
DAI7, respectively, the estimated number
of GFP?/TuJ1?cells was 1.40 ? 103at
DAI3 and 9.10 ? 103at DAI7 per GF-
DAI3 and DAI7 ( p ? 0.01), whereas the total number of GFP?
cells rather decreased to 86% during this period. This time-
dependent increase in the actual number of GFP?/neuronal
marker-positive cells reinforces the idea that such cells were un-
likely to be products of cell fusion between pre-existing neurons
and non-neuronal cells, or mere artifacts in histology. Further-
more, albeit that GF-treatment increased the number of GFP?
cells only 1.6-fold at DAI3 and 2.7-fold at DAI7, GFP?cells
animals. These results are consistent with the idea that GFs not
cells gradually decreased after DAI7, and they eventually disap-
peared by DAI28 (data not shown). In addition, as described
above, no GFP?cells were found to express NeuN, which fea-
tures a more mature phenotype of neurons, at any time points
examined when control viruses were used for infection (see
Unlike these neuronal cells, substantial fractions of GFP?
cells expressed glial cell markers GFAP (Fig. 4G) and GalC (Fig.
4H) without treatment with GFs, and their percentages were not
( p ? 0.160 for GFAP?cells and p ? 0.327 for GalC?cells) (Fig.
suggesting that GFP?/GalC?cells detected at DAI7 were newly
generated oligodendrocytes. In fact, it has been demonstrated
that immature oligodendrocytes are generated in both the intact
2000; Ishii et al., 2001; Watanabe et al., 2002, 2004; Talbott et al.,
expressing MBP or PLP, markers for myelin-forming oligoden-
drocytes, at any time points examined in either GF-treated or
peared to be limited in injured tissue (see below). Unlike these
cells in the oligodendrocyte lineage, many GFP?/GFAP?and
(Figs. 1I, 4I). Because mature astrocytes are known to retain the
the astrocyte lineage.
EnhancedneurogenesisbyNeurogenin2andBDNF in vitro
The above study demonstrated that the production of new neu-
rons from endogenous NPCs can be induced under certain con-
ditions. This, in turn, suggests the presence of certain mecha-
nisms that actively suppress the neurogenic potential of NPCs in
situ. We first addressed this issue using in vitro culture of NPCs.
To mimic the situation of virus-infected NPCs in vivo, growing
neurosphere cells were infected with pMXIG viruses, and subse-
quently, neuronal and glial differentiation of GFP?cells after
removal of GFs was examined (Fig. 5).
It has been shown that the expression of various cytokines is
significantly upregulated in the injured spinal cord (Nakamura
and Bregman, 2001; Setoguchi et al., 2001, 2004; Velardo et al.,
2004; Chen et al., 2005). Among them, BMPs and CNTF have
been shown to inhibit neuronal differentiation of NPCs both in
chi et al., 2004). Consistent with this, treatment of neurospheres
with BMP4 and CNTF significantly increased the percentage of
were compared with that of control virus-infected cells (open bars). C, Effect of Ngn2 on neuronal differentiation of NPCs.
Manipulation of neuronal differentiation of NPCs by Ngn2 in vitro. A, Neuronal and glial differentiation of GFP
11954 • J.Neurosci.,November15,2006 • 26(46):11948–11960Ohorietal.•RegenerationoftheInjuredSpinalCord
GFAP?astrocytes among total GFP?cells, and this occurred at
the expense of TuJ1?neurons and O4?oligodendrocytes ( p ?
significantly alter the rate of cell proliferation or death of either
GFP?or GFP?cells in culture (data not shown) and, thus, the
observed effects most likely reflected their actions on differenti-
ation of NPCs. Conversely, the extracellular BMP inhibitor nog-
gin decreased the fraction of GFAP?cells (Fig. 5A). Retrovirus-
mediated overexpression of Smad6 and Smad7, which block
5B). Likewise, a dominant-negative (dn) form of STAT3 (Ka-
makura et al., 2004), which inhibits the activity of endogenous
STAT3, the major intracellular signal transducer downstream of
CNTF receptors (Sun et al., 2001; Kamakura et al., 2004), in-
creased the percentages of TuJ1?and O4?cells ( p ? 0.001 for
TuJ1 and p ? 0.01 for O4) (Fig. 5B). These results suggest that
BMP4 and CNTF (or related cytokines) are expressed by NPCs
themselves and/or their progeny, and that such endogenous fac-
tors inhibit neurogenesis in an autocrine and/or paracrine man-
ner. This could be one of the mechanisms by which neuronal
differentiation of NPCs is attenuated in vivo. However, the effect
of blocking the actions of these endogenous cytokines on neuro-
genesis was rather weak: ?5% of total GFP?cells differentiated
attenuated by Smad6/7, dn-STAT3, or both (Fig. 5B) (data not
chi et al., 2004; Enzmann et al., 2005).
by NPCs. Our previous study suggested that signaling through
the cell-surface receptor Notch is involved in the inhibition of
neuronal differentiation of NPCs, and that overexpression of the
tion (Yamamoto et al., 2001b). A more recent study has also
shown that Ngn2 enhances neuronal differentiation of grafted,
exogenous NPCs in vivo (Hofstetter et al., 2005). Then, we tested
whether Ngn2 can also stimulate neurogenesis in the presence of
Ngn2-expressing retroviruses, 23.9 ? 1.7% of total GFP?cells
in the control culture ( p ? 0.0001; n ? 3)
(Fig. 5C). Under the same conditions, the
percentages of GFAP?astrocytes and
O4?oligodendrocytes were not signifi-
virus-infected cells (data not shown)
(Yamamoto et al., 2001b). Importantly,
the neurogenic action of Ngn2 was pre-
and CNTF. Even a higher percentage of
Ngn2-expressing NPCs differentiated into
neurons in the presence of BMP4 than in
its absence ( p ? 0.001), consistent with a
previous study using embryonic brain-
derived NPCs (Sun et al., 2001). More-
over, BDNF, which promotes differentia-
tion and survival of new neurons in the
adult CNS (Namiki et al., 2000; Coumans
et al., 2001; Chmielnicki et al., 2004), in-
creased the percentage of TuJ1?neurons
generated by Ngn2-expressing
(29.4 ? 1.0%; n ? 3; p ? 0.005).
StimulationofneurogenesisbyNgn2andBDNF in vivo
Based on these in vitro results, we next tested the activities of
Ngn2 viruses and BDNF in vivo. Unlike control virus-infected
cells, a small, but significant percentage of Ngn2 virus-infected
cells became HuC/D?(2.3 ? 3.2%; n ? 3) and NeuN?(3.0 ?
and 21.1 ? 2.3%, respectively; n ? 3 animals; p ? 0.01). In the
presence of GFs, however, the percentages of GFP?/HuC/D?
cells did not significantly differ between control and Ngn2 virus-
infected animals ( p ? 0.1404). Thus, GF treatment appeared to
exert a stronger effect than Ngn2 overexpression on the genera-
of Ngn2 and GFs showed a much stronger activity to induce
( p ? 0.01), suggesting that these two manipulations collaborate
to induce NeuN?neurons.
The coexpression of Ngn2 confirmed that GFP?/NeuN?
neurons were derived from Ngn2 virus-infected cells (Fig. 6B).
Moreover, many GFP?/NeuN?cells were also labeled with
BrdU administered between DAI0 and DAI2, indicating that
such cells were indeed generated by cells that proliferated in situ
or without GFs. Nevertheless, GFP?/NeuN?cells were detected
only in Ngn2 virus-infected animals. Thus, we conclude that the
possibility that the costaining of GFP and NeuN was caused by
certain artifacts is highly unlikely.
As shown in Figure 6, C and D, many GFP?cells in Ngn2
virus-infected tissues developed thick processes with intense
MAP2 staining. Their soma and processes were often associated
tons of surrounding preexisting neurons (Fig. 6D, arrows), sug-
gesting more mature properties of GFP?/NeuN?neurons than
those of GFP?/HuC/D?cells. Most (?95%) of these GFP?/
NeuN?neurons were positive for GABA (Fig. 6E), but negative
at DAI7. GFP?/HuC/D?cells were detected in dissociated single cells, whereas GFP?/NeuN?cells were detected in tissue
Ohorietal.•RegenerationoftheInjuredSpinalCordJ.Neurosci.,November15,2006 • 26(46):11948–11960 • 11955
example, GFP?/NeuN?cells detected in the anterior horn were
scattered within a cluster of large motor neurons and smaller
?m; n ? 8) (Fig. 6F). However, the morphology and location of
individual GFP?/NeuN?cells were highly variable depending
on their relative distance from the lesion epicenter and also
among treated animals. Moreover, none of these neurons ex-
pressed subtype-specific molecular markers examined such as
HB9, Islet1, Lim1, and Lim3 (Yamamoto et al., 2001b and refer-
ences therein), and therefore whether they differentiated into
specific neuronal subtypes remained undetermined.
alone, nor induced GFP?/NeuN?cells in control virus-infected
animals (no GFP?/NeuN?cells among 652 GFP?cells exam-
ined). When combined with Ngn2 and GFs, however, BDNF
significantly increased the percentage of GFP?/NeuN?cells
among total GFP?cells (28.2 ? 3.4%; n ? 3 animals; p ? 0.01
compared with animals without BDNF treatment) (Fig. 7A).
cells was significantly lower in both Ngn2/GF- and Ngn2/GF/
BDNF-treated animals compared with the control level (3.8 ?
0.9 and 3.7 ? 0.4% vs 6.3 ? 0.5%; p ? 0.01) (Fig. 7B). This
decrease alone, however, could not fully account for the much
larger increase of GFP?/NeuN?cells, suggesting that Ngn2 and
BDNF did not simply inhibit gliogenesis, but rather actively pro-
moted generation of neurons.
We further followed the survival of GFP?/NeuN?cells in
vivo. At DAI7, the estimated number of GFP?/NeuN?neurons
was 5.4 ? 0.5 ? 103(n ? 3) per spinal cord in Ngn2 virus-
infected/GF-treated animals (Fig. 7C). Their numbers, however,
were only 33 and 3% at DAI14 and DAI28, respectively, com-
pared with that detected at DAI7. Although the total number of
of NeuN?neurons among them also decreased over time (Fig.
7A). Thus, GFP?/NeuN?new neurons appeared to be elimi-
nated faster than other GFP?cell populations in injured tissue.
Silencing of the GFP transgene could partly explain the observed
loss of GFP-labeled new neurons (Vroemen et al., 2003). How-
to DAI28. Furthermore, we observed longer survival of Ngn2
virus-infected cells in other parts of the CNS (our unpublished
results). Thus, we favor the idea that the observed decrease re-
flected the actual loss of new neurons in injured spinal cords.
Consistent with this idea, when the neurotrophic factor BDNF,
which is thought to promote survival of neurons, increased the
number of GFP?/NeuN?cells 1.9-fold in Ngn2/GF-treated an-
imals at DAI7 (9.4 ? 0.2 ? 103; n ? 4; p ? 0.001 in two-tailed
unpaired t test) (Fig. 7C). Moreover, larger numbers of GFP?/
NeuN?cells remained at DAI14 and DAI28 in BDNF-treated
animals ( p ? 0.0001) (Fig. 7C). However, few GFP?/NeuN?
cells remained detectable at DAI56 or later time points (data not
appears to be very limited in the injured spinal cord.
We next tested the effect of another proneural transcription fac-
tor, Mash1, which has been implicated in both neurogenesis and
oligodendrogenesis during development (Parras et al., 2004).
When NPCs were isolated as neurospheres from Mash1 viruses-
infected tissue, significantly higher percentages of Mash1-
cytes, and conversely, a much smaller fraction became GFAP?
astrocytes compared with control virus-infected cells (Fig. 8A).
Unlike Ngn2, Mash1 did not change the percentage of TuJ1?
neurons among GFP?cells. Thus, Mash1 selectively increased
oligodendrocytes in culture of adult spinal cord NPCs.
As described above, a substantial fraction of control virus-
infected cells were GalC?in vivo (Fig. 4I). These results are con-
sistent with previous studies in which production of new oligo-
dendrocytes by NG2?cells was detected under various insult
conditions (McTigue et al., 1998, 2001; Ishii et al., 2001; Wa-
tanabe et al., 2002, 2004; Talbott et al., 2005; Zai and Wrathall,
2005; Yang et al., 2006). In line with this, we found that some
NG2?cells in injured tissue expressed endogenous Mash1 (Fig.
8B). This is in sharp contrast to endogenous Ngn2; we could not
injury (data not shown) (Yamamoto et al., 2001b). Such NG2?/
Mash1?cells, however, were small in number at DAI14, and
almost disappeared at DAI28. These results raise the possibility
that endogenous Mash1 is involved in the generation of new
oligodendrocytes, but its limited expression accounts for their
restricted generation and maturation in injured tissue.
To test this idea, we examined the effect of constitutive over-
expression of Mash1 together with GF treatment in vivo. Consis-
tent with the results of the above in vitro experiments, signifi-
cantly larger fractions of Mash1 virus-infected cells became
GalC?and GST-??oligodendrocytes compared with control
(Fig. 8F). Because few GFP?cells expressed these markers at
of new oligodendrocytes in situ. Furthermore, at DAI28, a small
but significant fraction of GFP?cells expressed RIP (Fig. 8D)
and PLP (Fig. 8E), markers for more mature, myelin-forming
11956 • J.Neurosci.,November15,2006 • 26(46):11948–11960Ohorietal.•RegenerationoftheInjuredSpinalCord
cells, which were never detected in control virus-infected tissues
cells at DAI7 and DAI28, respectively, in animals treated with
Mash1 viruses and GFs. The estimated number of GFP?/
Despite this relatively large number of immature cells detected
early, only 2.7% of them appeared to advance to PLP?cells at
DAI28 (510 GFP?/PLP?cells per spinal cord). Moreover,
GFP?/PLP?and GFP?/GST-??cells were barely detectable at
DAI56 and later time points (data not shown). Instead, the ma-
jority (50.8 ? 6.3%; n ? 3 animals) of Mash1-expressing cells
remained NG2?at DAI28. These results suggest that the major
immature cells and their maturation to myelin-forming cells.
Spontaneous tissue regeneration after damage is very limited in
the adult spinal cord. Many lines of recent studies have demon-
strated that such limitation is attributable to, at least in part,
see Q. Cao et al., 2002). In this study, we describe strategies to
overcome such restriction.
Retrovirus-mediatedgeneticmanipulationofNPCs in situ
We used GFP-expressing retroviruses to genetically manipulate
proliferative cells in the injured spinal cord. We found that a
fraction of virus-infected, GFP?cells grew as neurospheres and
differentiated into neurons and glia in culture, demonstrating
that they exhibited the properties of NPCs. Importantly, the ma-
jority (?80%) of GFP?cells that formed neurospheres were
expressing these markers were also the predominant cell type
among the whole neurosphere-forming cells derived from the
injured spinal cord.
NG2?cells in the adult CNS have originally been thought to
be glia-restricted progenitors (Horner et al., 2000; Dawson et al.,
2003). Previous studies, however, have revealed that a subpopu-
produce neurons (Belachew et al., 2003; Nunes et al., 2003).
cord (Ishii et al., 2001; McTigue et al., 2001; Watanabe et al.,
have demonstrated previously that NG2?cells are the predomi-
nant cell type that divides early after injury. Other studies have
fraction of proliferative cells, and that many of them coexpress
NG2 (Yamamoto et al., 2001b; Watanabe et al., 2004; Talbott et
al., 2005; Kitada and Rowitch, 2006). Consistent with these ob-
servations, GFP retroviruses administered immediately after in-
jury preferentially infected Olig2?/Nkx2.2?/NG2?cells. Horky
et al. (2006) also reported a similar result using a different virus
construct and injury paradigm. Given the observation that a sig-
nificant fraction of these cells differentiated into neurons or oli-
godendrocytes in GF-treated animals, these results suggest that
they represent at least a part of endogenous NPCs in the adult
spinal cord. We found, however, that only ?40% of NG2?cells
and Nkx2.2?cells contain both NG2?and NG2?cell popula-
tions (Watanabe et al., 2004; Talbott et al., 2005). Thus, cells
appears to be specific for NPCs. Moreover, although the vast
all of these cells formed neurospheres in vitro. This could be
because NPCs are only a fraction among cells expressing these
ditions do not support proliferation of all NPCs in vitro. More
control (open bars) and Mash1 (filled bars) viruses were subjected to neurosphere culture at
glial cell markers were quantified (mean ? SD; n ? 3–6 animals; *p ? 0.05; **p ? 0.01
compared with control virus-infected cells). B, Expression of endogenous Mash1 (green) in
markers GST- ? (C), RIP (D), and PLP (E) (red) in Mash1 virus-infected, GFP?cells (green,
ages of GFP?cells expressing oligodendroglial markers in spinal cords infected with control
examined using dissociated single cells, whereas GST-??and PLP?cells were detected in
Ohorietal.•RegenerationoftheInjuredSpinalCord J.Neurosci.,November15,2006 • 26(46):11948–11960 • 11957
studies are necessary to define the in vivo identity of NPCs in the
adult spinal cord.
Differentiation of NPCs into neurons and oligodendrocytes is
tightly restricted by the environment in the injured spinal cord.
Then, how do the manipulations described in this study over-
come such restriction? First, it is unlikely that otherwise non-
NPC cells transdifferentiated into NPC-like cells in response to
fraction of GFP?cells early after infection were essentially iden-
tical between manipulated and unmanipulated tissues, and
moreover, such phenotypes were preserved in GFP?cells-
derived neurospheres. Thus, pre-existing, endogenous NPCs
drocytes in vivo.
Previous studies reported various beneficial actions of GFs in
1999; Rabchevsky et al., 2000; Kojima and Tator, 2002; Meijs et
al., 2004). Their effects on neurogenesis by endogenous NPCs,
that direct administration of GFs into injured tissue can induce
the production of new neurons in the otherwise non-neurogenic
GFs also increased the number of GFP?/TuJ1?new neurons
between DAI3 and DAI7. These results are consistent with the
idea that GFs stimulated both proliferation and neuronal differ-
entiation of endogenous NPCs. GFs might have enhanced sur-
vival of NPCs and newborn neurons as well. GFs act as mitogens
for NPCs in vitro (Weiss et al., 1996; Kojima and Tator, 2002;
itory for their differentiation. Therefore, their neuron-inducing
lular molecules likely act simultaneously on NPCs in vivo so that
the outcome of their combinatorial actions could be different
from that observed in vitro. In fact, previous studies have shown
that exogenous GFs can enhance neurogenesis after brain injury
with other previous studies, suggest that the induction of new
neurons by GFs could be through interactions with multiple sig-
naling pathways such as those for Notch, BMPs, and CNTF
(Yamamoto et al., 2001b; Chojnacki et al., 2003; Mikami et al.,
2004; Setoguchi et al., 2004). In this context, GFs could either
directly act on NPCs, or indirectly modulate their activities
through acting on other cell types such as inflammatory cells
Yang et al., 2006). How GFs stimulate neurogenesis in the com-
plex environment of injured tissue remains to be clarified.
Our data suggest that maturation is another limiting step in
neuronal cell replacement in the injured spinal cord. Although a
significant fraction of GFP?cells became HuC/D?cells in GF-
treated animals, few cells were found to express NeuN that fea-
tures a more mature phenotype of neurons. Although the mech-
anisms underlying this inhibition are currently unknown, we
found that overexpression of Ngn2 can overcome this limiting
step. Although Ngn2 alone strongly stimulated neurogenesis by
neurons in vivo was rather weak in the absence of GFs. However,
even without GFs, a small, but significant number of Ngn2-
GFs, Ngn2 dramatically increased the number of GFP?/NeuN?
cells. Thus, the action of Ngn2 appeared to be distinct from that
of GFs, and their combination was most effective in inducing
neurogenesis in vivo.
In contrast, differentiation of GFP?cells into GalC?/
GST-??immature oligodendrocytes was detectable even in GF-
untreated animals. Yet, their maturation to MBP?/PLP?
myelin-forming cells did not occur at a detectable level. We
showed that overexpression of Mash1 can enhance the produc-
tion of GalC?/GST-??cells, and that at least some of these cells
proceed to more mature PLP?oligodendrocytes. These results
suggest that like neuronal cells, maturation and survival is a cru-
cord. This could be attributable to the absence of appropriate
trophic support and/or the presence of cell death-inducing sig-
nals (Nakamura and Bregman, 2001; Velardo et al., 2004). Thus,
a possible means to promote survival of new neurons and oligo-
dendrocytes could be a sustained supply of neurotrophic factors
2000; Coumans et al., 2001; Meijs et al., 2004; Cao et al., 2005).
Moreover, integration into the circuitry is probably important
for their maturation and survival in vivo (Dobkin and Havton,
2004). Thus, strategies to enhance regeneration of these cells lo-
In this study, we detected ?9400 NeuN?new neurons in Ngn2
parts of the CNS (Arvidsson et al., 2002; Nakatomi et al., 2002;
et al., 2001, 2002; Hofstetter et al., 2005). Considering that our
retrovirus-mediated method labeled only a small fraction of
enous NPCs is likely larger than this level. However, poor long-
term survival of new neurons is still the major issue common to
the strategies using endogenous and exogenous NPCs. Thus, in
terms of functional recovery, significance of supplying new neu-
rons at this level of quantity remains to be explored. In case of
transplantation of exogenous NPCs, many cell types other than
effects (Lu et al., 2003; Hofstetter et al., 2005). Under certain
circumstances, grafted cells appear to exert detrimental effects as
well (Enzmann et al., 2005; Hofstetter et al., 2005). Similar situ-
ations may also need to be considered in case of mobilizing en-
dogenous NPCs by growth factor treatment and genetic manip-
ulations. Additional improvement of such strategies may lead to
development of novel cell replacement therapy for spinal cord
Lois C, Morrison SJ, Alvarez-Buylla A (2003) Fusion of bone-marrow-
derived cells with Purkinje neurons, cardiomyocytes and hepatocytes.
Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O (2002) Neuronal re-
placement from endogenous precursors in the adult brain after stroke.
Nat Med 8:963–970.
(2003) Postnatal NG2 proteoglycan-expressing progenitor cells are in-
trinsically multipotent and generate functional neurons. J Cell Biol
Cao Q, Benton RL, Whittemore SR (2002) Stem cell repair of central ner-
vous system injury. J Neurosci Res 68:501–510.
11958 • J.Neurosci.,November15,2006 • 26(46):11948–11960 Ohorietal.•RegenerationoftheInjuredSpinalCord
Cao Q, Xu XM, DeVries WH, Enzmann GU, Ping P, Tsoulfas P, Wood PM,
Bunge MB, Whittemore SR (2005) Functional recovery in traumatic
spinal cord injury after transplantation of multineurotrophin-expressing
glial-restricted precursor cells. J Neurosci 25:6947–6957.
Cao QL, Zhang YP, Howard RM, Walters WM, Tsoulfas P, Whittemore SR
rat spinal cord are restricted to a glial lineage. Exp Neurol 167:48–58.
Cao QL, Howard RM, Dennison JB, Whittemore SR (2002) Differentiation
of engrafted neuronal-restricted precursor cells is inhibited in the trau-
matically injured spinal cord. Exp Neurol 177:349–359.
Chen J, Leong SY, Schachner M (2005) Differential expression of cell fate
determinants in neurons and glial cells of adult mouse spinal cord after
compression injury. Eur J Neurosci 22:1895–1906.
Cheng H, Cao Y, Olson L (1996) Spinal cord repair in adult paraplegic rats:
partial restoration of hind limb function. Science 273:510–513.
Chmielnicki E, Benraiss A, Economides AN, Goldman SA (2004) Adenovi-
rally expressed noggin and brain-derived neurotrophic factor cooperate
to induce new medium spiny neurons from resident progenitor cells in
the adult striatal ventricular zone. J Neurosci 24:2133–2142.
Chojnacki A, Shimazaki T, Gregg C, Weinmaster G, Samuel Weiss S (2003)
Glycoprotein 130 signaling regulates notch1 expression and activation in
the self-renewal of mammalian forebrain neural stem cells. J Neurosci
A, Fischer I (2000) Characterization and intraspinal grafting of EGF/
bFGF-dependent neurospheres derived from embryonic rat spinal cord.
Brain Res 874:87–106.
(2001) Axonal regeneration and functional recovery after complete spi-
nal cord transection in rats by delayed treatment with transplants and
neurotrophins. J Neurosci 21:9334–9344.
Dawson MR, Polito A, Levine JM, Reynolds R (2003) NG2-expressing glial
progenitor cells: an abundant and widespread population of cycling cells
in the adult rat CNS. Mol Cell Neurosci 24:476–488.
Dobkin BH, Havton LA (2004) Basic advances and new avenues in therapy
of spinal cord injury. Annu Rev Med 55:255–282.
Enzmann GU, Benton RL, Woock JP, Howard RM, Tsoulfas P, Whittemore
SR (2005) Consequencesofnogginexpressionbyneuralstem,glial,and
neuronal precursor cells engrafted into the injured spinal cord. Exp Neu-
Goldman SA (2004) Directed mobilization of endogenous neural progeni-
Mol Ther 6:466–472.
Han SS, Kang DY, Mujtaba T, Rao MS, Fischer I (2002) Grafted lineage-
restricted precursors differentiate exclusively into neurons in the adult
spinal cord. Exp Neurol 177:360–375.
Han SS, Liu Y, Tyler-Polsz C, Rao MS, Fischer I (2004) Transplantation of
glial-restricted precursor cells into the adult spinal cord: survival, glial-
specific differentiation, and preferential migration in white matter. Glia
Hauben E, Schwartz M (2003) Therapeutic vaccination for spinal cord in-
jury: helping the body to cure itself. Trends Pharmacol Sci 24:7–12.
Hill CE, Proschel C, Noble M, Mayer-Proschel M, Gensel JC, Beattie MS,
Bresnahan JC (2004) Acute transplantation of glial-restricted precursor
cells into spinal cord contusion injuries: survival, differentiation, and
effects on lesion environment and axonal regeneration. Exp Neurol
Hofstetter CP, Holmstrom NA, Lilja JA, Schweinhardt P, Hao J, Spenger C,
Wiesenfeld-Hallin Z, Kurpad SN, Frisen J, Olson L (2005) Allodynia
limits the usefulness of intraspinal neural stem cell grafts; directed differ-
entiation improves outcome. Nat Neurosci 8:346–353.
Horky LL, Galimi F, Gage FH, Horner PJ (2006) Fate of endogenous stem/
progenitor cells following spinal cord injury. J Comp Neurol
Horner PJ, Gage FH (2000) Regenerating the damaged central nervous sys-
tem. Nature 407:963–970.
Horner PJ, Power AE, Kempermann G, Kuhn HG, Palmer TD, Winkler J,
Thal LJ, Gage FH (2000) Proliferation and differentiation of progenitor
Ishii K, Toda M, Nakai Y, Asou H, Watanabe M, Nakamura M, Yato Y,
Fujimura Y, Kawakami Y, Toyama Y, Uyemura K (2001) Increase of
oligodendrocyte progenitor cells after spinal cord injury. J Neurosci Res
Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J (1999)
Identification of a neural stem cell in the adult mammalian central ner-
vous system. Cell 96:25–34.
Kamakura S, Oishi K, Yoshimatsu T, Nakafuku M, Masuyama N, Gotoh Y
(2004) Hes binding to STAT3 mediates crosstalk between Notch and
JAK-STAT signalling. Nat Cell Biol 6:547–554.
Kitada M, Rowitch DH (2006) Transcription factor co-expression patterns
indicate heterogeneity of oligodendroglial subpopulations in the adult
spinal cord. Glia 54:35–46.
KojimaA,TatorCH (2002) Intrathecaladministrationofepidermalgrowth
factor and fibroblast growth factor 2 promotes ependymal proliferation
and functional recovery after spinal cord injury in adult rats. J Neuro-
Leber SM, Sanes JR (1991) Lineage analysis with a recombinant retrovirus:
application to chick spinal motor neurons. Adv Neurol 56:27–36.
Lee TT, Green BA, Dietrich WD, Yezierski RP (1999) Neuroprotective ef-
fects of basic fibroblast growth factor following spinal cord contusion
injury in the rat. J Neurotrauma 16:347–356.
Lim DA, Tramontin AD, Trevejo JM, Herrera DG, Garcia-Verdugo JM,
Alvarez-Buylla A (2000) Noggin antagonizes BMP signaling to create a
niche for adult neurogenesis. Neuron 28:713–726.
I (1999) Transplants of fibroblasts genetically modified to express
BDNF promote regeneration of adult rat rubrospinal axons and recovery
of forelimb function. J Neurosci 19:4370–4387.
Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem cells consti-
tutively secrete neurotrophic factors and promote extensive host axonal
growth after spinal cord injury. Exp Neurol 181:115–129.
Martens DJ, Seaberg RM, van der Kooy D (2002) In vivo infusions of exog-
enous growth factors into the fourth ventricle of the adult mouse brain
cle and the central canal of the spinal cord. Eur J Neurosci 16:1045–1057.
McTigue DM, Horner PJ, Stokes BT, Gage FH (1998) Neurotrophin-3 and
brain-derived neurotrophic factor induce oligodendrocyte proliferation
and myelination of regenerating axons in the contused adult rat spinal
cord. J Neurosci 18:5354–5365.
McTigue DM, Wei P, Stokes BT (2001) Proliferation of NG2-positive cells
and altered oligodendrocyte numbers in the contused rat spinal cord.
J Neurosci 21:3392–3400.
Meijs MF, Timmers L, Pearse DD, Tresco PA, Bates ML, Joosten EA, Bunge
MB, Oudega M (2004) Basic fibroblast growth factor promotes neuro-
nal survival but not behavioral recovery in the transected and Schwann
cell implanted rat thoracic spinal cord. J Neurotrauma 21:1415–1430.
Mikami Y, Okano H, Sakaguchi M, Nakamura M, Shimazaki T, Okano HJ,
Kawakami Y, Toyama Y, Toda M (2004) Implantation of dendritic cells
in injured adult spinal cord results in activation of endogenous neural
stem/progenitor cells leading to de novo neurogenesis and functional
recovery. J Neurosci Res 76:453–465.
Mocchetti I, Rabin SJ, Colangelo AM, Whittemore SR, Wrathall JR (1996)
Increased basic fibroblast growth factor expression following contusive
spinal cord injury. Exp Neurol 141:154–164.
MoritaS,KojimaT,KitamuraT (2000) Plat-E:anefficientandstablesystem
for transient packaging of retroviruses. Gene Ther 7:1063–1066.
Nakamura M, Bregman BS (2001) Differences in neurotrophic factor gene
Exp Neurol 169:407–415.
M, Miyazono K, Kishimoto T, Kageyama R, Taga T (2001) BMP2-
mediated alteration in the developmental pathway of fetal mouse brain
cells from neurogenesis to astrocytogenesis. Proc Natl Acad Sci USA
Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N,
Tamura A, Kirino T, Nakafuku M (2002) Regeneration of hippocampal
pyramidal neurons after ischemic brain injury by recruitment of endog-
enous neural progenitors. Cell 110:429–441.
Namiki J, Kojima A, Tator CH (2000) Effect of brain-derived neurotrophic
factor, nerve growth factor, and neurotrophin-3 on functional recovery
and regeneration after spinal cord injury in adult rats. J Neurotrauma
Nunes MC, Roy NS, Keyoung HM, Goodman RR, McKhann G II, Jiang L,
Ohorietal.•RegenerationoftheInjuredSpinalCord J.Neurosci.,November15,2006 • 26(46):11948–11960 • 11959
KangJ,NedergaardM,GoldmanSA (2003) Identificationandisolation
of multipotential neural progenitor cells from the subcortical white mat-
ter of the adult human brain. Nat Med 9:439–447.
Parras CM, Galli R, Britz O, Soares S, Galichet C, Battiste J, Johson JE, Naka-
fuku M, Vescovi A, Guillemot F (2004) Mash1 specifies neurons and
oligodendrocytes in the postnatal brain. EMBO J 23:4495–4505.
Rabchevsky AG, Fugaccia I, Turner AF, Blades DA, Mattson MP, Scheff SW
ery following severe spinal cord injury to the rat. Exp Neurol
Schwab ME (2002) Repairingthe
Setoguchi T, Yone K, Matsuoka E, Takenouchi H, Nakashima K, Sakou T,
Komiya S, Izumo S (2001) Traumatic injury-induced BMP7 expression
in the adult rat spinal cord. Brain Res 921:219–225.
Setoguchi T, Nakashima K, Takizawa T, Yanagisawa M, Ochiai W, Okabe M,
Yone K, Komiya S, Taga T (2004) Treatment of spinal cord injury by
transplantation of fetal neural precursor cells engineered to express BMP
inhibitor. Exp Neurol 189:33–44.
Shihabuddin LS, Horner PJ, Ray J, Gage FH (2000) Adult spinal cord stem
cells generate neurons after transplantation in the adult dentate gyrus.
J Neurosci 20:8727–8735.
Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neu-
berg ME (2001) Neurogenin promotes neurogenesis and inhibits glial
differentiation by independent mechanisms. Cell 104:365–376.
Talbott JF, Loy DN, Liu Y, Qiu MS, Bunge MB, Rao MS, Whittemore SR
(2005) Endogenous Nkx2.2?/Olig2? oligodendrocyte precursor cells
astrocytes. Exp Neurol 192:11–24.
Teng YD, Mocchetti I, Taveira-DaSilva AM, Gillis RA, Wrathall JR (1999)
Basic fibroblast growth factor increases long-term survival of spinal mo-
tor neurons and improves respiratory function after experimental spinal
cord injury. J Neurosci 19:7037–7047.
Teramoto T, Qiu J, Plumier JC, Moskowitz MA (2003) EGF amplifies the
replacement of parvalbumin-expressing striatal interneurons after isch-
emia. J Clin Invest 111:1125–1132.
Torii M, Matsuzaki F, Osumi N, Kaibuchi K, Nakamura S, Casarosa S, Guil-
lemot F, Nakafuku M (1999) Transcription factors Mash-1 and Prox-1
delineate early steps in differentiation of neural stem cells in the develop-
ing central nervous system. Development 126:443–456.
TE, Muzyczka N, Reier PJ (2004) Patterns of gene expression reveal a
temporally orchestrated wound healing response in the injured spinal
cord. J Neurosci 24:8562–8576.
Vroemen M, Aigner L, Winkler J, Weidner N (2003) Adult neural progeni-
tor cell grafts survive after acute spinal cord injury and integrate along
axonal pathways. Eur J Neurosci 18:743–751.
Watanabe M, Toyama Y, Nishiyama A (2002) Differentiation of prolifer-
ated NG2-positive glial progenitor cells in a remyelinating lesion. J Neu-
rosci Res 69:826–836.
Watanabe M, Hadzic T, Nishiyama A (2004) Transient upregulation of
Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds
BA (1996) MultipotentCNSstemcellsarepresentintheadultmamma-
lian spinal cord and ventricular neuroaxis. J Neurosci 16:7599–7609.
Proliferation of parenchymal neural progenitors in response to injury in
the adult rat spinal cord. Exp Neurol 172:115–127.
Takebayashi H, Nabeshima Y, Kitamura T, Weinmaster G, Nakamura K,
Nakafuku M (2001b) Transcription factor expression and Notch-
dependent regulation of neural progenitors in the adult rat spinal cord.
J Neurosci 21:9814–9823.
Yang H, Lu P, McKay HM, Bernot T, Keirstead H, Steward O, Gage FH,
EdgertonVR,TuszynskiMH (2006) Endogenousneurogenesisreplaces
oligodendrocytes and astrocytes after primate spinal cord injury. J Neu-
Zai LJ, Wrathall JR (2005) Cell proliferation and replacement following
contusive spinal cord injury. Glia 50:247–257.
11960 • J.Neurosci.,November15,2006 • 26(46):11948–11960Ohorietal.•RegenerationoftheInjuredSpinalCord