Bone Morphogenetic Protein Signaling and Olig1/2 Interact to

Regulate the Differentiation and Maturation of Adult

Oligodendrocyte Precursor Cells
XIAOXIN CHENG,
a,b
YAPING WANG,
a,b,d
QIAN HE,
a,b
MENGSHENG QIU,
a,c
SCOTT R. WHITTEMORE,
a,b,c
QILIN CAO
a,b
a
Kentucky Spinal Cord Injury Research Center and Departments of
b
Neurological Surgery and
c
Anatomical Sciences
and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky, USA;
d
Department of
Anesthesiology, Second Xian-Ya Hospital of South Central University, Changsha, Hunan, Peoples Republic
of China
Key Words. Adult oligodendrocyte precursor cell Spinal cord Bone morphogenetic protein Transcription factor Differentiation
Remyelination
ABSTRACT
Promotion of remyelination is an important therapeutic
strategy for the treatment of the demyelinating neurological
disorders. Adult oligodendrocyte precursor cells (OPCs),
which normally reside quiescently in the adult central ner-
vous system (CNS), become activated and proliferative after
demyelinating lesions. However, the extent of endogenous
remyelination is limited because of the failure of adult OPCs
to mature into myelinating oligodendrocytes (OLs) in the
demyelinated CNS. Understanding the molecular mecha-
nisms that regulate the differentiation of adult OPCs could
lead to new therapeutic strategies to treat these disorders. In
this study, we established a stable culture of adult spinal
cord OPCs and developed a reliable in vitro protocol to
induce their sequential differentiation. Adult OPCs ex-
pressed bone morphogenetic protein (BMP) type Ia, Ib, and
II receptor subunits, which are required for BMP signal
transduction. BMP2 and 4 promoted dose-dependent astro-
cyte differentiation of adult OPCs with concurrent suppres-
sion of OL differentiation. Treatment of OPCs with BMP2
and 4 increased ID4 expression and decreased the expres-
sion of olig1 and olig2. Overexpression of olig1 or olig2
blocked the astrocyte differentiation of adult OPCs induced
by BMP2 and 4. Furthermore, overexpression of both olig1
and olig2, but not olig1 or olig2 alone, rescued OL differenti-
ation from inhibition by BMP2 and 4. Our results demon-
strated that downregulation of olig1 and olig2 is an important
mechanism by which BMP2 and 4 inhibit OL differentiation of
adult OPCs. These data suggest that blocking BMP signaling
combined with olig1/2 overexpression could be a useful thera-
peutic strategy to enhance endogenous remyelination and fa-
cilitate functional recovery in CNS demyelinated disorders.
STEM CELLS 2007;25:32043214
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
In spite of the ubiquitous distribution in the mature central
nervous system (CNS), the majority of oligodendrocytes (OLs)
originate from relatively discrete ventral regions along the neu-
ral axis during early embryogenesis. They subsequently migrate
laterally and dorsally to populate all parts of the developing
CNS before differentiating into myelin-forming OLs [1, 2]. The
secreted glycoprotein Sonic Hedgehog (Shh), released from the
notochord and floor plate, is primarily responsible for the initial
specification of oligodendrocyte precursor cells (OPCs) in the
ventral spinal cord [35]. More recent studies show that a
small number of OLs are generated from the dorsal spinal
cord or forebrain during later developmental stages in a
Shh-independent manner [68]. Irrespective of their ventral
or dorsal origin, however, two basic-helix-loop-helix (bHLH)
transcription factors, olig1 and olig2, are ultimately required
for OL development, since OLs completely fail to develop in
olig1 and olig2 double-null mice [9, 10]. During CNS devel-
opment, bone morphogenetic proteins (BMPs), members of
the transforming growth factor- protein family of extracel-
lular ligands, repress oligodendrogenesis while enhancing the
development of astrocytes [11, 12]. Implantation of noggin-
producing cells into the early developing chicken spinal cord
or anti-BMP antibody coated beads into developing Xenopus
promotes the subsequent appearance of OL progenitors in
dorsal neural tube [13, 14], suggesting that endogenous dor-
sally expressed BMPs inhibit oligodendrogenesis. Con-
versely, elevated BMP expression inhibits the appearance of
ventral OL progenitors [13, 14], and overexpression of BMP4
enhances astrocytic lineage commitment in vivo and signif-
icantly inhibits the generation of OLs [15]. In vitro, BMP
signaling promotes astrocytic differentiation at the expense
of OLs from embryonic or postnatal multipotential NSCs
[1620] or OPCs [21, 22]. However, the mechanism(s) by
which BMP signaling inhibits OL development remain to be
elucidated.
Correspondence: Qilin Cao, M.D., Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, MDR616, 511 South Floyd
Street, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA. Telephone: 502-852-0284; Fax: 502-852-5148;
e-mail: q0cao001@gwise.louisville.edu Received April 18, 2007; accepted for publication August 31, 2007; first published online in STEM
CELLS EXPRESS September 13, 2007. AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0284
TISSUE-SPECIFIC STEM CELLS
STEMCELLS 2007;25:32043214 www.StemCells.com
OPCs reside in the white matter of the adult CNS [23, 24].
Although these cells remain relatively quiescent in the normal
CNS, they become activated and proliferate in response to CNS
injury, including demyelination [2528]. More importantly,
these cells have the potential to differentiate into mature OLs
and myelinate axons both in vitro and in vivo [2931]. How-
ever, endogenous remyelination by adult OPCs is very limited in
a number of demyelinated disorders, including multiple sclero-
sis [32, 33] and spinal cord injury (SCI) [34, 35]. The failure of
endogenous remyelination may be due to the inhibition of adult
OPC differentiation and maturation into myelinating OLs. Al-
though OPCs are present in the demyelinated adult CNS and
even contact demyelinated axons, they fail to differentiate into
mature OLs that remyelinate these axons [28, 3638]. Under-
standing the molecular mechanism(s) that regulate the differen-
tiation of adult OPCs may lead to new therapeutic strategies for
demyelinated disorders. Most previous studies investigating the
response of OPCs to environmental factors present within de-
myelinating lesions have used embryonic and neonatal progen-
itor cells. However, the proliferation and differentiation of adult
and perinatal OPCs in response to growth factors are different
[3941]. The molecular mechanism(s) that control their differ-
entiation may also be different. For example, olig1 is not re-
quired for OL generation and myelination during CNS devel-
opment but is essential for remyelination following
demyelinating lesions in adult mice [42].
In the present study, we established an in vitro model to
directly study the proliferation and differentiation of adult spinal
cord OPCs. We provide evidence that the interaction between
BMP signaling and olig1 and olig2 regulates the differentiation
of adult OPCs.
MATERIALS AND METHODS
All animal care and surgical interventions were undertaken in strict
accordance with the Public Health Service Policy on Humane Care
and Use of Laboratory Animals and with the approval of the
University of Louisville Institutional Animal Care and Use Com-
mittee and the Institutional Biosafety Committee.
Isolation of OPCs from Adult Spinal Cord
OPCs were immunopanned from adult spinal cords of Fischer rats
(34 months old) with an O4 antibody using a protocol modified
from a previous study [39]. Briefly, the dissected spinal cords were
minced into 1-mm
3
pieces and incubated in Hanks balanced salt
solution containing 0.1% papain, 0.1% neutral protease, and 0.01%
DNase for 30 minutes at 37C. The digestion was stopped by the
addition of an equal volume of Dulbeccos modified Eagles me-
dium (DMEM) containing 20% fetal bovine serum (FBS). Tissues
were dissociated by repeated trituration with fire-polished Pasteur
pipettes and were filtered through 70-m nylon mesh. The cells
were incubated at 37C on an anti-RAN-2 antibody-coated dish for
30 minutes to deplete type 1 astrocytes and meningeal cells and then
transferred to an O4-coated dish for 45 minutes to select OPCs. The
purified OPCs were removed with trypsin and cultured in poly-L-
lysine/laminin (P/L)-coated dishes with DMEM/Hams F-12 me-
dium containing 1 N2 and 1 B27 supplements, 20 ng/ml
fibroblast growth factor 2 (FGF2), 10 ng/ml platelet-derived growth
factor aa (PDGFaa), 5 g/ml insulin, and 0.1% bovine serum
albumin. Cells were fed with fresh growth medium every other day.
In all cases, an aliquot of cells was analyzed immunohistochemi-
cally or by fluorescence-activated cell sorting the next day to
determine the efficiency of the immunopanning. Only those cell
preparations in which 95% of the bound cells expressed O4 were
used in the subsequent experiments.
OL Differentiation and Maturation of Adult OPCs
Passage 47 adult OPCs were plated on P/L-coated 60-mm culture
dishes for Western blot or 24-well plates for immunohistochemical
analyses. The following day, adult OPCs were induced to differentiate
by withdrawal of FGF2 and PDGFaa from the growth medium for 3
days with or without BMP2 and 4 (R&D Systems Inc., Minneapolis,
http://www.rndsystems.com). To examine the effects of BMP2 and 4
on OL maturation of adult OPCs, cells were allowed to differentiate
into O1 OLs by withdrawal of FGF2 and PDGFaa for 2 days and
then cultured for 3 more days with or without BMPs. The concentration
of BMP2 and 4 was 10 ng/ml except where otherwise indicated.
Olig1 and Olig2 Overexpression
Olig1, olig2, and enhanced green fluorescence protein (EGFP) cDNAs
were cloned into the LZRS retroviral vector [43]. To generate high titer
virus, NX cells (provided by Dr. Gary Nolan, Stanford University)
were transfected using GenePorter II (Gene Therapy Systems, Inc., San
Diego, http://www.genlantis.com), which routinely gave transfection
efficiencies of 50%65%. Selection with 2 g/ml puromycin began 48
hours later. To harvest viral supernatant, medium was changed to the
appropriate serum-free medium overnight and harvested the next day.
We routinely obtained titers of 5 10
5
to 5 10
6
transforming
units/ml. Cells were treated with 1 g/ml polybrene for 1 hour, fol-
lowed by a wash and subsequent replacement of half of the medium
with viral stock. After incubation at 37C for 6 hours, the medium was
changed to the appropriate growth medium. With this protocol, ap-
proximately 50% of the cells were labeled. Two days after infection,
adult OPCs were induced to differentiate and mature with or without
BMP2 and 4, as described above.
olig1 and olig2 Small Interference RNA
A retrovirus vector carrying small interference RNAs (siRNAs)
targeting the mouse olig1 or olig2 gene was prepared using the BD
Knockout RNAi System (Clontech, Palo Alto, CA, http://www.
clontech.com). The polyacrylamide gel electrophoresis-purified
complementary oligonucleotide pair for the hairpin siRNA was
synthesized to target the coding region of mouse olig1 or olig2
mRNA as follows: 5-gatccGCCACGAGTACAAACATCAATT-
CAAGAGATTGATGTTTGTACTCGTGGTTTTTTACGCGTg-
3, 5-aattcACGCGTAAAAAACCACGAGTACAAACATCAATC-
TCTTGAATTGATGTTTGTACTCGTGGCg-3, and 5-gatccGT-
ACAAAGATTGACTCCTTTTCAAGAGAAAGGAGTCAATCTT-
TGTACTTTTTTACGCGTg-3, 5-aattcACGCGTAAAAAAGTA-
CAAAGATTGACTCCTTTCTCTTGAAAAGGAGTCAATCTTT-
GTACg-3, respectively. A complementary pair of oligonucleotides
for a sense-only control RNA was also designed. These oligonucle-
otide pairs containing BamHI and EcoRI overhangs were annealed
and ligated to a linearized RNAi-Ready pSIREN-RetroQ-ZsGreen
vector digested with BamHI and EcoRI (Clontech). The RNAi-
Ready pSIREN-RetroQ-ZsGreen vector is a self-inactivating retro-
viral expression vector designed to express a small hairpin RNA
using the human U6 promoter. The resultant constructs were am-
plified, purified, and sequenced. High-titer retrovirus vectors ex-
pressing olig1 or olig2 siRNAs were produced in NX cells as
described above. The ZsGreen fluorescent marker yields a bright
green fluorescence, permitting direct monitoring of the infection
efficiency in adult OPCs.
Immunofluorescence In Vitro
To detect surface membrane antigens, cells cultured on 24-well
plates were incubated with the primary antibodies A2B5, O4, and
O1 (hybridoma supernatant, undiluted; American Type Culture
Collection, Manassas, VA, http://www.atcc.org) at 4C for 45 min-
utes. After fixation with 4% paraformaldehyde (PFA), cells were
incubated in fluorescein isothiocyanate- or Texas Red-conjugated
donkey anti-mouse IgM for 1 hour at room temperature (RT). For
recognition of other antigens, cells cultured on 24 well plates were
fixed with 4% paraformaldehyde (PFA). Mouse monoclonal anti-
bodies against 2,3-cyclic nucleotide phosphodiesterase (CNPase;
1:800; Sternberger Monoclonal, Covance, Princeton, NJ, http://
www.covance.com), myelin basic protein (MBP; 1:50; Chemicon,
Temecula, CA, http://www.chemicon.com), glial fibrillary acidic
3205 Cheng, Wang, He et al.
www.StemCells.com
protein (GFAP; 1:400; Sigma-Aldrich, St. Louis, http://www.
sigmaaldrich.com), or proteolipid protein (PLP; 1:200; Serotec,
Serotec, Raleigh, NC, http://www.serotec.com) or rabbit polyclonal
antibodies against olig1 (1:100; Chemicon), Olig2 (1:2,000; a gen-
erous gift from Dr. Charles Stiles, Harvard University), or chicken
polyclonal antibody anti-EGFP (1:500; Chemicon) were applied
overnight at 4C. Then, the appropriate fluorophore-conjugated
secondary antibodies (1:200; Jackson Immunoresearch Laborato-
ries, West Grove, PA, http://www.jacksonimmuno.com) were ap-
plied, and the nuclei were counterstained with 4,6-diamidino-2-
phenylindole. Negative control experiments performed with the
appropriate species-specific IgG or with inappropriate secondary
antibodies showed negligible background. Total cellular counts for
each experimental well were obtained in 10 fields with a 20
objective from three independent culture wells. The result for each
experimental condition was verified a minimum of three times.
Western Blot Experiments
Cells were harvested in ice-cold lysis buffer. Equivalent amounts of
total protein extract from each sample were mixed with sample buffer,
boiled, and loaded onto SDS polyacrylamide gels. Electrophoretic
separation of the extracts was typically performed on 7.5%15% (de-
pending on the molecular weight of the protein of interest) discontin-
uous acrylamide gels under denaturing conditions. Proteins were then
transferred to nitrocellulose membranes and probed with monoclonal-
specific antibodies raised against GFAP (Sigma-Aldrich), MBP, PLP,
or olig1 or with polyclonal antibodies raised against olig2, and phos-
phated Smad 1/5/8. In addition, an antibody against -actin (Sigma-
Aldrich) was used as a loading control. Appropriate secondary horse-
radish peroxidase-conjugated antibodies were used for detection with
chemiluminescence ECL reagents (Amersham Biosciences, Piscat-
away, NJ, http://www.amersham.com).
Spinal Cord Injury and Immunohistochemistry for
Tissue
After anesthetization with Nembutal (50 mg/kg, i.p.), adult female
Fischer 344 rats received a dorsal laminectomy at the ninth thoracic
vertebral level (T9) to expose the spinal cord, and then a 150 kdyn
contusive SCI using the Infinite Horizons (Lexington, KY) impactor
as described previously [44, 45]. After surviving 1 week or 1 month,
animals were perfused transcardially with 0.01 M phosphate-buff-
ered saline, pH 7.4, followed by 4% PFA in 0.1 M phosphate buffer.
The injured spinal cords were removed, cryoprotected in 30%
sucrose buffer overnight at 4C, and then transversely sectioned at
20 m on a cryostat. After blocking with 10% donkey serum in
Tris-buffered saline containing 0.3% Triton X-100 (TBST) for 1
hour at RT, the sections were incubated in TBST containing 5%
donkey serum, polyclonal rabbit anti-GFAP (1:500; Dako, Carpin-
teria, CA, http://www.dako.com)/monoclonal mouse anti-BMP2 or
anti-BMP4 (1:200; Chemicon), monoclonal mouse anti-olig1 (1:
100; a generous gift from Dr. Charles Stiles, Harward University)/
polyclonal anti-NG2 (1:200; Chemicon), or monoclonal mouse anti-
platelet-derived growth factor receptor (1:25; BD Pharmingen,
San Diego, http://www.bdbiosciences.com/index_us.shtml)/poly-
clonal rabbit anti-olig2 (1:2,000) overnight at 4C. Then, the ap-
propriate fluorophore-conjugated secondary antibodies (1:200;
Jackson Immunoresearch Laboratories) were applied. A Nikon C1
confocal microscope (Nikon, Tokyo, http://www.nikon.com) was
used to capture representative images. Photomicrographs were as-
sembled using Adobe Photoshop and Adobe Illustrator software
(Adobe Systems Inc., San Jose, CA, http://www.adobe.com).
RESULTS
Characterization of Adult OPC
In the presence of FGF2 and PDGFaa, O4 OPCs proliferated
and could be passaged many times. Proliferating OPCs dis-
played a characteristic adult OPC morphology, with several
small short processes emanating from a small round cell body
(Fig. 1A), as shown in the intact adult spinal cord by NG2
Figure 1. Isolation and differentiation of adult oligodendrocyte pre-
cursor cells (OPCs) in vitro. Adult OPCs were purified from the spinal
cord of adult rats by immunopanning with the O4 antibody. The pro-
liferating precursors displayed a characteristic adult OPC morphology
with several small short processes emanating from a small round cell
body (A). All cells expressed O4 (B), A2B5 (B), and NG2 (D, E). In the
presence of fibroblast growth factor 2 (FGF2) and platelet-derived
growth factor aa (PDGFaa), adult OPCs divided and are readily labeled
by BrdU (B, C). These OPCs proliferated for multiple passages without
changing phenotypes (F). Data in (F) were the mean SD of four
independent experiments. Three days after withdrawal of FGF2 and
PDGFaa, OPCs constitutively differentiated into OLs expressing O1
(G). Five days after differentiation, the majority of adult OPCs differ-
entiated into mature OLs expressing CNPase (H) and MBP (I). When
differentiated in the presence of 10% fetal bovine serum, more than 60%
of the cells differentiated into GFAPastrocytes (J) that also expressed
A2B5 and had a typical stellate morphology of type 2 astrocytes (J).
Scale bars 25 m (AC, G, J), 10 m (D), 20 m (E, I), and 50 m
(H). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; CNPase, 2,3-
cyclic nucleotide phosphodiesterase; GFAP, glial fibrillary acidic pro-
tein; MBP, myelin basic protein.
3206 BMP Signaling and olig1/2 Regulate OPCs
immunostaining [23, 46]. More than 95% of these cells were
stained by antibodies directed against O4 (Fig. 1B, 1F), A2B5
(Fig. 1C, 1F), or NG2 (Fig. 1D, 1F). Fewer than 5% of the adult
OPCs expressed the more mature OL-specific proteins O1 or
CNPase and they did not express MBP, PLP, GFAP, or vimentin
(data not shown). To examine their proliferative capacity, the
adult OPCs were cultured in serum-free medium containing
FGF2 and PDGFaa for 5 days, and then 10 M 5-bromo-2-
deoxyuridine (BrdU) was added overnight. More than 50% of
adult OPCs were BrdU (Fig. 1B, 1C), indicating that they
were mitotically active. After proliferating for 28 passages, the
percentage of adult OPCs expressing A2B5, NG2, or O4 did not
significantly change (Fig. 1F), indicating that adult OPCs can
proliferate extensively in vitro without phenotypic drift. When
cultured in serum-free medium containing insulin and ciliary
neurotrophic factor but lacking FGF2 and PDGFaa for 3 days,
more than 85% of cells differentiated to express O1 and devel-
oped the typical process-bearing morphology of OLs (Fig. 1G).
Moreover, after 6 days of differentiation, more than 80% of the
cells developed into CNPase (Fig. 1H) or MBP (Fig. 1I)
mature OLs with obvious membrane sheet. No GFAP astro-
cytes were observed (data not shown). In contrast, when cul-
tured in the same differentiation medium but containing 10%
FBS, more than 60% of the cells differentiated into GFAP
astrocytes that coexpressed A2B5 with typical stellate morphol-
ogy of type 2 astrocytes (Fig. 1J). These results showed that
adult spinal cord OPCs were bipotential and differentiated into
OLs in the absence of serum and into type 2 astrocytes in its
presence.
Expression of BMP Receptors by OPCs and OLs
OL differentiation in cell culture can be roughly divided into
three stages: A2B5/O4 OPCs, O1 immature OLs, and
MBP mature OLs. As described above, adult OPCs readily
differentiated into O1 and MBP OLs after withdrawal of
FGF2 and PDGFaa for 3 and 5 days, respectively. To determine
whether cells in the three stages of OL development can respond
to BMP, we examined their expression of BMP receptors Ia, Ib,
and II using immunohistochemistry and Western blot. Adult
OPCs expressed BMP receptors BMPR Ia (Fig. 2Aa), BMPR Ib
(Fig. 2Ab), and BMPR II (Fig. 2Ac). With slight downregula-
tion, immature O1 and mature MBP OLs still persistently
expressed these receptors (Fig. 2Ad). These findings suggest
that OPCs in the different stages of OL differentiation all have
the potential to respond to BMP.
BMP2 and BMP4 Suppress OL Differentiation and
Promote Astrocyte Differentiation by Adult OPCs
Previous studies showed that either BMP2 or BMP4 increased
astrocyte differentiation and inhibited OL differentiation of neu-
ral stem cells or OPCs isolating from embryonic or postnatal
developing CNS [1719, 21, 22]. Whether BMPs have similar
effects on adult OPCs has not been determined. When adult
OPCs were differentiated without BMP2 or BMP4 for 3 days,
almost all of the cells differentiated into O1OLs. No astrocyte
differentiation was observed (Fig. 2Ba). After addition of
BMP2, BMP4, or both in the differentiation medium, the num-
ber of OPCs that differentiated into O1 OLs dramatically
decreased, with concurrent increases of GFAPastrocytes (Fig.
2Bb2Bd). The number of OPCs that differentiated into mature
MBPOLs also decreased (Fig. 2Be2Bh). To determine dose-
dependent effects on the differentiation of adult OPCs, increas-
ing concentrations of BMP2, 4, or both 2 and 4 were added to
the medium. As shown in Figure 2C, when differentiated for 3
days in the presence of BMP2 at concentrations of 1, 2, 5, or 10
ng/ml, the percentage of adult OPCs that differentiated into
GFAP astrocytes significantly increased from 0.4% at control
to 14%, 18%, 25%, and 46%, respectively (p .001 at all four
concentrations). The percentage of O1 OLs decreased from
77% to 60%, 45%, 34%, and 19% (p .001), respectively, and
MBPOLs also were suppressed from 58% to 60%, 45%, 34%,
and 19% (p .01), respectively. BMP4 was more potent than
BMP2 in the inhibition of OL differentiation and promotion of
astrocyte differentiation. In the presence of BMP4 at 1, 2, 5, and
10 ng/ml, the number of OPCs that differentiated into GFAP
astrocytes were 35%, 44%, 66%, and 75%, respectively, all
being significantly increased compared with 0.4% in control
cultures (all p .001). The number of O1 and MBP OLs
were 20%, 13%, 6%, 1% and 21%, 9%, 2%, 1%, respectively,
all significantly decreased compared with 77% and 58% (all p
.001). In the presence of BMP2 and 4 at the same concentra-
tions, further increases in the number of GFAP astrocytes and
decreases of O1 or MBP OLs were observed (Fig. 2C).
These findings were confirmed by Western blot experiments
(Fig. 2D). Moreover, we found that the expression of pSmad1/
5/8 and ID4, two intracellular downstream targets of BMP
signaling, was also increased in proportion with the dosage of
BMP2, 4, or 2 and 4, suggesting that BMP signaling inhibits OL
differentiation and promotes astrocyte differentiation of adult
OPCs through Smad-dependent pathways.
BMP2 and BMP4 Downregulate the Expression of
olig1 and olig2 During Adult OPC Differentiation
To determine the potential mechanism through which BMPs
affect the differentiation of adult OPCs, we examined the ex-
pression of olig1 and olig2, two bHLH transcription factors
important for OL development. When differentiating for 3 days
in the absence of BMP2 and 4, the majority of cells developed
into OLs (Fig. 3Aa) that expressed olig1 (Fig. 3Ab) and olig2
(Fig. 3Ac). Addition of BMP2 and 4, however, significantly
decreased the number of olig1-expressing cells from 79% to
15% (p .001) and olig2-expressing cells from 76% to 15%
(p .001) in addition to dramatically increasing the number
of GFAP astrocytes and decreasing O1, CNPase, or
MBP OLs (Fig. 3Ad3Af, 3B). Western blot experiments
confirmed that addition of BMP2 and 4 in the differentiating
medium activated Smad1/5/8 to inhibit the expression of
olig1, olig2, and MBP and to promote the expression of
GFAP (Fig. 3C).
Previous studies have shown that adult OPCs change their
responses to growth factors after culturing for a long time in the
presence of FGF2 and PDGFaa [41]. To address this possibility,
we tested the effects of BMP2 and 4 on differentiation of freshly
isolated OPCs from adult spinal cord. As shown in supplemental
online Figure 1, all OPCs expressed A2B5 (supplemental online
Fig. 1A) and O4 (supplemental online Fig. 1B). After differen-
tiation without FGF2 and PDGFaa for 3 days, most OPCs
differentiated into OLs expressing O1 (supplemental online Fig.
2A), CNPase (supplemental online Fig. 2C), and MBP (supple-
mental online Fig. 2E). No GFAP astrocytes were observed
(supplemental online Fig. 2A). Addition of BMP2 and 4 dra-
matically increased the number of GFAP astrocytes (supple-
mental online Fig. 2B) and simultaneously decreased the
number of O1 (supplemental online Fig. 2B), CNPase (sup-
plemental online Fig. 2D), or MBP (supplemental online Fig.
2E) OLs. The number of cells expressing Olig2 (supplemental
online Fig. 2C, 2D) or Olig1 (supplemental online Fig. 2E, 2F)
was also significantly decreased by the treatment of BMP2 and
4. These data indicate that the effects of BMP2 and 4 on the
differentiation of freshly isolated adult OPCs are identical to
their effects on OPCs of passages 47, which we used in most
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of our present study. Thus, exposure to FGF2 and PDGFaa in
vitro does not change the responses of adult OPCs to BMP2
and 4.
BMP2 and 4 Inhibit OL Maturation
The findings that immature O1 OLs expressed BMP receptors
(Fig. 2) and that the number of MBP OL decreased after the
treatment with BMP2 and 4 suggested that BMP might also
inhibit the maturation of OL from immature O1 to more
mature MBP stages. To determine the effects of BMP2 and 4
on OL maturation, adult OPCs were allowed to differentiate into
O1 OLs by withdrawing FGF2 and PDGFaa for 2 days and
then continued to mature for 3 more days in the absence or
presence of BMP2 and 4. As shown previously, when differen-
tiating for 2 days after withdrawal of FGF2 and PDGFaa, more
than 75% of adult OPCs developed into O1 OLs; after 3 more
days, more than 95% of cells became O1 OLs (Fig. 4Aa), and
90% of OPCs matured into CNPase (Fig. 4Ab) or MBP
(Fig. 4Ac) OLs. When 10 ng/ml BMP2 and 4 were added for the
last 3 days of differentiation, a significant decrease in the
number of OLs expressing either MBP (from 95% to 64%) or
CNPase (from 93% to 58%; p .001) was observed (Fig.
4Ad4Af, 4B). However, the number of O1 OLs (from 98
89%; p .05) or GFAP astrocytes (from 1.1% to 5.6%; p
.05) did not significantly change (Fig. 4Aa, 4Ad, 4B). These
results showed that BMP2 and 4 inhibited the maturation of
O1 into MBP or CNPase OLs.
Figure 2. Inhibition of oligodendrocyte
(OL) differentiation and promotion of astro-
cyte differentiation of adult oligodendrocyte
precursor cells (OPCs) by BMP2 and 4.
Adult OPCs expressed BMPR Ia (Aa), Ib (Ab),
and II (Ac). Although they were slightly
downregulated, all three BMPRs were per-
sistently expressed in immature O1 and
more mature MBP OLs after Dif3d and
Dif5d, respectively (Ad). Treatment with
BMP2, 4, or both decreased the number of
O1 OLs, with concurrent increases in the
number of GFAP astrocytes (BaBd). The
number of MBP OLs was also decreased
(BeBh). Quantification of cells immuno-
stained for GFAP, O1, or MBP showed a
dose-dependent increase in the number of
GFAP astrocytes and decrease of O1 or
MBP OLs differentiated from adult OPCs
by BMP2, 4, or both, respectively (C). Data
represent the mean SD (n 4; one and
two stars represent p .05 and 0.01, respec-
tively). Western blot experiments confirmed
the dose-dependent activation of pSmad1/
5/8, ID4, the intracellular downstream target
of BMP signaling with the increasing and
decreasing expression of GFAP and MBP,
respectively, by BMP2, 4, or both (D). Scale
bar 20 m (AaAc) and 25 m (BaBh).
Abbreviations: BMP, bone morphogenetic
protein; BMPR, bone morphogenetic protein
receptor; DAPI, 4,6-diamidino-2-phenylin-
dole; Dif3d, differentiation for 3 days; Dif5d,
differentiation for 5 days; GFAP, glial fibril-
lary acidic protein; MBP, myelin basic protein;
Undif, undifferentiated.
3208 BMP Signaling and olig1/2 Regulate OPCs
We further examined the expression of olig1 and olig2
during the maturation of adult OPCs. When differentiating in the
presence of BMP2 and 4, the number of olig1 cells signifi-
cantly decreased from 79% at 5 days after differentiation with-
out BMP2 and 4 to 49% (p .01) (Fig. 4Ab, 4Ae, 4B, 4C). The
number of olig2 cells was also decreased from 80% at control
to 45% (p .05) (Fig. 4Ac, 4Af, 4B, 4C). BMP2 and 4 also
inhibited the development of both CNPase- or MBP-expressing
mature OLs, as shown by both immunohistochemistry (Fig. 4A,
4B) and Western blot (Fig. 4C). Our results showed that BMP2
and 4 specifically decreased the expression of both olig1 and
olig2 and subsequently inhibited OL differentiation and matu-
ration of adult OPCs.
Overexpression of olig1 and olig2 Blocks the BMP-
Mediated Inhibition of Adult OPC Differentiation
When differentiating in the presence of BMP2 and 4 for 3 days,
70% of EGFP-expressing OPCs became GFAP astrocytes
(Fig. 5A, 5D). Astrocyte differentiation in olig1- or olig2-
expressing OPCs was dramatically decreased to 25% (p .001)
and 32% (p .001), respectively (Fig. 5B5D). However, the
percentage of O1OLs derived from olig1- or olig2-expressing
OPCs (22% and 26%, respectively) after 3 days of differentia-
tion in the presence of BMP2 and 4 was slightly but not
significantly increased compared with 17% of EGFP-OPCs
(both p .05) (Fig. 5E5I). These results indicate that overex-
pression of olig1 or olig2 is sufficient to block the astrocyte
differentiation of adult OPCs induced by BMP2 and 4. How-
ever, overexpression of either olig1 or olig2 failed to block the
inhibition of BMP2 and 4 on OL differentiation of adult OPCs.
Since BMP2 and 4 downregulate the expression of both olig1
and olig2 in the adult OPCs, overexpression of both olig1 and
olig2, not olig1 or olig2 alone, may be necessary to overcome
the inhibition of BMP2 and 4 on OL differentiation of adult
OPCs. To test this hypothesis, we infected adult OPCs with
retroviruses encoding both olig1 and olig2 and then differenti-
ated the cells for 3 days in the presence of BMP2 and 4. As
shown in Figure 5H and 5I, the percentage of O1 OLs in
olig1/olig2-expressing adult OPCs was 63%, which was signif-
icantly increased compared with 17%, 22% or 26% in EGFP-,
olig1-, or olig2-expressing adult OPCs, respectively (all p
.001).
Expression of Both olig1 and olig2 Is Necessary for
OL Differentiation of Adult OPCs
To further elucidate the role(s) of olig1 and olig2 in the differ-
entiation of adult OPCs, we tested whether downregulation of
olig1 or olig2 by specific siRNA would inhibit OL differentia-
tion of adult OPCs. We first tested whether siRNAs were able to
efficiently downregulate the expression of olig1 or olig2 in the
adult OPCs. Expression of either olig2 (Fig. 6A6C) or olig1
(Fig. 6D6F) in adult OPCs infected with retroviruses encoding
control scrambled siRNA did not change. However, the expres-
sion of olig2 was dramatically decreased in these adult OPCs
infected with retroviruses encoding olig2 siRNA (Fig. 6A
6C). Expression of olig1, however, did not change in these
olig2 siRNA OPCs (data not shown). Similarly, adult OPCs
expressing olig1 siRNA significantly downregulated its expres-
sion of olig1 (Fig. 6D6F) but not olig2 (data not shown).
These results demonstrated that olig1 or olig2 siRNA specifi-
cally knocked down the expression of olig1 and olig2 in adult
OPCs, respectively. We then examined whether OL differenti-
ation of adult OPCs was inhibited following the downregulation
of olig1 or olig2 with specific siRNA. Adult OPCs infected with
retroviruses encoding either control scrambled, olig1, or olig2
siRNA were differentiated for 3 days. In the control group, more
than 90% of infected adult OPCs differentiated into O1 OLs
(Fig. 6G, 6J). O1 OLs, however, significantly decreased in
olig1 siRNA OPCs (35%; p .001) (Fig. 6I, 6J) or in olig2
siRNA OPCs (59%; p .05) (Fig. 6H, 6J). These results
Figure 3. BMP2 and BMP4 inhibit adult
oligodendrocyte precursor cell (OPC) differ-
entiation into oligodendrocytes (OLs) in
vitro by downregulating olig1 and olig2.
Three days after differentiation in vitro, al-
most all adult OPCs differentiated into OLs
expressing O1 (Aa) or CNPase (Ab). A few
cells expressed MBP (Ac). Addition of 10
ng/ml BMP2 and 4 dramatically decreased
the number of O1-, CNPase-, or MBP-ex-
pressing OLs while significantly increasing
the number of GFAP

astrocytes (AdAf).
BMP2 and 4 significantly decreased the
number of cells immunostained for olig1 and
olig2 (B). Data represent the mean SD
(n 4; stars indicate p .001). Western
blot experiments confirmed the immunohis-
tochemical results and further showed that
BMPs activate pSmad1/5/8 in adult OPCs
(C). Similar results were obtained in four
independent experiments. Scale bar 100
m (AaAf). Abbreviations: BMP, bone
morphogenetic protein; CNPase, 2,3-cyclic
nucleotide phosphodiesterase; DAPI, 4,6-
diamidino-2-phenylindole; Dif3d, differenti-
ation for 3 days; GFAP, glial fibrillary acidic
protein; MBP, myelin basic protein; Undif,
undifferentiated.
3209 Cheng, Wang, He et al.
www.StemCells.com
showed that both olig1 and olig2 were necessary for OL differ-
entiation of adult OPCs. No GFAP astrocyte differentiation
was observed in either EGFP, olig1, or olig2 siRNA OPCs after
3 days differentiation without BMP2 and 4 (data not shown).
Expression of BMP2 and 4 and olig1 and 2 in the
Injured Spinal Cord
Obvious expression of BMP2 or BMP4 was not observed in the
normal adult spinal cord (supplemental online Fig. 3A, 3D). At
7 days after contusion, expression of BMP2 and BMP4 was
significantly increased in the reactive hypotrophic astrocytes in
the injured spinal cord (supplemental online Fig. 3B, 3E). Their
expression in the astrocytes continued to increase in the spinal
cord at 1 month after contusion (supplemental online Fig. 3C,
3F). These results showed that expression of BMP2 and BMP4
was significantly increased after SCI.
Olig1 was localized in the nuclei of NG2 OPCs in the
normal spinal cord, and almost all NG2OPCs expressed olig1
(supplemental online Fig. 4A, inset, arrows). At 1 week (sup-
plemental online Fig. 4B) and 1 month (supplemental online
Fig. 4C) after the contusion, the number of NG OPCs signif-
icantly increased in the injured spinal cord, but the number of
olig1 cells decreased. Most of NG2 OPCs did not express
olig1 (supplemental online Fig. 4B, 4C, inset, arrowheads),
whereas some OPCs remained olig1-positive (supplemental on-
line Fig. 4B, 4C, inset, arrows). OPCs in the adult spinal cord
also expressed olig2 in their nuclei (supplemental online Fig.
4D, inset, arrow). At 1 week postinjury, the number of olig2
cells increased in the injured spinal cord, and its expression in
some cells was upregulated (supplemental online Fig. 4E).
However, many adult OPCs did not express olig2 (supplemental
online Fig. 4E, inset, arrowheads). The number of olig2 cells
and its expression level decreased in the injured spinal cord at 1
month postinjury compared with 1 week postinjury (supplemen-
tal online Fig. 4F). More OPCs did not express olig2 (supple-
mental online Fig. 4F, inset, arrowheads).
DISCUSSION
OPCs in the Adult Spinal Cord
We obtained highly purified OPCs from the adult rat spinal cord
that share many fundamental properties with OPCs from peri-
natal and adult optic nerve or subcortical white matter. They
express similar phenotype-specific antigens, O4, A2B5, and
NG2. OPCs cultured from adult rat optical nerve [47] or human
cortical white matter [48, 49] express O4. Approximately 80%
of acutely isolated cycling progenitor cells in the adult mam-
malian white matter are O4[50]. OPCs in the adult spinal cord
also express A2B5 and NG2, which is in accord with previous
results from adult optic nerve [39, 47]. In the intact adult spinal
cord, more than 70% of BrdU cycling precursor cells express
NG2 [23]. Therefore, the purified A2B5/NG2/O4 OPCs
in this study are the most actively proliferative precursor cells in
the normal adult spinal cord.
Consistent with present data with adult spinal cord OPCs,
OPCs from postnatal seven days rat optic nerve can proliferate
for many passages without differentiation in the presence of
PDGFaa and in the absence of thyroid hormone [51, 52]. FGF2
can inhibit the differentiation of OPCs and promote their pro-
liferation [49, 53] and can convert slowly dividing adult OPCs
to rapidly dividing cells with characteristics of perinatal OPCs
[41]. Moreover, the adult spinal cord OPCs differentiate into
OLs in the serum-free medium and into type 2 astrocytes in
medium containing serum, a property of OPCs from other CNS
Figure 4. BMP2 and BMP4 inhibit oligo-
dendrocyte (OL) maturation in vitro. Differen-
tiated for 5 days, almost all cells matured into
CNPase or MBP OLs expressing olig1 and
olig2 (AaAc, B). If they were differentiated
for 2 days without BMPs to allow adult oligo-
dendrocyte precursor cell to become O1OLs
and then differentiated for 3 more days with
BMPs, the number of cells expressing O1 did
not change (Ad, B). The number of cells ex-
pressing CNPase, MBP, olig1, and olig2, how-
ever, significantly decreased (AeAf, B). No
obvious GFAP astrocyte differentiation was
observed with or without BMPs (Aa, Ad, B).
Data represent the mean SD (n 4; stars
indicate p .01). Western blot experiments
confirmed the decreasing expression of
CNPase, MBP, olig1, and olig2 with increas-
ing pSMAD1/5/8 after BMP treatment (C).
Scale bar 25 m (AaAf). Abbreviations:
BMP, bone morphogenetic protein; CNPase,
2,3-cyclic nucleotide phosphodiesterase;
DAPI, 4,6-diamidino-2-phenylindole; GFAP,
glial fibrillary acidic protein; MBP, myelin ba-
sic protein.
3210 BMP Signaling and olig1/2 Regulate OPCs
regions [54, 55]. The fact that adult spinal cord OPCs can be
induced to differentiate into different developmental stages of
Figure 5. Overexpression of olig1 and olig2 inhibits astrocyte differ-
entiation and promotes OL differentiation of adult oligodendrocyte
precursor cells (OPCs). Adult OPCs were infected with retroviruses
encoding EGFP, Olig1, Olig2, or Olig1 and 2 and then differentiated for
3 days in the presence of 10 ng/ml BMP2 and 4. Many adult OPCs,
including EGFP-infected ones (A, D) differentiated into GFAP astro-
cytes. Overexpressing Olig1 (B, D), or Olig2 (C, D) significantly
decreased the number of adult OPCs differentiating into astrocytes (D).
Data represent the mean SD (n 4; stars indicate p .001). The
number of adult OPCs differentiating into O1 OLs was repressed in
the presence of BMP2 and 4 (E, I). Overexpression of olig1 (F, I) or
olig2 (G, I) failed to result in a significant increase of O1 OLs.
Overexpression of olig1 and olig2, however, overcame the inhibition of
BMP signaling to significantly increase OL differentiation from adult
OPCs (H, I). Data represent the mean SD (n 4; star indicates p
.01). Scale bars 25 m. Abbreviations: DAPI, 4,6-diamidino-2-
phenylindole; EGFP, enhanced green fluorescence protein; GFAP, glial
fibrillary acidic protein; OL, oligodendrocyte.
Figure 6. Inhibition of adult oligodendrocyte precursor cell (OPC) dif-
ferentiation into oligodendrocytes (OLs) following downregulation of Olig2
and Olig1. Olig2 and Olig1 siRNA downregulated the expression of Olig2
and Olig1 in adult OPCs. Adult OPCs were infected with retroviruses that
express either scrambled negative control (AF), Olig2 (AC), or Olig1
(DF) siRNA. Two days later, control adult OPCs did not alter their
expression of Olig2 ([AC], arrows) or Olig1 ([DG], arrows). However,
adult OPCs infected with retroviruses expressing either Olig2 or Olig1
siRNA downregulated their expression of Olig2 ([AC], arrows) or Olig1
([DG], arrows), respectively. Adult OPCs were differentiated for 3 days
following infection with retroviruses encoding negative control, Olig2, or
Olig1 siRNA. Uninfected OPCs ([GI], not green cells) and OPCs infected
with scrambled control siRNA ([G], green) differentiated into OLs express-
ing O1 ([GI], red). Adult OPCs expressing Olig2 siRNA ([H], green cells)
or Olig1 siRNA ([I], green cells) did not differentiate into O1 OLs. Data
represent the mean SD (n 4; one and two stars indicate p .05 and
0.01, respectively). Scale bar 25 m (AF, AF), 20 m (G, H), and
10 m (I). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; EGFP,
enhanced green fluorescence protein; siRNA, small interference RNA.
3211 Cheng, Wang, He et al.
www.StemCells.com
OLs by withdrawing FGF2 and PDGFaa for certain times (Fig.
1) establishes a stable adult spinal cord OPC model to investi-
gate potential molecular mechanism(s) by which adult OPCs
proliferate and differentiate in vitro.
BMP Signaling Inhibits Oligodendrogenesis and
Promotes Astrogliogenesis from Adult OPCs
In vitro, BMP signaling enhances astrocyte differentiation and
inhibits OL differentiation from cultured neural stem cells or
OPCs isolated from embryonic or perinatal CNS [16, 17, 21, 22,
56]. In vivo, BMP signaling also represses OL development [11,
13, 57]. Furthermore, overexpression of BMP4 in transgenic
mice under the control of the neuron-specific enolase promoter
results in a significant decrease in OLs, with a concurrent
increase in astrocytes [15]. The present study extends these
previous findings to show that BMP signaling also inhibits the
differentiation and maturation of adult OPCs, suggesting that
BMP signaling may play an important role in the regulation of
remyelination of the adult CNS following traumatic or demy-
elinating injuries.
Importantly, present data document the molecular mecha-
nism(s) by which BMP signaling inhibits OPC differentiation
into mature OLs. One possible mechanism is the inhibition of
olig1/2 activity by ID4 (Fig. 2), as suggested by a previous study
[20]. Although ID4 and ID2 are expressed in proliferating
OPCs, their expression decreases progressively as OPCs differ-
entiate and mature [20, 58, 59]. Overexpression of ID2 or ID4
in OPCs inhibits OL differentiation, whereas downregulation of
their expression induces premature differentiation in vitro [20,
58, 59]. ID2 and ID4 bind olig1 and olig2 in the cytoplasm of
OPCs to prevent their translocation to nucleus, thereby prevent-
ing olig1 and olig2 from binding target DNA [20]. The present
study further suggests that an additional mechanism by which
BMP signaling inhibits OL differentiation from adult OPC is the
downregulation of olig1 and olig2 (Figs. 25). That overexpres-
sion of olig1 and olig2 reverses the inhibitory effects of BMP
signaling on OL differentiation of OPCs further confirms the
suggestion that BMP signaling inhibits oligodendrogenesis from
OPCs by downregulating olig1 and olig2. Therefore, downregu-
lation of olig1 and olig2 expression and inhibition of olig1/2
activity by increased ID4 may work synergistically to inhibit OL
differentiation and to enhance astrogliogenesis (Fig. 7).
Olig1 and olig2 in the Differentiation of Adult OPCs
Transcriptional regulation orchestrates oligodendrogenesis dur-
ing CNS development [1, 2, 60]. Olig1 and olig2 play important
roles in generating OLs during embryogenesis [9, 10, 61, 62].
Olig2 is required for OL lineage specification during CNS
development, whereas olig1 contributes more to OL differenti-
ation and maturation [9, 10, 63, 64]. However, olig2 compen-
sates for the function of olig1 during OL development, since OL
differentiation and myelination are delayed but eventually reach
normal level in olig1-null mice [9, 10]. In contrast to embryonic
development, adult OPCs require both olig1 and olig2 for their
differentiation and maturation. Downregulation of either olig1
or olig2 by siRNA inhibits adult OPCs differentiation into OLs
(Fig. 6). Overexpression of olig1 or olig2 alone fails to over-
come the inhibition of BMP to promote OL differentiation since
BMP signaling decreases the expression of both olig1 and olig2.
However, overexpression of both olig1 and olig2 enhances OL
differentiation even in the presence of BMP2 and 4 (Fig. 5).
These results are consistent with data from Arnett et al. [42],
which show that olig1 is required not for the development of
OLs but for remyelination in adult mice following demyelina-
tion. Taken together, these data indicate that olig1 is required for
the differentiation and maturation of adult OPCs and endoge-
nous remyelination after demyelination in adult CNS. Whether
olig2 plays an important role in the differentiation and matura-
tion and myelination of OLs during development is not known,
since olig2-null mice die during the perinatal period. Interest-
ingly, olig2 expression is retained during OL maturation, sug-
gesting its possible role in OL differentiation. Consistent with
this idea, our in vitro analyses show that olig2 is also required
for OL differentiation and maturation from adult OPCs. These
data also suggest that it may have a potential role during
remyelination. Conditional olig2 knockout mice will be very
useful to study its effects on remyelination in vivo.
In addition to OL development, olig1 and olig2 also play an
important role in astrogliogenesis. Overexpression of olig1 or
olig2 significantly inhibits astrocyte differentiation of adult
OPCs induced by BMP signaling, suggesting that either olig1 or
olig2 is sufficient to repress astrogliogenesis. These results are
in good accord with the previous observation of Lu et al. [10],
which show that the null mutation of either olig1 or olig2 alone
is not sufficient to increase astrogliogenesis in the spinal cord.
The double-null mutation of olig1 and olig2, however, results in
an apparent increase in astrogliogenesis, with a complete failure
of OL development [9]. Thus, downregulation of both olig1 and
olig2 is required for OPCs to differentiate into astrocytes since
olig1 and olig2 could complement the functions of one another
sufficient to inhibit astrocyte differentiation. Consistent with
this notion, we observe that both olig1 and olig2 are dramati-
cally downregulated when astrocyte differentiation from adult
OPCs is induced by BMP signaling. Our results further show
that downregulation of olig1 or olig2 alone by siRNA fails to
result in enhanced astrocyte differentiation even though OL
differentiation is inhibited, suggesting that repression of OL differ-
entiation alone is not sufficient to cause the astrocyte differentiation
from adult OPCs if BMP signaling is not present. BMP signaling
activates Smad proteins, which translocate into the nucleus to
form a complex with p300 to directly activate the GFAP pro-
Figure 7. A schematic illustration of the regulation of adult oligoden-
drocyte precursor cell (OPC) differentiation by the interaction of BMP
signaling and olig1/2. BMPs upregulate or downregulate the expression
of ID4 and olig1/2, respectively, by Smad-dependent or -independent
pathways. Increasing the expression of ID4, which blocks the translo-
cation of olig1 and 2 to the nucleus, works synergistically with the
downregulation of olig1 and 2 to inhibit oligodendrocyte (OL) differ-
entiation and permit and/or potentiate astrocyte differentiation following
BMP signaling. Olig1 and 2 are necessary to promote OL differentiation
and sufficient to inhibit astrocyte differentiation by binding p300 to
interrupt the formation of the Smad/p300 complex, which activates the
GFAP promoter. Therefore, overexpression of olig1 and 2 overcomes
the effects of BMP signaling on adult OPCs to promote OL differenti-
ation and inhibit astrocyte differentiation. Abbreviations: BMP, bone
morphogenetic protein; BMPR, bone morphogenetic protein receptor.
3212 BMP Signaling and olig1/2 Regulate OPCs
moter [65, 66]. Olig2 interacts with p300 to interrupt the com-
plex of p300 and Smad1 and thereby represses the astrocyte-
specific GFAP promoter [67]. Because of their structural
similarity, it is possible that olig1 may use the same mechanism
to inhibit astrocyte differentiation. This would suggest that BMP
signaling is able to induce adult OPCs to differentiate into
astrocytes through the activation of GFAP promoter by the
Smad-p300 complex only when olig1 and olig2 are also down-
regulated (Fig. 7). Downregulation of both olig1 and olig2 is
required, but not sufficient, for astrocyte differentiation from
adult OPCs.
Implications for Remyelination
OPCs reside quiescently in the white matter of the adult CNS
[23, 24]. They become activated and proliferate in response to
CNS injury, including demyelination [2528]. However, adult
OPCs fail to differentiate and mature into OL capable of remy-
elinating demyelinated axons in multiple sclerosis lesions even
though endogenous OPCs are present in the demyelinated areas
and some even closely contact the affected axons [36, 38,
6870]. Inhibitory signals that prevent OPCs from differentia-
tion and maturation are present in the demyelinated lesions [71,
72]. We suggest that BMP signaling may be an important factor
that inhibits OL differentiation and maturation in the injured
CNS. In fact, expression of BMPs dramatically increases after
CNS injury [73, 74]. Moreover, neuronal and OL differentiation
from engrafted neural stem cells in the injured spinal cord is
increased after blocking BMP signaling by expressing noggin
[73]. Consistent with those findings, we also observed a signif-
icant increase of BMP2 and 4 in the hypertrophic astrocytes
after traumatic SCI (supplemental online Fig. 3), and expression
of olig1 and olig2 in the OPCs of the injured spinal cord
decreased (supplemental online Fig. 4). Manipulation of BMP
signaling may provide a new therapeutic strategy to enhance
remyelination from endogenous or/and grafted OPCs after mul-
tiple sclerosis or SCI.
ACKNOWLEDGMENTS
This work was supported by National Center for Research
Resources Grant 5P20 RR 015576 (to Q.C. and S.R.W.), NIH
R01 NS37717 (to M.Q.), the Kentucky Spinal Cord and Head
Injury Research Trust (to Q.C. and S.R.W.), Norton Healthcare,
the Commonwealth of Kentucky Research Challenge for Excel-
lence Trust Fund, and The Christopher Reeve Paralysis Foun-
dation (to S.R.W.). We thank George Harding of the Anatomical
Sciences and Neurobiology, University of Louisville School of
Medicine, for expert technical help.
DISCLOSURE OF POTENTIAL CONFLICTS
OF INTEREST
The authors indicate no potential conflicts of interest.
REFERENCES
1 Miller RH. Regulation of oligodendrocyte development in the vertebrate
CNS. Prog Neurobiol 2002;67:451467.
2 Rowitch DH. Glial specification in the vertebrate neural tube. Nat Rev
Neurosci 2004;5:409419.
3 Orentas DM, Hayes JE, Dyer KL et al. Sonic hedgehog signaling is
required during the appearance of spinal cord oligodendrocyte precur-
sors. Development 1999;126:24192429.
4 Soula C, Danesin C, Kan P et al. Distinct sites of origin of oligodendro-
cytes and somatic motoneurons in the chick spinal cord: Oligodendro-
cytes arise from Nkx2.2-expressing progenitors by a Shh-dependent
mechanism. Development 2001;128:13691379.
5 Poncet C, Soula C, Trousse F et al. Induction of oligodendrocyte pro-
genitors in the trunk neural tube by ventralizing signals: Effects of
notochord and floor plate grafts, and of sonic hedgehog. Mech Dev
1996;60:1332.
6 Cai J, Qi Y, Hu X et al. Generation of oligodendrocyte precursor cells
from mouse dorsal spinal cord independent of Nkx6 regulation and Shh
signaling. Neuron 2005;45:4153.
7 Vallstedt A, Klos JM, Ericson J. Multiple dorsoventral origins of oligo-
dendrocyte generation in the spinal cord and hindbrain. Neuron 2005;
45:5567.
8 Fogarty M, Richardson WD, Kessaris N. A subset of oligodendrocytes
generated from radial glia in the dorsal spinal cord. Development 2005;
132:19511959.
9 Zhou Q, Anderson DJ. The bHLH transcription factors OLIG2 and
OLIG1 couple neuronal and glial subtype specification. Cell 2002;109:
6173.
10 Lu QR, Sun T, Zhu Z et al. Common developmental requirement for Olig
function indicates a motor neuron/oligodendrocyte connection. Cell
2002;109:7586.
11 Hall AK, Miller RH. Emerging roles for bone morphogenetic proteins in
central nervous system glial biology. J Neurosci Res 2004;76:18.
12 Agius E, Soukkarieh C, Danesin C et al. Converse control of oligoden-
drocyte and astrocyte lineage development by Sonic hedgehog in the
chick spinal cord. Dev Biol 2004;270:308321.
13 Mekki-Dauriac S, Agius E, Kan P et al. Bone morphogenetic proteins
negatively control oligodendrocyte precursor specification in the chick
spinal cord. Development 2002;129:51175130.
14 Miller RH, Dinsio K, Wang R et al. Patterning of spinal cord oligoden-
drocyte development by dorsally derived BMP4. J Neurosci Res 2004;
76:919.
15 Gomes WA, Mehler MF, Kessler JA. Transgenic overexpression of
BMP4 increases astroglial and decreases oligodendroglial lineage com-
mitment. Dev Biol 2003;255:164177.
16 Gross RE, Mehler MF, Mabie PC et al. Bone morphogenetic proteins
promote astroglial lineage commitment by mammalian subventricular
zone progenitor cells. Neuron 1996;17:595606.
17 Mabie PC, Mehler MF, Kessler JA. Multiple roles of bone morphoge-
netic protein signaling in the regulation of cortical cell number and
phenotype. J Neurosci 1999;19:70777088.
18 Zhu G, Mehler MF, Zhao J et al. Sonic hedgehog and BMP2 exert
opposing actions on proliferation and differentiation of embryonic neural
progenitor cells. Dev Biol 1999;215:118129.
19 Nakashima K, Takizawa T, Ochiai W et al. BMP2-mediated alteration in
the developmental pathway of fetal mouse brain cells from neurogenesis
to astrocytogenesis. Proc Natl Acad Sci U S A 2001;98:58685873.
20 Samanta J, Kessler JA. Interactions between ID and OLIG proteins
mediate the inhibitory effects of BMP4 on oligodendroglial differentia-
tion. Development 2004;131:41314142.
21 Mabie PC, Mehler MF, Marmur R et al. Bone morphogenetic proteins
induce astroglial differentiation of oligodendroglial-astroglial progenitor
cells. J Neurosci 1997;17:41124120.
22 Grinspan JB, Edell E, Carpio DF et al. Stage-specific effects of bone
morphogenetic proteins on the oligodendrocyte lineage. J Neurobiol
2000;43:117.
23 Horner PJ, Power AE, Kempermann G et al. Proliferation and differen-
tiation of progenitor cells throughout the intact adult rat spinal cord.
J Neurosci 2000;20:22182228.
24 Scolding N, Franklin R, Stevens S et al. Oligodendrocyte progenitors are
present in the normal adult human CNS and in the lesions of multiple
sclerosis. Brain 1998;121:22212228.
25 Levine JM, Reynolds R. Activation and proliferation of endogenous
oligodendrocyte precursor cells during ethidium bromide-induced demy-
elination. Exp Neurol 1999;160:333347.
26 Reynolds R, Cenci di Bello I, Dawson M et al. The response of adult
oligodendrocyte progenitors to demyelination in EAE. Prog Brain Res
2001;132:165174.
27 Keirstead HS, Levine JM, Blakemore WF. Response of the oligodendro-
cyte progenitor cell population (defined by NG2 labelling) to demyeli-
nation of the adult spinal cord. Glia 1998;22:161170.
28 Talbott JF, Loy DN, Liu Y et al. Endogenous Nkx2.2/Olig2 oligo-
dendrocyte precursor cells fail to remyelinate the demyelinated adult rat
spinal cord in the absence of astrocytes. Exp Neurol 2005;192:1124.
29 Windrem MS, Roy NS, Wang J et al. Progenitor cells derived from the
adult human subcortical white matter disperse and differentiate as oli-
3213 Cheng, Wang, He et al.
www.StemCells.com
godendrocytes within demyelinated lesions of the rat brain. J Neurosci
Res 2002;69:966975.
30 Windrem MS, Nunes MC, Rashbaum WK et al. Fetal and adult human
oligodendrocyte progenitor cell isolates myelinate the congenitally dys-
myelinated brain. Nat Med 2004;10:9397.
31 Gensert JM, Goldman JE. Endogenous progenitors remyelinate demy-
elinated axons in the adult CNS. Neuron 1997;19:197203.
32 Franklin RJ. Why does remyelination fail in multiple sclerosis? Nat Rev
Neurosci 2002;3:705714.
33 Dubois-Dalcq M, Ffrench-Constant C, Franklin RJ. Enhancing central
nervous system remyelination in multiple sclerosis. Neuron 2005;48:
912.
34 Bunge RP, Puckett WR, Becerra JL et al. Observations on the pathology
of human spinal cord injury. A review and classification of 22 new cases
with details from a case of chronic cord compression with extensive focal
demyelination. Adv Neurol 1993;59:7589.
35 Totoiu MO, Keirstead HS. Spinal cord injury is accompanied by chronic
progressive demyelination. J Comp Neurol 2005;486:373383.
36 Chang A, Nishiyama A, Peterson J et al. NG2-positive oligodendrocyte
progenitor cells in adult human brain and multiple sclerosis lesions.
J Neurosci 2000;20:64046412.
37 Chang CF, Lin SZ, Chiang YH et al. Intravenous administration of bone
morphogenetic protein-7 after ischemia improves motor function in
stroke rats. Stroke 2003;34:558564.
38 Wolswijk G. Oligodendrocyte precursor cells in the demyelinated mul-
tiple sclerosis spinal cord. Brain 2002;125:338349.
39 Shi J, Marinovich A, Barres BA. Purification and characterization of
adult oligodendrocyte precursor cells from the rat optic nerve. J Neurosci
1998;18:46274636.
40 Wolswijk G, Munro PM, Riddle PN et al. Origin, growth factor re-
sponses, and ultrastructural characteristics of an adult-specific glial pro-
genitor cell. Ann N Y Acad Sci 1991;633:502504.
41 Wolswijk G, Noble M. Cooperation between PDGF and FGF converts
slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with
characteristics of O-2Aperinatal progenitor cells. J Cell Biol 1992;118:
889900.
42 Arnett HA, Fancy SP, Alberta JA et al. bHLH transcription factor Olig1
is required to repair demyelinated lesions in the CNS. Science 2004;306:
21112115.
43 Kinsella TM, Nolan GP. Episomal vectors rapidly and stably produce
high-titer recombinant retrovirus. Hum Gene Ther 1996;7:14051413.
44 Cao Q, Zhang YP, Iannotti C et al. Functional and electrophysiological
changes after graded traumatic spinal cord injury in adult rat. Exp Neurol
2005;191(suppl 1):S3S16.
45 Cao Q, Xu XM, DeVries WH et al. Functional recovery in traumatic
spinal cord injury after transplantation of multineurotrophin-expressing
glial-restricted precursor cells. J Neurosci 2005;25:69476957.
46 McTigue DM, Wei P, Stokes BT. Proliferation of NG2-positive cells and
altered oligodendrocyte numbers in the contused rat spinal cord. J Neu-
rosci 2001;21:33923400.
47 Wolswijk G, Noble M. Identification of an adult-specific glial progenitor
cell. Development 1989;105:387400.
48 Armstrong RC, Dorn HH, Kufta CV et al. Pre-oligodendrocytes from
adult human CNS. J Neurosci 1992;12:15381547.
49 Gogate N, Verma L, Zhou JM et al. Plasticity in the adult human
oligodendrocyte lineage. J Neurosci 1994;14:45714587.
50 Gensert JM, Goldman JE. Heterogeneity of cycling glial progenitors
in the adult mammalian cortex and white matter. J Neurobiol 2001;
48:7586.
51 Tang DG, Tokumoto YM, Raff MC. Long-term culture of purified
postnatal oligodendrocyte precursor cells. Evidence for an intrinsic
maturation program that plays out over months. J Cell Biol 2000;148:
971984.
52 Tang DG, Tokumoto YM, Apperly JA et al. Lack of replicative senes-
cence in cultured rat oligodendrocyte precursor cells. Science 2001;291:
868871.
53 Grinspan JB, Reeves MF, Coulaloglou MJ et al. Re-entry into the cell
cycle is required for bFGF-induced oligodendroglial dedifferentiation
and survival. J Neurosci Res 1996;46:456464.
54 Noble M, Wolswijk G, Wren D. The complex relationship between cell
division and the control of differentiation in oligodendrocyte-type-2
astrocyte progenitor cells isolated from perinatal and adult rat optic
nerves. Prog Growth Factor Res 1989;1:179194.
55 Raff MC, Miller RH, Noble M. A glial progenitor cell that develops in
vitro into an astrocyte or an oligodendrocyte depending on culture
medium. Nature 1983;303:390396.
56 See J, Zhang X, Eraydin N et al. Oligodendrocyte maturation is inhibited
by bone morphogenetic protein. Mol Cell Neurosci 2004;26:481492.
57 Wada T, Kagawa T, Ivanova A et al. Dorsal spinal cord inhibits oligo-
dendrocyte development. Dev Biol 2000;227:4255.
58 Wang S, Sdrulla A, Johnson JE et al. A role for the helix-loop-helix
protein Id2 in the control of oligodendrocyte development. Neuron
2001;29:603614.
59 Kondo T, Raff M. The Id4 HLH protein and the timing of oligodendro-
cyte differentiation. EMBO J 2000;19:19982007.
60 Ross SE, Greenberg ME, Stiles CD. Basic helix-loop-helix factors in
cortical development. Neuron 2003;39:1325.
61 Zhou Q, Wang S, Anderson DJ. Identification of a novel family of
oligodendrocyte lineage-specific basic helix-loop-helix transcription fac-
tors. Neuron 2000;25:331343.
62 Lu QR, Yuk D, Alberta JA et al. Sonic hedgehogregulated oligoden-
drocyte lineage genes encoding bHLH proteins in the mammalian central
nervous system. Neuron 2000;25:317329.
63 Lu QR, Cai L, Rowitch D et al. Ectopic expression of Olig1 promotes
oligodendrocyte formation and reduces neuronal survival in developing
mouse cortex. Nat Neurosci 2001;4:973974.
64 Takebayashi H, Nabeshima Y, Yoshida S et al. The basic helix-loop-
helix factor olig2 is essential for the development of motoneuron and
oligodendrocyte lineages. Curr Biol 2002;12:11571163.
65 Nakashima K, Yanagisawa M, Arakawa H et al. Synergistic signaling in
fetal brain by STAT3-Smad1 complex bridged by p300. Science 1999;
284:479482.
66 Fukuda S, Taga T. Cell fate determination regulated by a transcrip-
tional signal network in the developing mouse brain. Anat Sci Int
2005;80:1218.
67 Fukuda S, Kondo T, Takebayashi H et al. Negative regulatory effect of
an oligodendrocytic bHLH factor OLIG2 on the astrocytic differentiation
pathway. Cell Death Differ 2004;11:196202.
68 Chang A, Tourtellotte WW, Rudick R et al. Premyelinating oligoden-
drocytes in chronic lesions of multiple sclerosis. N Engl J Med 2002;
346:165173.
69 Wolswijk G. Chronic stage multiple sclerosis lesions contain a relatively
quiescent population of oligodendrocyte precursor cells. J Neurosci
1998;18:601609.
70 Wilson HC, Scolding NJ, Raine CS. Co-expression of PDGF alpha
receptor and NG2 by oligodendrocyte precursors in human CNS and
multiple sclerosis lesions. J Neuroimmunol 2006;176:162173.
71 Back SA, Tuohy TM, Chen H et al. Hyaluronan accumulates in demy-
elinated lesions and inhibits oligodendrocyte progenitor maturation. Nat
Med 2005;11:966972.
72 John GR, Shankar SL, Shafit-Zagardo B et al. Multiple sclerosis: Re-
expression of a developmental pathway that restricts oligodendrocyte
maturation. Nat Med 2002;8:11151121.
73 Setoguchi T, Nakashima K, Takizawa T et al. Treatment of spinal cord
injury by transplantation of fetal neural precursor cells engineered to
express BMP inhibitor. Exp Neurol 2004;189:3344.
74 Setoguchi T, Yone K, Matsuoka E et al. Traumatic injury-induced BMP7
expression in the adult rat spinal cord. Brain Res 2001;921:219225.
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3214 BMP Signaling and olig1/2 Regulate OPCs