Olig1 function is required for remyelination potential of transplanted neural
progenitor cells in a model of viral-induced demyelination
Lucia M. Whitmana,c,1, Caroline A. Blanca,c,1, Chris S. Schaumburga,
David H. Rowitchd, Thomas E. Lanea,b,c,⁎
aDepartment of Molecular Biology and Biochemistry, University of California, Irvine 92697–3900, USA
bSue and Bill Gross Stem Cell, Center, University of California, Irvine 92697–3900, USA
cMultiple Sclerosis Research Center, University of California, Irvine, 92697–3900 USA
dHoward Hughes Medical Institute, Department of Pediatrics, Department of Neurological Surgery, University of California, San Francisco 94143–0525, USA
a b s t r a c ta r t i c l ei n f o
Received 31 January 2012
Accepted 4 March 2012
Available online 17 March 2012
Neural progenitor cells
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in cu-
mulative neurologic deficits associated with progressive myelin loss. We have previously shown that trans-
plantation of neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse
hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells (OPCs)
that is associated with remyelination and axonal sparing. The current study examines the contributions
of the transcription factor Olig1 on NPC differentiation and remyelination. Under defined conditions, NPCs
preferentially differentiate into oligodendroglia whereas NPCs isolated from Olig1-deficient (Olig1−/−)
mice exhibit enhanced differentiation into astrocytes. Transplantation of Olig1−/− and Olig1+/+ NPCs
into JHMV-infected mice resulted in similar cell survival, proliferation, and selective migration to areas of de-
myelination. However, only recipients of wild type NPCs exhibited extensive remyelination compared to
mice receiving Olig1−/− NPCs. In vivo characterization of NPCs revealed that Olig1+/+ NPCs preferentially
differentiated into NG2-positive OPCs and formed processes expressing myelin basic protein that encircled
axons. In contrast, the majority of transplanted Olig1−/− NPCs differentiated into GFAP-positive cells consis-
tent with the astrocyte lineage. These results indicate that exogenous NPCs contribute to improved clinical
and histological outcome and this is associated with remyelination by this donor population. Further, these
findings reveal that Olig1function is required for the remyelination potential of NPCs after transplant, through
specification and/or maintenance of oligodendroglial identity.
© 2012 Elsevier Inc. All rights reserved.
An important clinical aspect related to the pathogenesis of the
human demyelinating disease multiple sclerosis is the eventual
remyelination failure in chronic demyelinated plaques by endoge-
nous neural progenitor cells (NPCs) that give rise to oligodendrocyte
precursor cells (OPCs) (Franklin and Ffrench-Constant, 2008). Such
failure in myelin regeneration could be due to multiple factors includ-
ing inflammation, inhibitor molecules present in the lesion or age-
related deficits in endogenous OPCs (Fancy et al., 2010). With this
in mind, cell-based therapy using exogenous NPCs or OPCs has
emerged as candidate therapies for promoting remyelination
(Pluchino et al., 2009; Sher et al., 2008). Most studies have utilized ei-
ther autoimmune models of neuroinflammatory-mediated demyelin-
ation, or chemical-induced gliotoxic demyelination to assess the
remyelination potential of NPCs. While such models have advantages
in capturing certain aspects of MS-like pathogenesis, they do not
capture the full range of possible causative factors.
We have focused on a model of persistent viral infection that is
correlative with chronic neuroinflammation and demyelination
(Hosking and Lane, 2010). Our laboratory has recently demonstrated
that transplantation of syngeneic mouse NPCs into mice persistently
infected with the neurotropic JHM strain of mouse hepatitis virus
(JHMV) is well tolerated and is associated with axonal sparing accom-
panied by extensive remyelination while not significantly dampening
either neuroinflammation or T cell responses (Hardison et al., 2006;
Totoiu et al., 2004). Evident from this work is i) the ability of
engrafted cells to migrate to regions of demyelination by responding
to the chemokine ligand CXCL12 (Carbajal et al., 2010) and ii) prefer-
ential differentiation of transplanted NPCs into oligodendrocyte
Experimental Neurology 235 (2012) 380–387
⁎ Corresponding author at: Department of Molecular Biology and Biochemistry, 3205
McGaugh Hall, University of California, Irvine, USA, CA 92697–3900. Fax: +1 949 824
E-mail addresses: email@example.com (L.M. Whitman), firstname.lastname@example.org
(C.A. Blanc), Schaumburg_Christopher@Allergan.com (C.S. Schaumburg),
email@example.com (D.H. Rowitch), firstname.lastname@example.org (T.E. Lane).
1These authors contributed equally to the work described herein.
0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
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progenitor cells (OPCs) ((Carbajal et al., 2010; Totoiu et al., 2004). The
experiments described here address the functional contributions of
exogenous NPCs to remyelination.
The genes Olig1 and Olig2 encode basic helix–loop helix transcrip-
tion factors that are expressed in neural progenitor cells (Zhou et al.,
2000) and are required for fundamental processes of CNS development
including oligodendrocyte formation (Lu et al., 2002a,b). Olig1 is espe-
cially involved in oligodendrocyte development as well as maturation
(Lu et al., 2002a,b). Previous studies have shown the implication of
Olig1 in differentiation and remyelination in toxin induced models of
demyelination (Arnett et al., 2004). However these models do not
take into account the potential effects of an inflammatory environment
on NPCs. To this end, we have compared the remyelination and differ-
entiation potential of competent and incompetent (Olig1−/−) NPCs
post-transplant into JHMV-infected mice.
Materials and methods
Mice and virus
Five-week-old male C57BL/6 mice were purchased from the Na-
tional Cancer Institute (Frederick, MD) and Olig1−/− male (Lu
et al., 2002a,b) mice (C57Bl/6 background) were bred in the UCI vi-
varium. For viral infection, mice were anesthetized by intraperitoneal
(i.p.) injection of ketamine 80–100 mg/kg (MP Biomedicals, OH) and
xylazine 5–10 kg/mg (Phoenix Pharmaceutical, MO). Mice were
infected i.c. with 200 plaque forming units (PFU) of JHMV (strain
2.2 V-1) suspended in 30 μl of sterile saline (Fleming et al., 1986).
Timed pregnant C57BL/6 mice (P14) were purchased from the Na-
tional Cancer Institute and perinatal pups were used for wild type
NPC cultures. C57BL/6-Tg (CAG-EGFP) 10sbJ mice (expressing green
fluorescent protein, GFP) were purchased from JAX (stock #003291)
(Lu et al., 2002a,b). C57BL/6-Tg (CAG-EGFP) 10sbJ mice were bred
with Olig1−/− mice to produce the GFP-positive Olig1−/− colony
(confirmed by PCR genotyping). All experiments were reviewed and
approved by the University of California, Irvine Institutional Animal
Care and Use Committee (IACUC).
Epidermal growth factor (EGF) responsive neurosphere cultures
were prepared from either EGFP-Olig1+/+ or EGFP-Olig1−/− mice.
Neurosphere cultures were prepared as previously described from the
brains of perinatal animals (Ben-Hur et al., 1998; Totoiu et al., 2004).
Briefly, dissected striata were razor minced and triturated in pre-
warmed 0.05% Trypsin (Invitrogen) for 10 min. Trypsin digestion was
halted with equal volume of 1× anti-trypsin (Invitrogen). Single cells
were resuspended in DMEM: F12 (Invitrogen) supplemented with 1×
B27 (Invitrogen), 1× Insulin–Transferrin–Selenium–X (Invitrogen),
T67407), and 20 ng/ml human recombinant EGF (Sigma-E9644) and
cultured for 5–6 days. Culture supernatant was replaced with fresh
media containing EGF on days 1, 3, and 5. After one week, mature neu-
rospheres (100–200 μm) were transferred to matrigel (BD Bioscience)
coated flasks (thin coat method, 1:30 dilution). Within 24 h individual
cells had spread out from attached spheres and formed a monolayer.
Following formation of a monolayer formed, cells were trypsinized
and suspended in sterile saline for transplant experiment.
40 ng/ml T3 (Sigma-
Differentiation of neurospheres
To assess differentiation potential, cells were grown on matrigel
coated imaging slides for a total of 4 days, fixed in 4% paraformalde-
hyde (Fisher Scientific, Fair Lawn, NJ) for 20 min and immunofluores-
cence staining was performed using standard protocols. Imaging
chambers were blocked with 10% normal goat serum (NGS) (Vector
Laboratories, Burlingame, CA) for 1 h at room temperature. Primary
antibodies (polyclonal rabbit anti-GalC, Chemicon, 1:50 dilution
in 10% NGS; polyclonal rabbit anti-GFAP, Invitrogen, 1:500 dilution
in 10% NGS; polyclonal rabbit anti-NG2, Chemicon, 1:200 dilution in
10% NGS or blocking solution (negative control, 10% NGS in PBS)
were applied to chambers overnight at 4 °C. Slides were rinsed
three times with PBS and fluorescent-conjugated secondary antibody
(Alexa 594, goat anti-rabbit) was applied and incubated for 1 h at
room temperature. Slides were rinsed three times in PBS and
mounted in vectashield (Vector Laboratories) with Dapi to visualize
cell nuclei. Cell quantification was conducted using a Nikon Eclipse
Ti microscope, 200× magnification. The percentage of immunoposi-
tive cells was determined by dividing the total number of immunopo-
sitive cells by the total number of Dapi-positive cells in five images,
multiplied by 100.
JHMV-infected mice develop demyelination associated with clini-
cal disease 10–14 post-infection (p.i.) (Fleming et al., 1986; Glass
et al., 2002). Transplant experiments were performed on days 14 p.i.,
when replicating virus is reduced below detectable levels and there
is evidence of demyelinating lesions. Clinical severity was assessed
using a previously described 4-point scale (Lane et al., 2000). Only an-
imals that developed partial-to-complete hind limb paralysis were
used for transplantation. For transplant experiments, recipient mice
with comparable clinical disease received either GFP-Olig1+/+ or
GFP-Olig1−/− NPCs, or vehicle control (sterile saline). Anaesthetized
animals received laminectory at T9–T10 to expose the spinal cord. An-
imals were then transplanted with 2.5 μl of NPC (250,000 cells) or
2.5 μl of sterile saline using a 10 μl Hamilton syringe (Hamilton) with
a silicon-coated pulled glass tip affixed in a stereotactic arm as
previously described (Nistor et al., 2005; Totoiu et al., 2004).
Transplanted recipient animals were euthanized at 21 and 35 days
post-infection (p.i.) (7 and 21 days post-transplant) and tissue was
fixed by intracardiac perfusion with 4% paraformaldehyde in PBS
(pH 7.4). Intact spinal columns were removed and fixed overnight
in 4% paraformaldehyde at 4 °C. The bone was removed to expose
the fixed spinal cord and the tissue 8 mm anterior and 8 mm posteri-
or from the injection site was divided into twelve tissue pieces
(1 mm). To evaluate GFP+NPC migration and differentiation
in vivo, tissue sections were cryoprotected in 30% sucrose for 7 days
and embedded in OCT (Tissue-Tek). Seven-micron thick transverse
sections were cut and used for immunofluorescence staining or
stained with luxol fast blue (LFB) in combination with H&E (hema-
toxylin and eosin) to determine the extent of demyelination. To eval-
uate the remyelination potential of transplanted NPC, even tissue
pieces were processed and embedded in resin while odd tissue pieces
were processed and embedded in OCT. For resin sectioning, tissue
pieces were exposed to 1% Osmium tetroxide (Electron Microscopy
Sciences), dehydrated in ascending alcohols, and embedded in Spurr
resin (Electron Microscopy Sciences) according to standard protocols.
Transverse semi-thin (1 μm) sections were cut from each block,
stained with alkaline toluidine blue, cover slipped, and examined by
light microscopy using an Olympus BX-60 microscope, 600× magnifi-
cation. The myelination of axons was determined by assessing the
thickness of the myelin sheath in relation to the axons diameter
(Guy et al., 1989; Hildebrand and Hahn, 1978). Demyelinated axons,
remyelinated axons and normally myelinated axons were counted
within an area equal to 10% of the total area of demyelination. The
quantitative assessment of remyelination was conducted throughout
the region 8 mm caudal and rostral to the transplant site. The number
demyelinated axons, remyelinated axons, the total number of axons
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
and the percent remyelinated axons was determined for each of the
four regions on each tissue block, averaged, then averaged across an-
imals within each group for each tissue block as previously described
(Totoiu et al., 2004).
The total numbers of GFP-positive cells was determined in each of
the twelve sampled locations surrounding the transplant site by
counting all GFP-positive cells co-localized with Dapi-positive nuclei.
Cell migration was represented in graphs of the number of GFP-
positive cells versus distance from transplant site (mm), and the ap-
proximate number of GFP-positive NPC 21 days post-transplant was
analyzed by area under the curve calculation performed in GraphPad
Prism (GraphPad Software) (Behrstock et al., 2008).
To assess in vivo differentiation of GFP-positive NPC 21 days after
OCT, and blocked for 1 h at room temperature with 10% goat serum
in PBS. Immunofluorescence staining was performed using standard
protocols. The following primary antibodies were added overnight at
4 °C: rabbit anti-MBP 1:200 (Chemicon, cat#AB980), rabbit anti-
GST-p 1:1000 (MBL, cat#311), rabbit anti-NG2 1:200 (Chemicon,
cat#AB5320), rabbit anti-GFAP 1:11000 (Invitrogen, cat#18-0063),
and rabbit anti-NF-150 (Chemicon, cat#AB1991). Appropriate conju-
gated goat secondary antibodies were used for visualization (Invitro-
gen). Slides were mounted in vectashield (Vector Laboratories) with
DAPI to visualize cell nuclei and to preserve fluorescence. Cell quantifi-
nification. The percentage of immunopositive cells was determined by
dividing the number of immunopositive cells by the number of Dapi-
positive nuclei, multiplied by 100.
All data is presented as average±SEM. Statistically significant dif-
ferences were assessed by one-way ANOVA, and p values less than
0.05 were considered significant.
Olig1 enhances NPC oligodendroglial commitment
To investigate the importance of Olig1 in oligodendrocyte lineage
commitment, NPCs were cultured from the brains of EGFP-Olig1+/+
(WT) and EGFP-Olig1-deficient mice (Olig1−/− mice). NPCs were
cultured on matrigel-coated slides for 5 days to induce differentiation
under defined conditions at which point defined cellular antigens
were used to identify lineage commitment by immunocytochemical
staining. By 5 days post-differentiation, the majority of cells cultured
from WT mice expressed antigens NG2 and/or GalC that are markers
associated with cells of the oligodendrocyte lineage (Fig. 1A and C).
Approximately 5% of cultured WT cells expressed the astrocyte-
associated marker GFAP (Fig. 1D and F). In contrast, genetic ablation
of Olig1 in NPCs resulted in reduced expression of both NG2 and
GalC (Fig. 1B and C) while there was an ~2-fold increase in GFAP ex-
pression when compared to WT cells (Fig. 1E and F). Immunocyto-
chemical staining of differentiated WT and Olig1−/− NPCs revealed
GalC-positive cells displaying an arborized morphology consistent
with oligodendroglia (Fig. 1A and B). GFAP-positive cells derived
from either WT or Olig1−/− NPCs exhibited a more flat or stellate
morphology (Fig. 1D and E).
GFP-NPC transplantation does not modulate the severity of
To avoidtoxicity andinduction of senescence associatedwithBrdU
labeling of NPC (Ross et al., 2008), we generated GFP-Olig1−/− mice
and used NPC cultured from these mice for subsequent transplanta-
tion into mice. NPC cultured from GFP-C57BL/6 mice were used as
WT controls. Two weeks prior to transplantation, recipient C57BL/6
mice were infected by i.c. injection of 200 PFU of JHMV, which is a
sufficient viral dose to induce immune-mediated demyelination.
Transplantation consisted of a single intraspinal injection of 2.5×105
cells (or vehicle control, HBSS) at thoracic vertebrae 9 and recipient
animals were sacrificed one week and three weeks post-transplant
to evaluate the extent of exogenous cell migration and engraftment.
Luxol fast blue (LFB) staining of a representative JHMV-infected
mouse 14 days p.i. indicated that at the time of transplantation,
% GFAP+ cells
% + cells
Fig. 1. Differentiation of neural progenitor cells derived from wild type and Olig1 deficient mice yields oligodendrocyte and astrocyte enriched cell populations in vitro respective-
ly.Neural progenitor cells were cultured from the brains of wild type and Olig1 deficient mice, 1-day postnatal pups, in the presence of EGF using the neurosphere culture method.
(A) Immunofluorescence staining of Galc-positive oligodendrocyte following 5 days in vitro differentiation of wild type NPC and (B) Olig1−/− NPC. (C) Quantification of immuno-
fluorescence staining for Galc and NG2 was performed 4–5 days following the simultaneous removal of growth factors and transfer of neurospheres to matrigel coated slides. (D)
Immunofluorescence staining of GFAP-positive astrocytes following 5 days in vitro differentiation of wild type NPC and (E) Olig1−/− NPC. (F) Quantification of immunofluores-
cence staining for GFAP. Data are presented as average±SEM and are representative of results of three independent cultures.
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
white matter demyelination was localized in the ventral white matter
in the vicinity of the central canal (Fig. 2A). Three weeks post-
transplant of either WT or Olig1−/− NPCs, demyelinated lesions
were found to extend from the ventral to the lateral white matter
(Fig. 2A). Quantification of demyelination in experimental mice indi-
cated a similar level of myelin damage in recipients of either WT or
Olig1−/− NPCs compared to vehicle control (Fig. 2B). Importantly,
these findings are consistent with earlier results and indicate that
transplantation of NPCs neither exacerbate nor ameliorate JHMV-
induced immunopathology and this was associated with no attenua-
tion in neuroinflammation (Hardison et al., 2006; Totoiu et al., 2004).
Similar migration of WT and Olig1−/− NPCs
Surgically engrafted NPCs preferentially migrate and accumulate
within areas of white matter damage in JHMV-infected mice
(Hardison et al., 2006; Totoiu et al., 2004). Moreover, we have deter-
mined that migration is mediated through CXCR4 expressed on trans-
planted NPCs responding to CXCL12 that is expressed within
demyelinating lesions (Carbajal et al., 2010). Transplanted WT and
Olig1−/− NPCs exhibited similar migration both rostral and caudal
to the site of implantation at 1 and 3 weeks post-transplant indicating
that Olig1 function does not regulate migration (Fig. 3A). In addition,
similar numbers of WT and Olig1−/− NPCs were detected in trans-
planted mice indicating that replication is not negatively affected in
the absence of Olig1 (Fig. 3B). Importantly, positional migration of
transplanted NPCs was not affected in the absence of Olig1 as both
populations of NPCs preferentially migrated into ventral and lateral
white matter columns of JHMV-infected mice (Fig. 3C). Collectively,
these findings provide compelling evidence that Olig1 function is
not required for NPC migration, proliferation, or preferential accumu-
lation within areas of white matter damage.
Olig1−/− NPCs do not promote remyelination
We next evaluated the extent of remyelination in JHMV-infected
recipients of either WT or Olig1−/− NPCs at 3 weeks post-
transplant. Spinal cord sections were used for either tracking migra-
tion of GFP-labeled NPCs or measuring remyelination to ensure that
assessment of histopathology was performed within areas in which
transplanted cells were present (Fig. 4A). A representative spinal
cord from a non-infected, non-transplanted mouse is shown to dem-
onstrate normal myelin thickness (Fig. 4B). Numerous demyelinated
axons were present among vacuoles, myelin debris, and activated
macrophages in JHMV-infected mice receiving either vehicle control
(Fig. 4C) or Olig1−/− NPCs (Fig. 4D). In contrast, infected mice trans-
planted with WT NPCs exhibited numerous axons with thin myelin
sheaths consistent with remyelination (Fig. 4E). Quantification of
remyelination in experimental groups of mice revealed an overall in-
crease in the frequency of remyelinated axons in mice transplanted
with WT NPCs when compared to either Olig1−/− NPCs or vehicle
control (Fig. 4F) (Totoiu et al., 2004). Furthermore, immunofluores-
cent staining in combination with confocal microscopy revealed GFP
signal overlying with MBP staining (Fig. 5A) as well as GFP-positive
wraps surrounding numerous axons (defined by neurofilament stain-
ing) (Fig. 5B) in mice receiving WT NPCs yet this was absent in recip-
ients of Olig1−/− NPCs. These findings support and extend previous
work from our laboratory indicating that transplantation of NPCs into
JHMV-infected mice results in extensive remyelination (Carbajal
et al., 2010; Totoiu et al., 2004). The overall paucity in remyelinated
axons in Olig1−/− NPCs suggests that although these cells are able
to migrate and accumulate within areas of pathology they are not
able to remyelinate demyelinated axons. Importantly, the demonstra-
tion of transplanted NPC-derived myelin wraps surrounding axons in
WT NPC recipients provides compelling evidence that transplanted
cells directly remyelinate axons.
In vivo differentiation of transplanted WT and Olig1−/− NPCs
Evaluation of differentiated WT and Olig1−/− NPCs at 3 weeks
post-transplant in JHMV-infected mice was performed by immuno-
fluorescence staining. Consistent with previous findings (Carbajal
et al., 2010), approximately 40% of the engrafted WT NPCs differenti-
ated to an oligodendrocyte lineage as determined by NG2 and GST-π
staining (Fig. 6A). The majority of WT NPCs had differentiated into
NG2-positive OPCs (27.78%±3.31) rather than mature oligodendro-
cytes as evidenced by GST-π staining (11.83%±0.72). In marked con-
trast, very few (b5%) of Olig1−/− NPCs expressed either NG2
(4.11%±1.92) or GST-π (2.7%±0.55) indicating these cells did not
preferentially differentiate into a myelin-competent cell (Fig. 6A). In-
deed, staining for the astrocyte marker GFAP revealed that the major-
ity (>70%) of transplanted Olig1−/− NPCs were GFAP-positive
(73.04%±3.1) and displayed a morphology consistent with an acti-
vated astrocyte (Fig. 6B). However, fewer than 20% of transplanted
WT NPCs differentiated in GFAP-positive cells (17.05%±3.57)
JHMV 14 day p.i.
Fig. 2. Transplantation of wild type and Olig1 deficient NPC does not modulate JHMV-induced demyelination.(A) Representative OCT embedded spinal cords stained with luxol fast
blue (intact myelin is bright blue) in combination with H&E staining. The intact myelinated white matter of sham-infected mouse (upper left) stains bright blue while demyelinated
lesions (dotted outline) are present in the ventral white matter 14 day p.i. JHMV infection (upper right). Representative tissue from mice that received wild type GFP+NPC trans-
plant (lower left) or GFP+Olig1−/− NPC transplant (lower right) indicate that demyelination spread evenly through the ventral white matter during the three-week survival pe-
riod. (B) Quantification of the overall percent demyelination following transplantation of wild type NPC, Olig1−/− NPC or vehicle control indicates that all three groups had
equivalent average. A black bar indicates the average of each group, while shapes represent individual animals. The percent demyelination for each individual animal is the average
demyelination from 12 spinal cord sections spaced over 16 mm, 8 mm anterior and 8 mm posterior from the transplant injection site. Percent demyelination=(area of demyelin-
ation/total area white matter)×100. Results are representative of two independent experiments.
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
There are no effective therapies available for patients with pro-
gressive forms of MS. For this and other devastating human white
matter disorders, exogenous NPC and OPC transplantation are of
great interest as having potential therapeutic roles (Conti et al.,
2006; Fancy et al., 2010; Kim and de Vellis, 2009; Ligon et al., 2006;
Pluchino and Martino, 2005; Sher et al., 2008; Tirotta et al., 2010;
Zhao et al., 2008). The use of a viral model of demyelination to evalu-
ate the remyelination potential of NPCs has unique features that
make this a relevant experimental model system. First, the etiology
of the human demyelinating disease is enigmatic with both genetic
factors and environmental influences considered important in initia-
tion and maintenance of disease. Viral infection has long been viewed
as a potential triggering mechanism involved in demyelination and
numerous human viral pathogens have been suggested to be involved
in eliciting myelin-reactive lymphocytes and/or antibodies that sub-
sequently infiltrate the CNS and damage the myelin sheath
(Ascherio and Munger, 2007; Ebers et al., 1995; Sospedra and
Martin, 2005). Therefore, viral models of demyelination are clearly
relevant and have provided important insight into mechanisms asso-
ciated with disease initiation, neuroinflammation and demyelination.
Given the possibility of viral infection in initiating demyelination as
well as the fact that numerous neurotropic viruses exist that are capa-
ble of persisting within the CNS, it is imperative to evaluate the
remyelination potential of stem cells in the presence of a persistent
viral infection that is correlative with chronic neuroinflammation
The present study sought to determine the importance of Olig1 in
NPC-mediated remyelination in JHMV-infected mice with established
demyelination. To address this, we used NPCs derived from mice de-
ficient in the transcription factor Olig1. The genes Olig1 and Olig2 en-
code basic helix–loop helix transcription factors that are expressed in
neural progenitor cells (Zhou et al., 2000) and are required for funda-
mental processes of CNS development including oligodendrocyte for-
mation (Lu et al., 2002a,b). The Olig1-deficient mice we employed are
Number GFP+ NPC
distance from injection (mm)
distance from injection (mm)
+6+2 +4 -4-2
Number GFP+ NPC
Olig1-/- NPC WT NPC
1 week post-transplant 3 week post-transplant
+6 +2 +4-6 -4-2 -60
1 week post-transplant3 week post-transplant
GFP+ WT NPC
GFP+ Olig1-/- NPC
Fig. 3. Wild type and Olig1 deficient neural progenitor cells migrate to demyelinated white matter after intra-spinal transplant.JHMV-infected mice with established demyelination
were transplanted with wild type GFP+NPC, GFP+Olig1−/− NPC, or vehicle control (HBSS) by intra-spinal injection. Animals were sacrificed 1 week and 3 weeks following trans-
plant and the 16 mm region of spinal cord surrounding the transplant site was removed, cut into 12 even pieces, and embedded in OCT for analysis. (A) Quantification of the num-
ber of wild type GFP+cells and GFP+Olig1−/− cells per tissue section at one-week and three-weeks post-transplant show anterior and posterior migration of NPC away from the
site of injection. The distribution and extent of migration of wild type and Olig1−/− GFP+NPC overlap at both timepoints. (B) An approximation of total numbers of transplanted
cells one-week and three-weeks after transplanted was calculated using area under the curve analysis of cell number/section data shown in (A). This analysis indicated that wild
type and Olig1−/− NPC have similar survival and proliferation in vivo. Results are representative of two independent experiments. (C) Representative images of GFP+wild type
(left) and Olig1−/− (right) NPC indicate that the transplanted cells migrate to ventral white matter. The anatomical distribution of GFP+NPC mirrors the distribution of demye-
lination at the time of transplant (14 days post-JHMV infection), Fig. 2A. Images are from animals sacrificed 21 days post-transplant. n=4 to 5. Results are representative of two
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
viable and have mildly delayed myelination during development (Lu
et al., 2002a,b). However, they showed a profound deficiency in
remyelination, indicating a nonredundant role for Olig1 in remyelina-
tion due to a defect in OPC differentiation to oligodendrocytes after
white matter injury (Arnett et al., 2004). We note that this contrasts
findings with a different Olig1-null line that shows a lethal develop-
mental hypomyelination phenotype (Xin et al., 2005). Thus, Olig1 is
critical for normal myelination and remyelination.
We took advantage of this genetic requirement of Olig1 in repair
to determine if intraspinal transplantation of Olig1−/− NPCs
resulted in remyelination. Our findings reveal that engrafted Olig1
−/− NPCs were well tolerated, replicated, and migrated to a similar
extent along white matter spinal cord tracts compared to WT NPCs.
However, there was a marked reduction in the number of remyeli-
nated axons in comparison to recipients of control cells. Paucity of
remyelination from Olig1−/− donor cells was associated with an
apparent change in lineage fate commitment, as Olig1-null mice
preferentially differentiated into GFAP-positive cells whereas WT
cells exhibited commitment to an oligodendrocyte lineage. This
was confirmed by our in vitro studies showing the importance of
Olig1−/− to enhance oligodendroglial lineage commitment, as the
absence of this transcription factor seemed to favor the differentia-
tion into astrocytes by NPCs.
In all recipients, WT NPCs migrated to demyelinated white matter
and formed MBP-containing processes that encircle neurofilament
positive axons. These findings are consistent with several possibili-
ties. First, Olig1 may be required to regulate cell fate differentiation
of NPCs into OPCs; alternatively, Olig1 may be required to maintain
OPC fate and in the absence of Olig1 function OPCs will “trans-differ-
entiate” to astrocytes. We favor the former argument as in Olig1−/−
mice OPC fate and myelination proceeds to a level comparable to
wildtype mice demonstrating Olig1 is not strictly required to main-
tain OPC fate. Therefore, our findings indicate that Olig1 is not re-
quired for migration into lesions but is important in selecting an
oligodendrocyte lineage fate. These findings differ from, but are not
in conflict, with Arnett et al. (2004) where OPCs were recruited into
demyelinating lesions induced by lysolethicin treatment but failed
to mature to participate in remyelination. Of note, in this study no in-
creased astrogliosis in lesions was observed. Similarly, transplanted
Olig1−/− NPCs migrated yet did exhibit differentiation primarily
Fig. 4. Transplantation of Olig1−/− NPC does not promote remyelination.JHMV-infected mice with established demyelination were transplanted with wild type NPC, Olig1−/−
NPC, or vehicle control (HBSS) by intra-spinal injection. Animals were sacrificed 3 weeks following transplant and the 16 mm region of spinal cord surrounding the transplant
site was removed, cut into 12 even pieces, and the even pieces were embedded spur resin to evaluate remyelination following NPC transplant. (A) Toluidine blue-stained transverse
spinal cord section 4 boxed areas indicating the 4 regions of remyelination analysis. For each even section, the number of normal, demyelinated and remyelinated axons were
counted and summed. (B) Resin embedded toluidine-blue stained spinal cord from an uninfected animal showing normal axons with thick myelin sheaths. Resin embedded
toluidine-blue stained spinal cord from MHV-infected mice transplanted with (C) vehicle control, (D) Olig1−/− NPC, or (E) wild type NPC. Note the predominance of demyelinated
axons (asterisk), infiltrating cells and tissue damage in the vehicle control and Olig1−/− NPC recipient mice. In contrast, the majority of the axons in the mouse transplanted with
wild type NPC have thin myelin sheaths (black arrow) indicative of remyelination. Demyelinated axons, asterisk; remyelinated axons, black arrow. (F) Quantification of the percent
remyelination by group shows that transplantation of wild type NPC results in 50% remyelinated axons compared to 19% and 18% remyelinated axons in the vehicle control and
Olig1−/− NPC groups respectively. (B–E) Scale bar represents 10 μm.
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
into astrocytes. An important difference in these results may reflect
differences in model systems utilized. While lysolethicin induces
focal demyelinating lesions in the absence of infiltration of activated
T lymphocytes and monocyte/macrophages, JHMV-induced demye-
lination is characterized by the presence of activated T lymphocytes
as well as other inflammatory cells that results in the secretion of nu-
merous proinflammatory cytokines/chemokines (Bergmann et al.,
2006; Hosking and Lane, 2010; Lane and Hosking, 2010). Therefore,
one intriguing possibility is that the inflammatory microenvironment
may tailor NPCs fate decision through an Olig1-regulated mechanism.
In addition, these findings are consistent with other studies highlight-
ing the importance of Olig1 in contributing to myelin repair following
experimental demyelination (Burton, 2005; Ligon et al., 2006; Maire
et al., 2010; Tsiperson et al., 2010).
Our findings suggest that exogenous NPCs actively participate in
remyelination following engraftment. This is supported by counting
remyelinated axons in experimental animals as well as the presence
of GFP-positive wraps encircling axons following transplantation of
wild type NPCs. Our findings are consistent with earlier studies by
Cummings et al. (2005) that demonstrated engraftment of human
NPCs promoted locomotor recovery in a rodent model of spinal cord
injury. Importantly, recovery was abolished by selective ablation of
engrafted cells suggesting that the therapeutic benefit was mediated
primarily by engrafted cells. However, a recent report indicated that
transplanted neural progenitors were shown to enhance proliferation
of host OPCs (Einstein et al., 2009). This was associated with in-
creased remyelination in a model of cuprizone-mediated demyelin-
ation indicating that transplanted neural progenitors stimulated
endogenous cells to participate in repair. Differences between exper-
imental outcomes most likely reflect differences within the model
systems employed as cuprizone represents an acute and focal model
of demyelination with a limited role for activated lymphocytes in par-
ticipating in myelin destruction and this is dramatically different
compared to JHMV-induced demyelination. Therefore, the environ-
mental signals encountered by engrafted cells will modulate the abil-
ity of the engrafted cell to home, differentiate, and participate in
repair. With this in mind, our results reveal insight into the impor-
tance of Olig1 within the context of engrafted NPCs into an ongoing
immune-mediated demyelinating disease initiated by viral infection:
i) Olig1 does not influence NPC positional migration, ii) Olig1 is re-
quired for differentiation into OPCs/oligodendrocytes, and iii) prefer-
ential differentiation into oligolineage cells is associated with
Role of the funding source
The funding source had no involvement in study design or in the
collection, analysis and interpretation of data as well as in writing of
the report and decision of submission for publication.
MBP GFP DAPI
NF GFP DAPI
Fig. 5. Wild type NPC derived cells form processes that encircle axons in vivo.Wild type GFP+NPCs form thin circular GFP+processes in vivo. (A) MBP staining of tissue from rep-
resentative recipient of GFP+wild type NPC showing overlap between MBP and GFP+process. (B) Neurofilament staining of tissue from representative recipient of GFP+wild
type NPC showing GFP+process encircling neurofilament labeled axons.
% positive GFP+ cells
% GFAP+ GFP+ cells
Fig. 6. Wild type GFP+NPCs differentiate into oligodendrocytes and Olig1 deficient
NPCs differentiate into astrocytes in vivo.Analysis of in vivo differentiation of trans-
planted cells into NG2+ oligodendrocyte progenitor cells, GST-π+oligodendrocytes
and GFAP+astrocytes in frozen OCT embedded spinal cord, 3 weeks post-transplant.
(A) Immunofluorescence staining for NG2 of spinal cord from a recipient that received
wild type GFP+NPC shows that 27.5% of the transplanted cells express the NG2 pro-
tein. In contrast, in a recipient that received GFP+Olig1−/− NPC, only 10% of the
transplanted cells express NG2. The overall percent of NG2 positive and GST-π positive
transplanted cells from each group was determined from 6 sections distributed evenly
in the 16 mm region surrounding the injection site. This analysis showed that 3 weeks
after transplant, about 40% of the wild type NPCs differentiated into cells of the oligo-
dendrocyte lineage, compared to less than 10% of Olig1−/− NPCs. n=4. *, pb0.01; **,
pb0.001. (B) The overall percent of GFAP positive transplanted cells from each group
was determined from 6 sections distributed evenly in the 16 mm region surrounding
the injection site. This analysis showed that 3 weeks after transplant, the majority of
Olig1−/− NPC (73±3) differentiates in GFAP expressing astrocytes while less than
20% of transplanted wild type NPCs express GFAP. n=4. **, pb0.001.
L.M. Whitman et al. / Experimental Neurology 235 (2012) 380–387
Acknowledgments Download full-text
This work was funded by National Institutes of Health (NIH) Grant
R01 NS041249, National Multiple Sclerosis Society (NMSS) Grant
RG3857A5, and a Collaborative Center Research Award from NMSS
to T.E.L. D.H.R. is an Investigator of the Howard Hughes Medical Insti-
tute and is also funded by NIH R01 NS040511. L.M.W. and C.S.S. were
supported by California Institute for Regenerative Medicine Training
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