Advances in Bioscience and Biotechnology, 2012, 3, 567-573 ABB
http://dx.doi.org/10.4236/abb.2012.35073 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
Olig1 and Olig2 promote oligodendrocyte differentiation of
neural stem cells in adult mice injured by EAE
Tamara L. Adams, Joseph M. Verdi*
Maine Medical Center Research Institute, 81 Research Drive, Scarborough, USA
Received 2 June 2012; revised 28 July 2012; accepted 28 August 2012
ABSTRACT
Investigating neural stem cell plasticity in the hippo-
campal niche, we demonstrate that retroviral forced
expression of Mash1 (Mammalian Achaete-Scute Ho-
molog 1), Olig1 (Oligodendrocyte transcription factor
1), and Olig2 (Oligodendrocyte transcription factor 2)
genes, transcription factors involved in enhanced oli-
godendrogenesis, can contribute to directing the dif-
ferentiation of adult subventricular zone neural stem
cells to functional oligodendrocytes. We found that
Mash1, Olig1 and Olig2 all induced oligodendrocyte
differentiation. However, Olig1 and Olig2 induction
resulted in an elevated number of generated oligoden-
drocytes without a significant production of other cell
lineages, unlike Mash1. These newly differentiated
cells are also capable of migration and possible myeli-
nation, showing that targeting oligodendrocyte pro-
duction and possible remyelination is a viable thera-
peutic strategy for restoration of neuronal function.
Keywords: Mash1; Olig1; Olig2; Oligodendrocyte; NSC;
EAE/MS
1. INTRODUCTION
Identifying signaling and pathways that govern oli-
godendrocyte production would allow for the generation
of a surplus of oligodendrocyte cells that are impervious
from immune rejection and could be used as a therapeu-
tic measure in diseases such as Multiple Sclerosis (MS)
where myelination is compromised. In order to ensure
the generation of sufficient number of oligodendrocytes
needed for effective cell therapy, it would be advanta-
geous to learn how these cells are derived from redi-
rected granule cell precursors and whether induction of
transcription factors involved in oligodendrocyte differ-
entiation would improve the number of differentiated
oligodendrocytes ([1] Raff et al., 1998, [2] Raff et al.,
2001, [3] Stemple et al., 1992).
In this study, we investigated neural stem cell plastic-
ity in the hippocampal niche resulting in neuronal repair
using a retroviral induction approach of multiple tran-
scription factors to direct NSCs differentiation into oli-
godendrocytes in an inflammatory microenvironment
analogous to that in MS [4] (Navikas et al., 1996). Our
intent was to produce a sizeable number of oligodendro-
cytes from mouse hippocampal neural stem cells in Ex-
perimental Autoimmune Encephalomyelitis (EAE) dis-
ease model, widely used to study pathogenesis of auto-
immunity, demyelination, cell trafficking, and tolerance
induction [5] (Olsson et al., 1995).
2. MATERIALS AND METHODS
2.1. Cell Culture
Forebrains were removed from15-day old mouse fetuses,
mechanically dissociated then digested in 0.05% trypsin
(Mediatech, Manassas, VA) for 15 min at 37˚, then
washed with 1× DPBS (Mediatech, Manassas, VA) and
passed gently through a 70 μm mesh. The cells were then
centrifuged then the pellet was resuspended in DMEM/
10% FBS/0.28% BSA. 14 mm diameter glass coverslips
were coated with poly L-lysine (Sigma, St. Louis, MO)
and placed in 24 well plates (CellstarOne, Monroe, North
Carolina). The cells were plated at 5 × 104 cells per cov-
erslip in 10 μl of media then placed at 37˚ with 5% CO2
for 30 minutes to allow attachment. 500 μl of culture
medium was added to each well for 2 weeks, media was
replaced twice a week [6] (C. Lubetzki et al., 1993).
2.2. Generation of EAE Mice
C57B/6, both male and female, age 10 weeks old, were
purchased from Taconic (Hudson, NY) and acclimated to
our vivarium. EAE was induced via injections of a com-
mercially available kit from Hooke’s Laboratories (San
Diego, CA, kit # EK-0115 (MOG 35-55/CFA Emulsion
PTX). This was used according to manufactures protocol.
The mice were monitored daily for changes (trembling,
limping, partial or full paralysis, and other signs of EAE
*Corresponding author.

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 568
induction) and were scored using Hooke’s scale of 0 - 5.
Using this system we were able to induce EAE as de-
fined by 24-hour partial paralysis in greater than 70% of
the mice within 14 days of the final injection of pertussis
toxin. All mouse studies were done with IACUC ap-
proval with minimal discomfort to animals.
2.3. Retroviral Production
The GFP, Mash1, Shh, and Olig1 and Olig2 virus parti-
cles were made by cloning each gene into the AP2-GFP
retrovirus plasmid via the Xho1 cut site. 293-GPG cells,
grown in DMEM media with 10% FBS and 200 ug/ml
G418, were used to obtain the retrovirus conditioned
medium. These cells were transfected with each retrovi-
rus construct using Invitrogen Lipofectamine 2000 Trans-
fection Reagent (Invitrogen, Carlsbad, CA). The condi-
tioned medium was taken every 24 hours. The medium
was then concentrated using the ViraBind Retrovirus
Concentration and Purification Kit Item #VPK-131 (Cell
Biolabs Inc., San Diego, CA) as protocol required to
obtain concentrated virus at <10'5 CFU.
2.4. Stereotactic Virus Injections
Mice were injected with Avertin at a concentration of
250 mg/kg to anesthetize the mice. Then each mouse was
stereotactically injected using a Lab Standard Stereotaxic
Instrument with Stoelting manual Stereotaxic injector
(Stoelting, Wood Dale, IL) at –2 anterior/posterior +1.5
medial/laeral and –2.3 dorsal/ventral from the skull with
concentrated virus.
2.5. Histological Preparation of Sections
Mice were then euthanized via a CO2 chamber and brains
and spinal cord taken for sectioning. Tissue was fixed in
4% paraformaldehyde prior to sectioning. The specimens
were processed using a Leica TP1020 Automated Tissue
Processor and a Fisher HistoCenter embedding station,
then sectioned at 5 μm with a Microm microtome.
2.6. Immunohistochemistry
Histological and cell culture sections were performed as
previously described in our lab [6] (Matluk et al., 2010).
The primary antibodies used: Mouse anti Olig (RIP, De-
velopmental Studies Hybridoma Bank, University of
Iowa), Mouse anti-MOG (#MAB5680, Chemicon of
Millipore, Billerica, MA), Mouse anti GFAP (Glial fi-
brillary acidic protein, #MAB360, Chemicon of Milli-
pore, Billerica, MA), Rabbit anti Neurofilament (#04-
1032, Chemicon of Millipore, Billerica, MA). Secondary
antibodies were either Alexafluor546 Goat anti-Rabbit
IgG, Alexafluor647 Goat anti-Rabbit IgG or Alexafluor546
Goat anti-Mouse IgG (Molecular probes of Invitrogen,
Carlsbad, CA). Pictures were taken using a Ziess Axio-
vert 200 inverted microscope.
3. RESULTS
3.1. In Vitro Induction of Oligodendrocyte
Differentiation
We focused on transcription factors that have been shown
regulate oligodendrocyte differentiation from NSCs (Fig-
ure 1(a)). We performed AP2 [4] (Galipeau et al., 1999)
retroviral induction of Mash1 [Mammalian Achaete-
Scute Homolog 1 ([7-9]], Shh [Sonic Hedgehog [10])],
Olig1 [Oligodendrocyte transcription factor ([11,12] and
Olig2 [Oligodendrocyte transcription factor 2 ([13] Du et
al., 2006, [14]), (Figure 1(b)) on isolated mouse neural
stem cells in culture to analyze their effects on the oli-
godendrocyte development using previously established
methods [15] (C. Lubetzki et al., 1993). We found that
Olig2 had the greatest effect on oligodendrocyte differ-
entiation and these newly generated oligodendrocytes
were making an effort to myelinate the axons of neurons
in cell culture (Figures 2A-F). We demonstrate that
NSCs induced with Olig2 retrovirus (Figure 2A) differ-
entiate into oligodendrocytes (Figure 2B) which migrate
toward neurons (Figure 2C) and initiate myelination
(Figures 2E and F). We found that both Olig1 and Olig2
induced oligodendrocyte differentiation, however, Olig2
induction resulted in a higher and more consistent aver-
age of generated oligodendrocytes (Figure 2G). We did
not observe a significant synergistic effect of Olig1 and
Olig2 induction (Figure 2G). Mash1 and Shh unlike
Olig1 and Olig2 produced a small number of oligoden-
drocytes when induced alone or in combination (Figure
2G). Interestingly, induction of NSCs with combinations
of Mash1 or Shh with Olig1 or Olig2 caused a decrease
in oligodendrocyte differentiation as compared to Olig1
or Olig2 alone (Figure 2G).
3.2. In Vivo Induction of Oligodendrocyte
Differentiation
We examined forced retroviral expression of Mash1,
Olig1, or Olig-AP2 in differentiating subventricular zone
neural stem cells and its effect on oligodendrocyte fate in
an inflammatory enriched microenvironment, we exam-
ined EAE C57B/6 mice that underwent concentrated
retrovirus particles (<10'5 CFU) injections in the sub-
ventricular zone, following a previously published pro-
tocol ([14] Jessberger et al., 2009). At week four, we ex-
amined histological sections of the brain to evaluate ret-
rovirally induced cell differentiation to oligodendrogenic,
neuronal, and glial lineages using specific lineage mark-
ers. Mash1 differentiate stem cells to oligodendrocytes
upon viral induction, but concomitantly we found a pro-
liferation of virally induced neurons and astrocytes

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 569
(a)
(b)
Figure 1. Schematic outline of forced oligodendrocyte differentiation in EAE induced mice: (a) Our in vivo schematic model depicts
forced induction of differentiated subventricular zone neural stem cells to oligodendrocytes in EAE induced adult mice that begin to
wrap neurons and migrate down the spinal cord; (b) Plasmid map of Olig2-AP2-GFP retrovirus constructs used in viral transduction.
This map is representative of all retroviral constructs used in this study.

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 570
Figure 2. In vitro forced oligodendrocyte differentiation using Olig2 retrovirus. (A) Forced expression of Olig2-AP2-GFP in differ-
entiated oligodendrocytes, Green depicts positive Olig2 retroviral induction, 40×; (B) Positive staining for MBP, a specific marker
for oligodendrocyte, 40×; (C) Positive staining for neurofilament depicts neuron in close proximity to oligodendrocyte, 40×; (D)
Dapi staining for nuclei; (E) Oligodendrocyte differentiated from SVZ NSCs using Olig2-AP2-GFP retroviral induction, phase con-
trast, 40×; (F) Overlayed image of (A-E) depicts growth of generated oligodendrocyte that makes contact with neuron and can poten-
tially myelinate the axon of neuron.

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 571
J
Figure 3. Mash1, Olig1, and Olig2 direct the differentiation of SVZ progenitor cells to oligodendrocytes. (A) Immunohisto-
chemical analysis of Mash1-AP2-GFP positive cells (Green) that was overlapped with staining of RIP, a marker for oli-
godendrocytes. Yellow depicts co-expression; (B) Mash1-AP2-GFP positive cells (Green) merged with overlap of GFAP,
marker for astrocyte lineage (Red). Yellow depicts co-expression; (C) Mash1-AP2-GFP positive cells (Green) merged with
overlap of neurofilament, marker for neurons (Red). Yellow depicts co-expression; (D) Immunohistochemical analysis of
Olig1-AP2-GFP positive cells (Green) merged with overlap of RIP (Red). Yellow depicts co-expression; (E) Olig1-AP2-
GFP positive cells (Green) merged with overlap of GFAP (Red). Yellow depicts co-expression; (F) Olig1-AP2-GFP positive
cells (Green) merged with overlap of neurofilament (Red). Yellow depicts co-expression; (G) Immunohistochemical analysis
of Olig2-AP2-GFP positive cells (Green) merged with overlap of RIP (Red). Yellow depicts co-expression; (H) Olig2-
AP2-GFP positive cells (Green) merged with overlap of GFAP (Red). Yellow depicts coexpression; (I) Olig2-AP2-GFP
positive cells (Green) merged with overlap of neurofilament (Red). Yellow depicts co-expression; (J) Chart illustrating the
range of cell numbers found to co-express virus and antibody for each virus (N = 12 for each virus).

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 572
Figure 4. Virus induced oligodendrocytes appear to myelinate neurons in vivo. (A) Mash1 induced oligodendrocytes appear to mye-
linate local neurons. Mash1-AP2-GFP (Green) Neurofilament (Red); (B) Olig1 induced oligodendrocytes appear to myelinate local
neurons. Olig1-AP2-GFP (Green) Neurofilament (Red); (C) Olig2 induced oligodendrocytes appear to myelinate local neurons.
Olig2-AP2-GFP (Green) Neurofilament (Red).
Figure 5. Virus induced oligodendrocytes migrate down the spinal cord. (A) Mash1 induced oligodendrocytes migrate to 2 cm down
the spinal cord. Mash1-AP2-GFP (Green) Neurofilament (Red); (B) Olig1 induced oligodendrocytes migrate to 3 cm down the spinal
cord. Olig1-AP2-GFP (Green), Neurofilament (Red); (C) Olig2 induced oligodendrocytes migrate to 3 cm down the spinal cord.
Olig2-AP2-GFP (Green), Neurofilament (Red).
(Figures 3A-C). Previous work implicates that Olig1
and Olig2 genes have positive effects on the oligoden-
drocyte differentiation process [16] (Alberta et al., 2001,
[15] Du ZW et al., 2006, and [17] Maire CL et al., 2010).
Our findings revealed that Olig1 (Figure 3D) and Olig2
(Figure 3G) induction resulted in a significant increase
of differentiated oligodendrocytes as compared to Mash1.
In contrast to Mash1, cells from neuron and astrocyte
lineages were not abundant in either Olig1 or Olig2 in-
duction (Figures 3E, F, H, I). These findings, when
compared to the GFP-AP2 virus control, suggest that
Olig1 and Olig2 induction results in an increased popula-
tion of differentiated oligodendrocytes without a signifi-
cant production of other cell lineages (Figure 3J).
3.3. Oligodendrocytes Derived from SVZ
Progenitors Possibly Myelinate and
Migrate to Spinal Cord
We next examined whether differentiated oligodendro-
cytes from the SVZ in EAE mice would myelinate neu-
rons or migrate within the central nervous system. We
observed that Mash1, Olig1, and Olig2-AP2 viral ex-
pressing oligodendrocytes were abundant and close to
neurons detected with antibody for neurofilament protein
NF with the prospective to myelinate (Figures 4A-C).
These newly differentiated stem cells show great poten-
tial in their ability to function as mature oligodendro-
cytes in an adult mouse model by making contact with
nearby neurons. Using a specific marker for oligoden-
drocytes (RIP) we investigated these differentiated oli-
godendrocytes migrating a distance of several centime-
ters within the central nervous system of EAE induced
C56B/6 mice. To show their migrating ability, we ana-
lyzed spinal cord sections up to 3 cm down each spine
and found that virus induced differentiated oligodendro-
cytes were observed to have migrated the entire length of
these sections (Figures 5A-C), giving support to the plas-
ticity of adult mouse neural stem cells. These observa-
tions along with the significant increase in oligodendro-
cyte production from SVZ-derived cells in the brain in-
jured by EAE provide additional evidence that predomi-
natly Olig1 and Olig2 expression in SVZ cells directs
them to a mature oligodendrocyte fate.
4. DISCUSSION
We have demonstrated that retroviral forced expression

T. L. Adams, J. M. Verdi / Advances in Bioscience and Biotechnology 3 (2012) 567-573 573
of Mash1, Olig1, and Olig2 genes, involved in enhanced
oligodendrogenesis, can contribute to directing the dif-
ferentiation of adult subventricular zone neural stem cells
to functional oligodendrocytes. Mash1 alone or with Shh
produced oligodendrocytes, but it was Olig1 and Olig2
that gave us a much larger increase in oligodendrocyte
numbers while creating fewer cells from the neuronal
lineage. These oligodendrocytes are also capable of mi-
gration and possible myelination. These findings could
allow us to design small molecules that would be used to
force ones own neural stem cells to make oligodendro-
cytes and remyelinate the damaged axons and conse-
quently restore neuronal function.
These reports show that targeting oligodendrocyte
production and possible remylination is a viable thera-
peutic strategy for restoration of neuronal function. We
envision that results of the proposed study may extend to
several neurological disorders where cellular replace-
ment is needed. Future work should be done to find
whether all the oligodendrocytes formed are mature
enough to partake in full remyelination of axons that
have loss of function due to EAE.
5. ACKNOWLEDGEMENTS
The authors would like to thank Dr. Jennifer Rochira and Dr. Aldona
Karaczyn for their knowledge and insight for experimental design.
Thank you to Dr. Josephine Nalbantoglu for sharing the AP2 retrovirus
construct. T.A. carried out experiments, analyzed data, and wrote the
manuscript. J.M.V. conceived the experimental design. Authors have
no conflict of interest.
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