Enhanced green fluorescent protein-expressing human mesenchymal stem
cells retain neural marker expression
David Gordona,⁎, Colin P. Gloverb, Andria M. Merrisona, James B. Uneyb, Neil J. Scoldinga
aMS Labs, Burden Centre, University of Bristol Institute of Clinical Neurosciences, Frenchay Hospital, Bristol, BS16 1JB, UK
bHenry Wellcome Laboratories for Integrative Neuroscience & Endocrinology, University of Bristol, Bristol, UK
Received 20 August 2007; received in revised form 17 September 2007; accepted 12 October 2007
Mesenchymal stem cells (MSCs) have the potential to play a role in autologous treatment of central nervous system injury or disease. Here we
transduced human MSCs with enhanced green fluorescent protein (EGFP). We compared the capacity of control and EGFP-positive cells to
proliferate under normal culture conditions, as well as express neural markers following trans-differentiation. EGFP-positive cells proliferated
comparably to controls, retained EGFP expression over the course of multiple passages, and retained neural marker expression at levels
comparable to control MSCs. Further neurogenic capacity of EGFP-positive human MSCs was examined by growth as neural stem cell-like
neurospheres. No significant difference was observed in the ability of control or EGFP-positive cells to generate primary neurospheres or to
expand during passage. When examined by immunostaining for the presence of neuroectodermal markers, neurosphere-derived cells similarly
expressed neural markers. We show that human MSCs expressing EGFP represent an attractive and practical source of stem cells for the study of
repair and regeneration in neurological models.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Culture; Neural differentiation; Stem cells; EGFP
Stem cells have enormous potential as therapeutic tools in
the treatment of neurological diseases as diverse as multiple
sclerosis (MS), stroke, trauma and Parkinson's disease
(Bjorklund, 2000; Cao et al., 2002; Park et al., 2002; Weissman,
2000). Most studies have focused on the use of embryonic
tissue as the primary stem cell source, but this raises significant
practical, immunological and ethical concerns. It is now well
accepted that adult stem cells are present in a variety of different
tissue types and that these tissue-specific cells have the capacity
to differentiate into a wider range of cell types than previously
thought (Clarke and Frisen, 2001; Poulsom et al., 2002; Preston
et al., 2003; Serafini and Verfaillie, 2006). The multipotentiality
of adult stem cells has generated much interest in their use as
autologous treatments for neurological disease, since it provides
a way to circumvent many of the difficulties arising from the use
of embryonic stem cells (Clarke and Frisen, 2001; Poulsom
et al., 2002; Prockop, 2002; Scolding, 2001).
An attractive source of adult stem cells for autologous stem
cell replacement therapies in the central nervous system (CNS)
is bone marrow (Chopp and Li, 2002; Jiang et al., 2002; Krause
et al., 2001; Pittenger et al., 1999). Human bone marrow-
derived mesenchymal stem cells (MSCs) can be extracted
relatively simply from adult tissue and maintained in cell
culture. When cultured under the appropriate conditions MSCs
are able to generate cells specific to the mesenchymal lineage in
vitro, specifically: chondrocytes (cartilage), osteoblasts (bone)
and adipocytes (fat) (Jiang et al., 2002; Pittenger et al., 1999). It
has further been demonstrated that both human and rodent
MSCs display remarkable phenotypic plasticity, trans-differ-
entiating into neural cells in vitro in the presence of appropriate
growth factors (Deng et al., 2001; Kim et al., 2002; Sanchez-
Ramos et al., 2000; Woodbury et al., 2000). More recently it has
been shown that human MSCs cultured in the presence of
Journal of Neuroimmunology 193 (2008) 59–67
⁎Corresponding author. Fax: +44 117 9186655.
E-mail address: D.Gordon@bristol.ac.uk (D. Gordon).
0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
epidermal growth factor (EGF) and basic fibroblast growth
factor (bFGF) can be induced to grow as neural stem cell
(NSC)-like neurospheres in vitro (Hermann et al., 2004). The
cells within these neurosphere-like structures express high
levels of early neuroectodermal markers, such as the proneural
genes and nestin (Hermann et al., 2004).
Such studies have provided the impetus for a range of
experimental investigations exploring the potential reparative or
neuroprotective effects of MSCs in models of neurological or
neurodegenerative disease (Akiyama et al., 2002a; Akiyama
et al., 2002b; Azizi et al., 1998; Brazelton et al., 2000).
However if such studies are to successfully lead to future
translational studies in patients, then human rather than rodent
cells must be investigated. Furthermore, all such studies are
complicated by the difficulty of identifying transplanted cells
and their derived progeny. One obvious possibility is to
transduce cells prior to implantation with markers such as
enhanced green fluorescent protein (EGFP). In the present study
we show that virally-transduced human MSCs ubiquitously
expressing EGFP retain their capacity to proliferate comparably
to control cells, as well as preserving the ability to express
neural markers when trans-differentiated under the appropriate
conditions. Moreover, EGFP-transduced human MSCs were
able to form and expand as NSC-like neurospheres. Our
findings indicate that human MSCs expressing EGFP represent
an attractive and practical source of stem cells for the study of
repair and regeneration in neurological models.
2. Materials and methods
2.1. Human MSC culture
Bone marrow was obtained by informed consent from
patients undergoing hip replacement surgery. Patient marrow
was used with the consent of the University of Bristol ethics
committee. The femoral neck was retained and stored overnight
at room temperature prior to processing. Marrow was homo-
genized with sterile scalpel blades in Hanks balanced salt
solution (HBSS; Sigma-Aldrich). Marrow samples were layered
over 25 ml of Ficoll (Lymphoprep, Axis-Shield PoC, Norway)
in 50 ml tubes and then centrifuged at 1200 g for 30 min. The
mononuclear cell layer was aspirated from the marrow/Ficoll
interface and made up to 30 ml with HBSS. The resulting
suspension was centrifuged at 300 g for 8 min. The pellet was
resuspended in 10 ml of red blood cell lysis buffer (0.83%
NH4Cl, 0.1% KHCO3and 0.004% EDTA in distilled H20) for
10 min at 4 °C, followed by centrifugation at 300 g for 10 min.
The final cell preparation was seeded at 1×107cells/25 cm2
flask (BD Falcon) in MSC growth medium, which comprised
Dulbecco's modified eagle's medium (DMEM; Sigma-
Aldrich), pH 7.2, containing 10% fetal bovine serum (FBS;
Stem Cell Technologies Inc.), 2 mM L-glutamine (Sigma-
Aldrich), 1 ng/ml recombinant human basic fibroblast growth
factor (bFGF; Sigma-Aldrich) and 50 U/ml penicillin/strepto-
mycin (Gibco BRL). Cells were maintained at 37 °C/5% CO2,
with media changed after 24 h to remove non-adherent cells,
and subsequently every 3 d afterwards.
Cultures were normally passaged after 7–10 d when cells
were confluent. Old MSC growth medium was removed from
flasks and cells rinsed with several ml of phosphate buffered
saline (PBS), pH 7.2. 1 ml of Accutase (PPA Laboratories) was
added to each culture flask/25 cm2and flasks incubated at 37 °C
for 8 min to harvest adherent cells. The resultant cell suspension
was centrifuged at 300 g for 8 min. The pellet was resuspended
in MSC growth medium, cell numbers established and MSCs
plated at 1×105cells/25 cm2flask, 5×105cells/75 cm2flask or
1.2×106cells/175 cm2flask in MSC growth medium at 37 °C/
5% CO2.The mesenchymal nature of cultures was confirmed by
flow cytometry for CD marker expression, and differentiation
into adipogenic, osteogenic or chondrogenic cells using routine
methods (data not shown) (Pittenger et al., 1999).
2.2. Viral transduction of human MSCs
The EGFP-CMV34 lentivirus construct was generated by
C.P. Glover and J. B. Uney (Glover et al., 2002). The EGFP
reporter gene was driven off the human cytomegalovirus pro-
moter (hCMV), while the woodchuck hepatitis virus post-
transcriptional regulatory element (WPRE) was incorporated
to enhance EGFP expression (Glover et al., 2002). To
transduce human MSCs, cells were passaged as normal to
generate a cell suspension. Cells were then plated out at
3×104cells/well of a 6-well plate (BD Falcon) in MSC
medium containing 8 μg/ml hexadimethrine bromide (Sigma-
Aldrich) and viral vector at 1, 10 or 20 multiplicities of
infection (MOI). The medium was changed after 8 and 24 h.
Cells were examined for fluorescence 48 h after initial virus
addition, then passaged as normal after 7–10 d.
2.3. Flow cytometry of EGFP-positive MSCs
Control and EGFP-positive MSCs were harvested from
tissue culture flasks as normal and divided into aliquots of
5×104cells. Suspensions were rinsed with sterile PBS and
centrifuged at 300 g for 8 min, then rinsed briefly with
propidum iodide solution (Sigma-Aldrich) to label dead cells.
Cells were centrifuged at 300 g for 8 min, resuspended in 1 ml
PBS, then analysed on a Beckman Coulter Epics XL.
2.4. Neuronal and glial induction
To induce trans-differentiation, human MSCs were grown to
confluence on PLL-coated coverslips for 7–10 d in MSC
growth medium. Cells were then incubated for a further 10–
14 d in MSC growth medium, neuronal induction medium or
glial induction medium (Hermann et al., 2004). Neuronal
induction medium was comprised of neurobasal medium
(Sigma-Aldrich), 1% FBS, 5% normal horse serum (Vector),
5 μM retinoic acid (Sigma-Aldrich), 1% N2 supplement
(Sigma-Aldrich) and 10 ng/ml recombinant human brain
derived neurotrophic factor (rhBDNF; Peprotech EC). To
make glial induction medium rhBDNF was replaced with
10 ng/ml recombinant human platelet-derived growth factor BB
(rhPDGF-BB; R & D Systems).
60 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
2.5. NSC-like neurosphere culture
MSCs were harvested as normal and plated in low-
attachment tissue culture 12-well plates (Nunc) at 1×104
cells/well in NS-A medium (Euroclone) containing 1% N2
supplement, 2 mM L-glutamine, 25 μg/ml insulin, 20 ng/ml
EGF (Sigma-Aldrich), 10 ng/ml bFGF and 2 ng/ml leukemia
inhibitory factor (Sigma-Aldrich). Cultures were left for 7 d in
vitro (7 DIV) at 37°C and 5% CO2. Passage was carried out by
collecting neurospheres by centrifugation (100 g for 10 min)
and mechanically dissociating them into a single cell suspension
after Accutase treatment for 10 min at 37 °C. For immunostain-
ing following differentiation, cells were plated at 1×104cells/
PLL-coated coverslip for 7–10 d in MSC growth medium,
neuronal induction medium or glial induction medium. For
proliferation studies, cells were replated as described above for
a further 7 DIV to generate secondary neurospheres, and then
passaged again for tertiary neurospheres.
Neurospheres were collected by centrifugation at 100 g for
10 min, and fixed in suspension with 4% PFA in PBS (pH 7.4)
for 30 min at room temperature. The neurosphere suspension
was rinsed 3×with PBS prior to staining. MSC coverslip-
cultures were similarly fixed for 30 min with 4% PFA in PBS
(pH 7.4) at room temperature, prior to PBS rinsing. Immunos-
taining of NSC-like neurospheres and differentiated cells on
Fig. 1. Viral transduction of human MSCs with EGFP. A schematic diagram detailing the lentiviral vector used in this study (A). EGFP is driven off the human
cytomegalovirus promoter (hCMV).The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) enhances EGFP expression. Phase contrast image
of cells 7 d after transduction with lentiviral vector at an MOI of 20 (B). Cells have morphology identical to non-transduced controls. Fluorescent imaging of human
MSCs 7 d after transduction indicate cells express EGFP at multiple MOIs. Shown are representative images of cells from MOI 1 (C) and MOI 20 (D). The number of
EGFP-transduced cellsincreaseswithincreasingMOI (E). WhileMOIs of 1(15.7%±3.2)and10 (18.9%±4.7)hadrelativelylowernumbersofcells expressingEGFP,
those cells transduced with an MOI of 20 displayed the highest levels of EGFP-expressing cells 7 d after initial transduction (45.2%±5.4). Growth curves of control
and transduced cells indicate no significant difference in expansion capabilities between control and transduced cells over the course of 6 passages (F). Scale bars
61 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
coverslips followed essentially the same protocol, although
neurospheres were maintained in suspension throughout. NSC-
like neurospheres and cells were incubated in 4% normal goat
serum (Vector) and 0.1% Triton-X-100 (Sigma-Aldrich) in
PBS, pH 7.2, for 60 min at room temperature. Following PBS
washing, neurospheres were stained with 1:500 mouse anti-
nestin (Chemicon International) and 1:100 mouse anti-O4
(Chemicon International.) overnight at 4°C. After a PBS wash,
neurospheres were incubated for 1 h at room temperature with
goat anti-mouse IgG-Alexa 488 (Molecular Probes). Spheres
were mounted between spacers in Vectashield containing DAPI
(Vector). Progenitors, neurons and astrocytes were identified by
staining with 1:500 mouse anti-nestin, 1:500 rabbit anti-βIII-
tubulin (Sigma-Aldrich) and 1:500 mouse ant-glial fibrillary
acidic protein (GFAP; Chemicon International), and identified
with goat anti-mouse or -rabbit IgG-Alexa 488 (Molecular
Probes) and goat anti-mouse or -rabbit-Alexa 546 (Molecular
probes). All images were generated on an inverted Leica
2.7. Statistical analysis
All statistical analyses were carried out using Prism 4
(GraphPad Software). All data was analysed by one way
analysis of variance (ANOVA) or the Mann–Whitney U test for
comparing two medians, and expressed as mean±standard error
of the mean (S.E.M.).
3.1. EGFP-transduced MSCs proliferate comparably to control
To examine the effect of EGFP expression on human MSCs,
we used a lentiviral vector to transduce cultured cells (Fig. 1A).
EGFP-expressing cells displayed morphology identical to
control cells when examined under phase contrast (Fig. 1B).
This similarity to control cells was observed for cells transduced
at multiplicities of infection (MOIs) of 1, 10 or 20. EGFP
expression was observed in cells fixed in situ ranging from an
MOI of 1 (Fig. 1C) to an MOI of 20 (Fig. 1D). EGFP
of the cell. When quantified over n=3 transduction experiments
itwas observedthat atMOIsof1and10, relativelylow numbers
of cells expressed EGFP 7 d after the initial addition of vector,
15.7%±3.2 and 18.9%±4.7 respectively (Fig. 1E).
Conversely, at an initial transduction MOI of 20, an average
of 45.2%±5.4 of cells expressed EGFP after 7 d (Fig. 1E).
Following viral transduction at differing MOIs, cells were
examined for their capacity to expand over 6 passages (Fig. 1F).
Control human MSCs displayed a cumulative fold expansion of
10. Cells transduced at MOIs of 1, 10 and 20 displayed
cumulative fold expansion of 8.1, 6.8 and 6.5 respectively.
When examined by ANOVA no significant difference was
observed between all cell groups at Pb0.05 over the course of
passage. To confirm that cells could be stably transduced with
lentiviral introduced EGFP, cells were examined over the course
of these 6 passages for the presence of EGFP (Fig. 2A). No
difference was observed (ANOVA at Pb0.05) between cells
initially transduced at MOI 1 or 10 over the course of 6
passages, with averages of 15.6%±0.5 and 17.6%±0.5 EGFP-
expressing cells, respectively. Cells transduced at an MOI of 20
retained a much higher number of cells positive for EGFP over
the course of passage, with an average of 41.4%±0.79 over 6
passages. When MSCs were examined after 4 passages by flow
cytometry (Fig. 2B), it was confirmed that MSCs retained
fluorescence. In transduced cells, a significant fluorescent shift
could be observed compared to control cells.
Fig. 2. Human MSCs retain EGFP fluorescence over the course of passage.
EGFP expression at different MOIs remained stable over the course of 6
passages (A), with no difference observed (ANOVA at Pb0.05) between cells
initially transduced at MOI 1 or 10 over the course of 6 passages, with averages
of 15.6%±0.5 and 17.6%±0.5 EGFP-expressing cells, respectively. Cells
originally transduced at MOI 20 retain a higher number of EGFP-positive cells
over the course of passage, with an average of 41.4%±0.79. Transduced MSCs
were analysed for EGFP expression 4 passages after initial viral transduction by
flow cytometry (B). Compared to control cells (top), EGFP-positive cells
(bottom, MOI 20 shown) displayed a significant fluorescent shift to the right
compared to controls.
62 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
3.2. EGFP-expressing human MSCs retain neurogenic
EGFP-expressing human MSCs were examined for their
neurogenic potential several passages after initial viral trans-
duction. Cells were examined for marker expression in cells
originally transduced at MOIs of 1, 10 and 20. All transduced
cells were equally capable of expressing neural markers,
independent of MOI. Representative cells from MOI 20 are
shown in Fig. 3. When examined for nestin-expression, both
control (Fig. 3A) and EGFP-expressing (Fig. 3B) cells retained
this capacity. Similarly, βIII-tubulin expression was identified
in control (Fig. 3C) and EGFP-transduced cells (Fig. 3D).
Expression of GFAP was likewise recapitulated between control
(Fig. 3E) and EGFP-expressing cells (Fig. 3F). When quantified
it was observed that neural marker expression was highly
dependent on culture in either neuronal or glial induction media
(Fig. 3G), with only a very small number of cells identified as
expressing nestin (3.1%±0.8 of control and 3.6%±1.5 of
EGFP-positive cells) or βIII-tubulin(1.3%±0.5 of control cells)
in MSC growth medium. The number of cells immunoreactive
for neural markers increased dramatically when cells were
cultured in induction medium (Fig. 3G). In neuronal induction
medium, 37.2%±2.3 of control and 34.2%±4 EGFP-positive
MSCs were nestin-positive, while 22.2%±2.4 of control MSCs
and 26.3%±2.0 of EGFP-positive cells were identified as
nestin-positive in glial induction medium. When βIII-tubulin
expression was examined by immunostaining in neuronal
induction medium, 27.5%±2.0 of control and 31.5%±1.6
EGFP-positive MSCs were βIII-tubulin positive, while 31.6%±
1.8 of control MSCs and 32.7%±2.3 of EGFP-positive cells in
glial induction medium were identified as βIII-tubulin positive.
When GFAP expression was examined in neuronal induction
medium, 31.8%±1.5 of control and 31.1%±2.0 of EGFP-
positive MSCs were identified as GFAP positive. In each of
these cases, no significant difference was observed in the
numbers of nestin, βIII-tubulin or GFAP positive cells between
control and EGFP-transduced MSCs. A significant difference
Fig. 3. GFP-transduced human MSCs express neural markers comparably to control cells. All images are representative images from control or cells originally
transduced at MOI 20, and grown in neuronal induction media. When stained for nestin, both controls (A) and EGFP-expressing cells can be positive for nestin (B;
arrow). Similarly both control (C) and EGFP-positive cells (D; arrow) express βIII-tubulin. The majority of βIII-tubulin positive cells exhibit a bipolar, spindly
phenotype. GFAP expression was predominantly observed in cells with a large flattened, fibroblast-like morphology in both controls (E) and EGFP-positive cells (F).
Quantification of human MSCs differentiated in MSC growth medium, neuronal induction medium or glial induction medium by immunostaining for the specified
neural markers (G). Graphs represent mean±S.E.M. from 3 separate experiments.⁎, Pb0.05 when control cells are compared to EGFP-positive cells in glial induction
medium. Scale bars 20 um.
63 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
(Pb0.05, Mann–Whitney U test) was only observed between
control (28.0%±2.2) and EGFP-positive (33.0%±2.7) cells
when they were cultured in glial induction medium. Further-
more, there was no significant difference in neuronal- versus
glial- induction medium in terms of cells positive for βIII-
tubulin or GFAP, suggesting that while expression of these
markers was dependent on growth in induction medium, it was
irrespective of which induction medium was utilized.
3.3. EGFP-transduced and control MSCs form NSC-like
To further examine the neurogenic capacity of human MSCs
with or without EGFP expression, cells were cultured in both
EGF and bFGF under low-attachment conditions (Fig. 4).
Under these conditions, it has previously been reported that
MSCs can expand and form NSC-like neurospheres with
neurogenic potential (Hermann et al., 2004). After 4 d in culture
single human MSCs proliferated to form small balls of cells
(Fig. 4A). After 8 d in culture, human MSCs formed NSC-like
neurospheres (Fig. 4B). This proliferative capacity was retained
in cells transduced with EGFP.NSC-like neurospheres (Fig. 4C)
from EGFP-positive cells retained EGFP fluorescence after 8 d
growth in culture (Fig. 4D). When the neurogenic ability of
EGFP-positive cells was examined by growth as NSC-like
neurospheres, it was clearly observed that the number of
primary neurospheres generated from human MSC cultures did
not significantly differ (ANOVA at PN0.05) between controls
or MSCs transduced at MOIs of 1, 10 or 20 (Fig. 4E). To
examine the effect of EGFP transduction on the ability of NSC-
like neurospheres to proliferate, the number of cells was
recorded over the course of 3 passages (Fig. 4F). No significant
difference was observed between control and transduced cells
over the course of passage (ANOVA at PN0.05). Immunostain-
ing coverslip-grown cells derived from control or EGFP-
expressing NSC-like neurospheres indicated that neural
Fig. 4. EGFP-transduced human MSCs undergo NSC-like neurosphere proliferation. To further quantify the neurogenic capacity of human MSCs expressing EGFP,
cellswereculturedinboth EGFandbFGFunderlow-attachmentconditions.After 4d inculturea singlehumanMSChadexpandedtoforma smallproliferativeball of
cells (A). Following 8 d in culture, NSC-like neurospheres were present in culture (B). Similar proliferative capacity is observed in EGFP-transduced cells. After 8 d
growth in EGF and bFGF, human MSCs have formed NSC-like neurospheres (C) that retain EGFP fluorescence (D). When cultured in EGF and bFGF, control and
GFP-positive cells from all MOIs produce comparable numbers of primary neurospheres (E). No significant difference is observed between cell transduced at MOIs of
1, 10 or 20 at PN0.05 by ANOVA. Furthermore, when primary NSC-like neurospheres are passaged, little difference can be observed between control and transduced
cells over the course of 3 passages (ANOVA at PN0.05) (F). Scale bars 40 um.
64 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
markers could be identified (Fig. 5) following culture in
neuronal induction medium. Immunostaining identified cells
immunoreactive for nestin (Fig. 5A), βIII-tubulin (Fig. 5B) and
GFAP (Fig. 5C). These NSC-like neurosphere-derived cells had
similar morphologies to those examined from control cells or
neurospheres. Intact NSC-like neurospheres immunostained in
suspension similarly contained cells positive for the presence of
neural markers on the surface, including nestin (Fig. 5D) and the
mature oligodendrocyte marker O4 (Fig. 5E).
Transduction of human MSCs with EGFP had no significant
impact on the ability of these cells to expand during normal
passage, nor did it appear to alter cell morphology in any
appreciable way. The unaltered population dynamics and
behavior of transduced MSCs on reaching confluence strongly
implies that cellular factors involved in cell-division were not
affected. Furthermore, EGFP expression did not appear to
impair the neural response of human MSCs to neural induction
media when examined by immunostaining. Human MSCs
grown in neural induction media expressed the early neuronal
marker nestin, as well as markers of more mature neural
phenotypes (βIII-tubulin, GFAP and O4), identifying their
capacity to trans-differentiate into neuronal and astrocyte-like
cells (Zhang et al., 2004). While a very small number of cells
examined by immunostaining were observed to express nestin
and βIII-tubulin constitutively in normal growth media
(Tondreau et al., 2004), the growth of human MSCs in neuronal
or glial induction media was necessary to see significant
numbers of cells expressing these neural markers (Hermann
et al., 2004). The further characterization of EGFP-expressing
cells was carried out by culture in the presence of EGF and
bFGF, to effectively generate NSC-like neurospheres in a
similar manner to those methods used to culture bona fide
NSCs (Hermann et al., 2004; Reynolds et al., 1992; Rietze et al.,
2001). No significant difference could be observed between
non-transduced controls and EGFP-expressing MSCs to gen-
erate primary NSC-like neurospheres, or to undergo expansion
over the course of passage. Intact NSC-like neurospheres, or
cells derived from them during passage, retained expression of
markers such as nestin, βIII-tubulin, GFAP and O4 (Hermann
et al., 2004).
MSC trans-differentiation into neuronal and glial cells has
been shown by previous workers for both rodent and human
MSCs, although there is still some controversy whether this
represents true neural differentiation (Deng et al., 2001; Krabbe
et al., 2005; Ortiz-Gonzalez et al., 2004; Sanchez-Ramos et al.,
2000; Woodbury et al., 2002; Woodbury et al., 2000). Using
FACS analysis, Western blotting and RT-PCR, other workers
have reported that over 80% of cultured MSCs constitutively
expressed native neuronal proteins such as nestin and βIII-
tubulin (Tondreau et al., 2004). Furthermore, there was
increased expression of more mature neuronal/glial proteins
(TH, MAP-2, and GFAP) after exposure to neural induction
medium, thus confirming the differentiation of MSCs into
neurons and astrocytes (Tondreau et al., 2004). Others have
demonstrated that neural cells derived from trans-differentiated
MSCs are physiologically active when examined by electro-
physiology (Wislet-Gendebien et al., 2005; Wislet-Gendebien
Fig. 5. Control and EGFP-positive NSC-like neurospheres express neural markers. Growth of EGFP-positive neurosphere-derived cells in neuronal induction medium
results in cells positive for the neural markers nestin (A), βIII-tubulin (B) and GFAP (C). The neurogenic capacity of NSC-like neurospheres was further examined by
immunostaining intact neurospheres. After 8 d in culture, the expression of both nestin (C) and the late oligodendrocyte marker O4 (D) was observed. Immunoreactive
cells are primarily located on the surface of intact NSC-like neurospheres (arrows), and appear to extend process-like extensions. Scale bars 40 um.
65D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
et al., 2003; Zhao et al., 2004). Further neurogenic capacity of
MSCs is demonstrated by their ability to form neurosphere-like
structures containing cells expressing neuroectodermal markers
(Hermann et al., 2004).
The ability of human MSCs to undergo differentiation along
mesenchymal or neural lineages makes them valuable as tools
for regenerative or reconstructive medicine (Pittenger et al.,
1999). If human MSCs are to be utilized in studies to examine
their role in CNS repair in vivo, then their continued ability to
differentiate along these developmental pathways following in
vitro manipulation must be preserved. Other workers have
demonstrated that human and porcine MSCs successfully
following transduction with adenoviral vectors containing GFP
or lacZ (Bosch et al., 2006; Conget and Minguell, 2000; Hung
et al., 2004). Although the capacity of MSCs to undergo trans-
differentiation orgrowth as neurosphere-like structures has been
documented previously (Hermann et al., 2004; Kim et al., 2006;
Tondreau et al., 2004), this ability in EGFP-transduced human
MSCs has not previously been well established.
Stem cell-based therapies provide a promising approach to
the treatment of neurological disease in humans. Recent reports
suggest that MSCs are an attractive non-embryonic or non-fetal
source ofstemcellssuitable forcellreplacementstrategiesinthe
treatment of CNS disorders (Chopp and Li, 2002; Jiang et al.,
2002; Krause et al., 2001; Pittenger et al., 1999). To explore the
cells labeled with EGFP may provide a useful mechanism for
identifying cells injected in vivo. Furthermore, such labeled
MSCs provide a powerful tool for identifying cells following
any trans-differentiation into neuronal or glial cells during the
course of CNS repair. We propose that human MSCs expressing
EGFP provide an attractive and practical source of stem cells for
the study of repair and regeneration in disease models and we
have shown that, at least in vitro, transducing these cells with
EGFP does not appear to alter their differentiate behavior.
This work was supported by the UK Multiple Sclerosis
Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J.D., 2002a. Remyelination of
the spinal cord following intravenous delivery of bone marrow cells. Glia.
Akiyama, Y., Radtke, C., Kocsis, J.D., 2002b. Remyelination of the rat spinal
cord by transplantation of identified bone marrow stromal cells. J. Neurosci.
Azizi, S.A., Stokes, D., Augelli, B.J., DiGirolamo, C., Prockop, D.J., 1998.
Engraftment and migration of human bone marrow stromal cells implanted
in the brains of albino rats—similarities to astrocyte grafts. Proc. Natl. Acad.
Sci. U S A 95, 3908–3913.
Bjorklund, A., 2000. Cell replacement strategies for neurodegenerative
disorders. Novartis Found. Symp. 231, 7–15 discussion 16–20.
Bosch, P., Fouletier-Dilling, C., Olmsted-Davis, E.A., Davis, A.R., Stice, S.L.,
2006. Efficient adenoviral-mediated gene delivery into porcine mesenchy-
mal stem cells. Mol. Reprod. Dev. 73, 1393–1403.
Brazelton, T.R., Rossi, F.M., Keshet, G.I., Blau, H.M., 2000. From marrow to
brain: expression of neuronal phenotypes in adult mice. Science 290,
Cao, Q., Benton, R.L., Whittemore, S.R., 2002. Stem cell repair of central
nervous system injury. J. Neurosci. Res. 68, 501–510.
Chopp, M., Li, Y., 2002. Treatment of neural injury with marrow stromal cells.
Lancet Neurol. 1, 92–100.
Clarke, D., Frisen, J., 2001. Differentiation potential of adult stem cells. Curr.
Opin. Genet. Dev. 11, 575–580.
Conget, P.A., Minguell, J.J., 2000. Adenoviral-mediated gene transfer into ex
vivo expanded human bone marrow mesenchymal progenitor cells. Exp.
Hematol. 28, 382–390.
Deng, W., Obrocka, M., Fischer, I., Prockop, D.J., 2001. In vitro differentiation
of human marrow stromal cells into early progenitors of neural cells by
conditions that increase intracellular cyclic AMP. Biochem. Biophys. Res.
Commun. 282, 148–152.
Glover, C.P., Bienemann, A.S., Heywood, D.J., Cosgrave, A.S., Uney, J.B.,
2002. Adenoviral-mediated, high-level, cell-specific transgene expression:
a SYN1-WPRE cassette mediates increased transgene expression with no
loss of neuron specificity. Mol. Ther. 5, 509–516.
Hermann, A., Gastl, R., Liebau, S., Popa, M.O., Fiedler, J., Boehm, B.O.,
Maisel, M., Lerche, H., Schwarz, J., Brenner, R., Storch, A., 2004. Efficient
generation of neural stem cell-like cells from adult human bone marrow
stromal cells. J. Cell Sci. 117, 4411–4422.
Hung, S.C., Lu, C.Y., Shyue, S.K., Liu, H.C., Ho, L.L., 2004. Lineage
differentiation-associated loss of adenoviral susceptibility and Coxsackie-
adenovirus receptor expression in human mesenchymal stem cells. Stem
Cells 22, 1321–1329.
Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-
Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J.,
Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A., Verfaillie, C.M.,
2002. Pluripotency of mesenchymal stem cells derived from adult marrow.
Nature 418, 41–49.
Kim, B.J., Seo, J.H., Bubien, J.K., Oh, Y.S., 2002. Differentiation of adult bone
marrow stem cells into neuroprogenitor cells in vitro. NeuroReport 13,
Kim, S., Honmou, O., Kato, K., Nonaka, T., Houkin, K., Hamada, H., Kocsis,
J.D., 2006. Neural differentiation potential of peripheral blood- and bone-
marrow-derived precursor cells. Brain Res. 1123, 27–33.
Krabbe, C., Zimmer, J., Meyer, M., 2005. Neural transdifferentiation of
mesenchymal stem cells—a critical review. APMIS. 113, 831–844.
Krause, D.S., Theise, N.D., Collector, M.I., Henegariu, O., Hwang, S., Gardner,
R., Neutzel, S., Sharkis, S.J., 2001. Multi-organ, multi-lineage engraftment
by a single bone marrow-derived stem cell. Cell 105, 369–377.
Ortiz-Gonzalez, X.R., Keene, C.D., Verfaillie, C.M., Low, W.C., 2004. Neural
induction of adult bone marrow and umbilical cord stem cells. Curr.
Neurovasc. Res. 1, 207–213.
Lachyankar,M.B., Redmond,D.E.,Snyder, E.Y.,2002.Globalgeneandcell
replacement strategies via stem cells. Gene Ther. 9, 613–624.
potential of adult human mesenchymal stem cells. Science 284, 143–147.
Poulsom, R., Alison, M.R., Forbes, S.J., Wright, N.A., 2002. Adult stem cell
plasticity. J. Pathol. 197, 441–456.
Preston, S.L., Alison, M.R., Forbes, S.J., Direkze, N.C., Poulsom, R., Wright,
N.A., 2003. The new stem cell biology: something for everyone. Mol.
Pathol. 56, 86–96.
Prockop, D.J., 2002. Adult stem cells gradually come of age. Nat. Biotechnol.
Reynolds, B.A., Tetzlaff, W., Weiss, S., 1992. A multipotent EGF-responsive
striatal embryonic progenitor cell produces neurons and astrocytes.
J. Neurosci. 12, 4565–4574.
Rietze, R.L., Valcanis, H., Brooker, G.F., Thomas, T., Voss, A.K., Bartlett, P.F.,
2001. Purification of a pluripotent neural stem cell from the adult mouse
brain. Nature 412, 736–739.
Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., Hazzi, C., Stedeford, T.,
Willing, A., Freeman,T.B., Saporta, S., Janssen,W., Patel, N., Cooper, D.R.,
66 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67
Sanberg, P.R., 2000. Adult bone marrow stromal cells differentiate into Download full-text
neural cells in vitro. Exp. Neurol. 164, 247–256.
Scolding, N., 2001. New cells from old. Lancet 357, 329–330.
Serafini, M., Verfaillie, C.M., 2006. Pluripotency in adult stem cells: state of the
art. Semin. Reprod. Med. 24, 379–388.
Tondreau,T.,Lagneaux,L., Dejeneffe, M., Massy,M., Mortier, C.,Delforge, A.,
Bron, D., 2004. Bone marrow-derived mesenchymal stem cells already
express specific neural proteins before any differentiation. Differentiation
Weissman, I.L., 2000. Translating stem and progenitor cell biology to the clinic:
barriers and opportunities. Science 287, 1442–1446.
Wislet-Gendebien, S., Leprince, P., Moonen, G., Rogister, B., 2003. Regulation
of neural markers nestin and GFAP expression by cultivated bone marrow
stromal cells. J. Cell Sci. 116, 3295–3302.
Wislet-Gendebien, S., Hans, G., Leprince, P., Rigo, J.M., Moonen, G., Rogister,
B., 2005. Plasticity of cultured mesenchymal stem cells: switch from nestin-
positive to excitable neuron-like phenotype. Stem Cells 23, 392–402.
Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and
human bone marrow stromal cells differentiate into neurons. J. Neurosci.
Res. 61, 364–370.
Woodbury, D., Reynolds, K., Black, I.B., 2002. Adult bone marrow stromal
stem cells express germline, ectodermal, endodermal, and mesodermal
genes prior to neurogenesis. J. Neurosci. Res. 69, 908–917.
Zhang, H., Wang, J.Z., Sun, H.Y., Zhang, J.N., Yang, S.Y., 2004. The effects of
GM1 and bFGF synergistically inducing adult rat bone marrow stromal cells
to form neural progenitor cells and their differentiation. Chin. J. Traumatol.
Zhao, L.X., Zhang, J., Cao, F., Meng, L., Wang, D.M., Li, Y.H., Nan, X., Jiao,
W.C., Zheng, M., Xu, X.H., Pei, X.T., 2004. Modification of the brain-
derived neurotrophic factor gene: a portal to transform mesenchymal stem
cells into advantageous engineering cells for neuroregeneration and
neuroprotection. Exp. Neurol. 190, 396–406.
67 D. Gordon et al. / Journal of Neuroimmunology 193 (2008) 59–67