1 Center for Cellular and Molecular Neurobiology, University of Liège, 17
Place Delcour, B-4020 Liège, Belgium
2 Department of Neurology, University of Liège, C.H.U. (B35) Sart Tilman,
B-4000 Liège, Belgium
* Author for correspondence (e-mail: s.wislet{at}ulg.ac.be)
Accepted 28 April 2003
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Summary |
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Key words: Nestin, Bone marrow stromal cells, GFAP, Neural stem cells, Glial differentiation
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Introduction |
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Stem cells located in continuously renewing tissues such as skin, gut or
bone marrow, are able to regenerate or repair these tissues throughout life.
However, in the non-regenerating adult central nervous system (CNS), neural
stem cells (NSC) have a poor capacity to generate new neurons or
oligodendrocytes to replace cells lost after injury or degeneration. In
nervous system disorders in which specific neuronal cell loss occur (e.g.
Parkinson's disease) transplantation of neural cells allows for the
replacement of lost cells and recovery of some degree of function
(Freed et al., 2001).
Embryonic stem cells are totipotent and are thus able to differentiate in
any kind of cell type present during development and in adulthood. At present,
there is no evidence that adult stem cells are totipotent, but some may have
the capacity to differentiate into phenotypes that belong to tissues other
than the one from which they originated, a property usually referred to as
plasticity or transdifferentiation. A recently reported example of such
plasticity is the finding that, after intravascular delivery of genetically
labelled adult mouse bone marrow into lethally irradiated adult hosts, donor
cells expressing neuronal markers were found in the host CNS
(Brazelton et al., 2000). In
vitro, a tiny fraction (2-5%) of bone marrow stromal cells cultured in the
presence of EGF or BDNF express nestin, glial fibrillary acidic protein (GFAP)
and neuron-specific nuclear protein (NeuN)
(Sanchez-Ramos et al., 2000
).
The addition of dibutiryl cyclic AMP has been reported to induce the
differentiation of human mesenchymal stem cells (MSCs) into early progenitors
of neural cells (Deng et al.,
2001
).
During the development of the CNS, proliferating neuroepithelial cells
express nestin (neural stem cells protein), an intermediate filament protein
(Lendahl et al., 1990), which
is also expressed by NSC in adult mammals and then used to identify adult
neural progenitors in culture (Dahlstrand,
et al., 1995
). Although nestin is not a specific marker of neural
stem cells because it is also transiently expressed in muscle progenitors and
in some epithelial derivatives (Mokry and
Nemecek, 1998
), the analysis of neurospheres obtained from ES
cells (which are known to be nestin-negative in vivo) demonstrates that all
the cells within those spheres express nestin, suggesting that nestin
expression is correlated to or is coincident with the initiation of sphere
formation (Tropepe et al.,
2001
).
The use of MSCs in auto-graft protocols in neurological diseases necessitates the identification of the molecular events that are important for the induction of neural differentiation of MSCs. We found that the presence of serum in the culture medium represses nestin expression by rat stromal cells (rSCs). Moreover, only nestin-positive rSCs are able to form aggregates in suspension when they are transferred to NSCs culture conditions. But, when those spheres or aggregates were placed in culture conditions known to favour neural differentiation of NSCs, only modifications of cell shape were observed. In contrast, when nestin-positive rSCs were grown in co-culture with mouse neural stem cells (mNSCs), heterogenous spheres formed which, when plated on polyornithine-coated surfaces, released 40% rSCs that differentiated into GFAP-positive cells. Nestin-negative rSCs cells, when grown in the same condition, gave rise to less than 5% GFAP-positive rSCs. Nestin expression by rSCs should thus be regarded as a first step in their progression to the neural lineage.
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Materials and Methods |
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Preparation and culture of mNSCs
Green C57BL/6 mice embryos (Jackson Immunoresearch Laboratory, West Grove,
PA, USA) were used as a source of mNSC. In the green mouse, GFP expression is
under the ß-actin promoter activity and NSC can therefore be identified
by their green fluorescence (Okabe et al.,
1997). The day of conception was determined by the presence of a
vaginal plug (embryonic day 0). E15 striata were isolated and triturated in
DEM/F12 (Invitrogen) with a sterile Pasteur pipette. The cell suspension was
filtered with a 70 µm-pore filter and viable cells were estimated by trypan
blue exclusion. The cells were plated (1x106 cells/75-cm
tissue culture flask) in DEM/F12 supplemented with epidermal growth factor
(EGF, 20 ng/ml, Sigma), N2 and B27 (Neurobasal medium, Invitrogen) which are
two multicomponent cell culture supplements devoid of any growth factor. When
the size of neurosphere reached approximately 50 cells, they were dissociated
to a single cell suspension by trituration and replated in fresh culture
medium.
Immunological characterization of rSCs
rSCs have been characterized by immunocytochemical labelling for CD45
(Pharmingen, The Netherlands; 1:200), CD11b (Pharmingen; 1:200), Thy1.1
(Chemicon, Wevelgem, Belgium; 1:200) and P75 NGF-R (Chemicon; 1:200)
(Goodell et al., 1997).
Fluorescence-activated cell sorting of first passage rSCs was performed with
anti-CD45 and anti-CD11b antibodies. Briefly, the cells were suspended with 10
mM ice-cold EDTA (Fluka, Bornem, Belgium) and 500,000 cells were washed in 3
ml of PBS containing 1% foetal calf serum. After centrifugation, the pellet
was suspended in 200 µl of the primary antibodies solutions, for 1 hour at
room temperature. They were then washed 3 times in PBS containing 1% foetal
calf serum and incubated with anti-mouse IgG (1:500) secondary antibody
coupled to FITC (Jackson Immunoresearch Laboratory) for 1 hour at room
temperature and in the dark. Before analysis using FACS, the cells were fixed
by a 15-minute incubation in 1% paraformaldehyde solution. The analysis was
performed with a FACSort instrument (Becton Dickinson) and the results were
analyzed using the Cellquest program (Becton Dickinson).
Functional characterization of rSCs
The adipogenic differentiation of rSCs was induced by treatment with
1-methyl-3-isobutylxanthine (0.5 mM, Sigma, Belgium), dexamethasone (1 µM,
Sigma), bovine insulin (0.01 mg/ml) and indomethacin (0.2 mM, Sigma). rSCs
were placed in the above adipocyte induction medium for 24 days. The
differentiation was evaluated by accumulation of lipid vacuoles and staining
with Oil Red O (Sigma) following fixation with 4% paraformaldehyde. To induce
osteocyte differentiation, the rSCs were incubated in DEM containing
dexamethasone (0.1 µM, Sigma), ascorbate (0.05 mM, Sigma) and
ß-glycerophosphate (10 mM, Sigma) for 12 days. A significant increase in
alkaline phosphatase activity was measured with the alkaline phosphatase
colorimetric test, following the manufacturer's instructions (Sigma).
Chondrogenic differentiation was induced with DEM medium containing
dexamethasone (0.1 µM, Sigma), sodium pyruvate (1 mM, Janssen Chemica),
ascorbic-2-phosphate acid (0.15 mM, Sigma), proline (0.35 mM, Sigma), bovine
insulin (0.25 µg/ml), selenic acid (6.25 µg/ml, Sigma) and linoleic acid
(5.35 µg/ml, Sigma). Chondrocytes were obtained when rSCs were grown as a
pellet in the induction medium for 20 days. The cell aggregates were fixed
with 4% paraformaldehyde, paraffin-embedded, cut at 5 µM sections and
stained with toluidine blue.
Induction of sphere formation by rSCs
After being induced to express nestin, rSCs were trypsinized and suspended
in DEM/F12 containing N2 and B27 supplements for 24 hours. During this time,
the cells aggregated. These aggregates were plated onto polyornithine-coated
dishes for 5 days in the same medium and were then processed for
immunocytochemistry as described below.
Co-culture rSCs and mNSC
Nestin-positive rSCs were trypsinized and were co-incubated with
GFP-positive mNSC (1x106 mNSC and 1x104
rSCs) for 48 hours in DEM/F12 containing EGF, N2 and B27 supplements. During
this time, the cells aggregated, forming heterogenous spheres. For a good
observation of the presence of rSCs into the heterogenous spheres, the rSCs
were colored in red with the DiD VybrantTM cell-labelling solutions
(Molecular Probes) following the manufacturer's instructions. These spheres
were plated on polyornithine-coated dishes for 5 days, in DEM/F12 containing
N2 and B27 supplements and were processed for immunocytochemistry as described
below. Nestin-negative rSCs were trypsinized and directly replated with
GFP-positive mNSC onto polyornithine-coated dishes for 5 days following the
same culture condition as described for the nestin-positive cells. The cells
were then processed for immunocytochemistry.
Immunocytochemistry
The cultures were fixed with 4% (v/v) paraformaldehyde for 15 minutes at
room temperature and washed 3 times in TBS buffer. They were then
permeabilized in 1% Triton X-100 (v/v) for 15 minutes and washed 3 times in
TBS buffer. Non-specific binding was blocked by a 1-hour treatment in TBST
(TBS buffer with 0.1% Tween) containing fat-free milk powder (30 mg/ml). The
cells were then incubated for 1 hour at room temperature with either
anti-p75/NGF-R, or anti-Thy1.1, or anti-nestin (Rat401, Pharmingen; mouse IgG,
dilution 1:1500), or anti-GFAP (Dako; mouse IgG, dilution 1:500), or anti-M2
(Developmental Studies Hybridoma Bank; rat IgG, dilution 1:500), or anti-GLAST
(Shibata et al., 1997) (rabbit
IgG, 1:4000) primary antibodies (diluted in blocking buffer). After 3 washes,
cells were incubated in FITC- or Cy5-conjugated anti-mouse IgG (Jackson
Immunoresearch; 1:500) or rhodamine-conjugated anti-rat IgG (Jackson
Immunoresearch, 1:500) for 1 hour at room temperature and in the dark. The
nuclei were stained with ethidium homodimer (0.2 µM, Sigma). The
preparations were then mounted in FluoprepTM (bioMérieux, Marcy
L'Etoile, France) and observed using a Bio-Rad MRC1024 laser scanning confocal
microscope.
Western blot
Total protein extracts were obtained from confluent cells that had been
cultured in the different media. The cells were harvested by scraping the dish
in 500 µl lysis buffer (0.6 M KCl, 5 mM EGTA, 5 mM EDTA, 1% Triton X-100
and 1 mM PMSF in PBS). Extracts were then fractioned into pelletable
(insoluble) and non-pelletable (soluble) proteins by centrifugation at 30,000
g for 15 minutes at 4°C. The pellet was resuspended in
loading buffer (glycerol 10% v/v; Tris 0.05 M, pH 6.8; SDS 2%, bromophenol
blue and 2.5% v/v ß-mercaptoethanol) and the suspension was centrifuged
at 30,000 g for 15 minutes at 4°C. The supernatant was
used for protein concentration measurement using the RC DC Protein Assay
(Bio-Rad). The same protein quantities in each lane were separated by
electrophoresis using the Phastgel 4-15% SDS gradient (Amersham Pharmacia
Biotech) and transferred to a PVDF membrane. The membranes were saturated with
3% gelatin (BioRad), incubated for 1 hour with a monoclonal antibody against
rat nestin (Pharmingen, 1:1,000) or ß-actin, as control for protein
loading (Sigma, 1:5000) at room temperature and then washed several times with
PBS-0.1% Tween. The membrane was then incubated in biotinylated goat
anti-mouse antibody (Boehringher Mannheim; 1:5000) for 1 hour at room
temperature. After several washes in PBS-0.1% Tween, the membrane was
incubated with peroxidase-coupled streptavidin (1:100,000, Sigma) at 37°C
for 1 hour. Radial glial cells were used as a positive control for nestin
expression.
DNA ploidy
DNA content per cell was determined by FACS analysis after staining the
cells with propidium iodide. After 5 days of co-culture, rSCs and mNSCs were
trypsinised and fixed into 70% ethanol, at 4°C during 16 hours. The cells
were stained with propidium iodide (400 µg/ml, Sigma) just before FACS
analysis.
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Results |
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Differentiation of rSCs into Nestin-positive cells
To induce neural differentiation of rSCs in long-term cultures, we placed
them in a classical NSC culture medium (DEM/F12 supplemented with N2 and B27).
After 72 hours most rSCs expressed nestin as revealed by immunocytochemistry
(Fig. 2A). When rSCs are placed
in DEM/F12 (Fig. 2B),
DEM/F12+N2 (Fig. 2C) and
DEM/F12+B27 (Fig. 2D), the same
result is obtained, suggesting that the removal of serum from the culture
medium was actually responsible for the induction of nestin expression by
rSCs. These results were confirmed by western blotting
(Fig. 2F). In all of these
experiments, no alteration in the morphology of nestin-positive SCs was
observed. We demonstrated that the absence of serum was necessary but not
sufficient for the induction of nestin expression. Indeed, the number of
passages of rSCs in vitro also regulates their ability to express nestin when
grown in serum-free condition (Fig.
3A-E): a minimum of ten passages is needed before rSCs are able to
express nestin when placed in serum-free culture conditions. However, the
capacity of these cells to differentiate into adipocytes or osteocytes did not
change as a function of the number of passages
(Fig. 3F,H).
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Induction of sphere formation by rSCs
Recently, Tropepe et al. (Tropepe et
al., 2001) suggested that nestin expression by ES cells is
correlated with the capacity to form neurospheres and that ES cells have to go
through a nestin-expression stage before differentiating into neural cells.
When nestin-positive and nestin-negative rSCs were trypsinised and replaced in
an NSC growth medium (DEM/F12, N2 and B27) in non-adherent conditions, we
observed that passage 15 nestin-positive rSCs aggregated in suspension
(Fig. 4A). In contrast, passage
4 nestin-negative rSCs which had been cultivated in serum-free conditions for
48 hours prior to being transferred to NSCs growth medium, remained in
suspension and did not form spheres or aggregates
(Fig. 4B). Passage 15 rSCs that
did not express nestin (because they had been cultivated in serum-containing
medium) adhered spontaneously to the dish
(Fig. 4C). We then plated the
nestin-positive rSCs aggregates on a polyornithine-coated surface for 5 days
as is done for neurospheres in order to stimulate cell differentiation. The
cell morphology changed from the flat and elongated shape of MSCs
(Fig. 4D) to more rounded
morphology (Fig. 4E), but
immunocytological labelling with antibodies recognizing astrocytic (anti-GFAP,
anti-GLAST), oligodendroglial (anti-O4, anti-A2B5) and neuronal markers
(anti-NeuN, anti-NSE, anti-Tuj1, anti-MAP2B) were all negative (data not
shown). Moreover, after 5 days in these conditions, nestin expression
decreased from 80% to 15% (Fig.
4F).
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Co-culture of rSCs and mNSCs
As nestin expression by rSCs appears insufficient to stimulate their
differentiation into neural cells, we have co-cultivated nestin-positive rSCs
with NSCs because the neural differentiation of MSC has been observed mainly
in vivo when cells had been grafted in newborn mice brain. When we
co-cultivated nestin-positive red-labelled rSCs for 5 days with NSCs obtained
from E15 `green mouse' striata (Okabe et
al., 1997), we observed that nestin-positive rSCs formed
heterogenous spheres (Fig. 5M)
with the NSC. These heterogenous spheres were then transferred on
polyornithine-coated dishes for 5 days to allow cellular differentiation.
Immunological labeling revealed the differentiation of 40±2.39%
nestin-positive rSCs into GFAP-expressing cells (n=3, representing
3000 cells) (Fig. 5A-C). A
similar percentage of GLAST-positive rSCs was also observed in these
conditions (Fig. 5G-I).
However, no nestin-positive rSCs expressed neuronal (anti-NeuN, anti-NSE,
anti-Tuj1, anti-MAP2B) or oligodendroglial markers (anti-O4, anti-A2B5) (data
not shown). Moreover, passage 4 nestin-negative rSCs were unable to form
heterogenous spheres, and when plated directly with mNSC onto a
polyornithine-coated surface, only 4% of them expressed GFAP
(Fig. 5D-F). Three
justifications could be formulated to explain the GFAP expression by rSC: 1) a
decrease of GFP expression in mNSCs, 2) a rSC-mNSC fusion event, and 3)
phenotypic plasticity of the rSCs. To exclude a GFP downregulation, we
cultivated GFP-positive mNSC alone in the same conditions, and a GFAP
immunoreactivity was observed within GFP-positive cells
(Fig. 5J-L). To exclude a cell
fusion process, we first used antibody M2, which specifically recognizes
mouse-specific astrocytes (Lagenaur and
Schachner, 1981
) to demonstrate that the GFP-negative astrocytes
that developed under these conditions were of rat origin: no GFP-negative,
GFAP-positive cells were recognized by the M2 antibody, but the GFAP- and
GFP-positive cells were (Fig.
5N,O). Furthermore, we analyzed the ploidy of the co-cultivated
cells in order to determine if rSCs adopt the astroglial phenotype of the
recipient cells by a hypothetical cell fusion event. We compared DNA ploidy of
the rSCs maintained in co-culture with mNSC for 5 days
(Fig. 5P) with the DNA ploidy
of rSCs and mNSC cultivated separately for the same period, and we found the
same DNA content in the three cultures, thus excluding a cell fusion
event.
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Discussion |
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Several recent reports suggest that bone marrow stromal cells could be a
non-embryonic or non-foetal source of stem cells suitable for cell replacement
strategies in the treatment of CNS disorders
(Kopen et al., 1999;
Akiyama et al., 2002a
;
Akiyama et al., 2002b
;
Brazelton et al., 2000
). An
understanding of the molecular regulation of such a `mesenchymal-neural'
transition may be very important when considering the use of SCs in the
treatment of CNS disorders.
In our study, rSCs have been isolated from adult rat bone marrow using a
differential adhesion procedure and selected by their capacity of rapid
proliferation. We demonstrate that these stromal cells are able to express
nestin. This expression is dependent on two factors. The first one is the
absence of serum-derived components in the culture medium. Indeed, in our
study using western blot analysis and cell counting, higher levels of nestin
expression were found in serum-free conditions, although RT-PCR and western
blot demonstrate a faint signal for nestin even in the presence of serum. The
second factor is the number of cell passages. A minimum of ten passages is
required for nestin expression by 75% of rSCs. However, the capacity of rMSC
to differentiate into adipocytes, osteocytes or chondrocytes does not change
as a function of the number of passages. This requirement of 10 passages
(which correspond to 25 doubling populations) in vitro was reproducible in 4
independent experiments. Recently, Jiang et al.
(Jiang et al., 2002),
demonstrated that rare cells within murine bone marrow MSC cultures can
differentiate not only into the mesenchymal lineage cells but also into
endothelium, ectoderm and endoderm. These rare cells, which have been named as
multipotent adult progenitor cells (or MAPCs), can be expanded for more than
100 population doublings, bringing about an enrichment in MAPCs in MSCs
culture. The increase in the percentage of nestin-positive MSCs as a function
of the number of passages could possibly be explained by the presence of these
MAPCs in our cultures and their increase in number with additional
passages.
We show that only rSCs from nestin-positive cultures are able to form clusters or aggregates in the non-adherent conditions used to cultivate NSCs. Nestin expression by rSCs and their ability to grow in suspension in such defined culture conditions bring them nearer to the NSCs phenotype. However, when nestin-positive rSCs spheres were plated onto an adherent surface, no glial and/or neuronal differentiation was observed. It seems that the complete neural differentiation of MSCs observed in vivo, may require the involvement of several induction signals which have not been reproduced in vitro. It is for this reason that rSCs and NSCs were co-cultivated. It was hoped that such an experiment would reproduce in vitro some of the complex molecular interactions that are required to induce a full neural differentiation of mesenchymal cells in vivo.
Under these co-culture conditions, nestin-positive rSCs were able to
express GFAP, the astroglia-specific intermediate filament protein, but also
GLAST, another marker of astroglia
(Shibata et al., 1997). When
nestin-negative rSCs were co-cultivated with NSCs under identical conditions,
a low percentage of GFAP-positive cells of mesenchymal origin was observed,
suggesting that nestin expression is a prerequisite for rSCs differentiation
into a GFAP- or a GLAST-positive cell type. This observation is in agreement
with the notion that an ordered succession of stimuli is needed to promote
such a differentiation.
Given the fact that it has been reported that 0.2-1 of ES cells per
105 bone marrow cells can fuse with an other cell-type and thus
mimic a differentiation and/or plasticity
(Terada and al., 2002), we
have excluded this possibility by two methods: first, we could not find any
mouse-specific antigenic labeling on GFAP-positive cells of rSCs origin, and
we exclude a tetraploidy in our co-culture by FACS analysis. So,
GFAP-expression by rSCs are independent of cell-fusion events or GFP
downregulation in mNSC, but well a neural phenotypic plasticity of rSCs.
In conclusion, nestin expression by rSCs should be regarded as a first step in their progression to the neural lineage. A better knowledge of the regulation of their differentiation into astrocytes and the definition of appropriate culture conditions to obtain their differentiation into neurons and/or oligodendrocytes is still needed before considering MSCs as an appropriate cellular material to be used for cell replacement therapies in CNS disorders.
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Acknowledgments |
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