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The FASEB Journal express article 10.1096/fj.04-1517fje. Published online April 14, 2004.
Bone marrow stem cells have the ability to populate the
entire central nervous system into fully differentiated
parenchymal microglia
Alain R. Simard and Serge Rivest
Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy
and Physiology, Laval University, Québec, Canada G1V 4G2
Corresponding author: S. Rivest, Laboratory of Molecular Endocrinology, CHUL Research
Center and Department of Anatomy and Physiology, Laval University, 2705 Laurier boul,
Québec, Canada G1V 4G2. E-mail: Serge.Rivest@crchul.ulaval.ca
ABSTRACT
Pluripotent stem cells can differentiate into a variety of cell types during tissue development and
regeneration. However, it is still unclear whether bone marrow-derived stem cells can migrate
across the blood-brain barrier in many regions of the central nervous system (CNS) and if these
cells can readily differentiate into functional parenchymal microglia. We thus studied the
differentiation fate of bone marrow stem cells upon immigration into the CNS. To this end, we
systemically transplanted stem cells that express green fluorescent protein (GFP) into lethally
irradiated mice and found that these cells immigrated into the brain parenchyma of many regions
of the CNS. Nearly all of the infiltrating cells had a highly ramified morphology and colocalized
with the microglial marker iba1. Moreover, these cells expressed high levels of the protein
CD11c, indicating that microglia of bone marrow origin may be potent antigen presenting cells.
These data suggest that microglia of blood origin could activate cells of the adaptive immune
system and cause harm to the CNS. Therefore, these results may have great clinical relevance for
both immune-derived neuronal disorders and cancer patients undergoing allogeneic
hematopoietic stem-cell transplantation.
Key words: Chimeric mice
● GFP mice ● gliogenesis ● inflammation ● innate immunity ● antigen-
presenting cells
● macrophages ● CD11c
t is a well-accepted fact that stem cells are capable of differentiating into multiple cell types
(1-3). In the central nervous system (CNS), however, neural stem cells have the ability to
give rise to astrocytes and oligodendrocytes but not microglia (4, 5). It has been suggested
that microglia are replenished partly by division of resident cells and partly by immigration of
circulating monocytes (6), though evidence for the latter was based on many assumptions. Two
previous studies have found many cells with microglial morphology associated with blood
vessels and were thus identified as perivascular microglial cells. However, both papers reported
that bone marrow-derived cells rarely crossed the blood-brain barrier (BBB) and
transdifferentiated into parenchymal microglia (7, 8). Moreover, this specific type of cell was
only consistently observed in the cerebellum (8). Therefore, the question still remains whether
I
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circulating stem cells can readily cross the BBB and differentiate into parenchymal microglia in
other regions of the brain.
To test this hypothesis, we transplanted GFP-expressing bone marrow cells into lethally
irradiated mice. We then evaluated the fate of the donor-derived cells in the CNS. Many of these
cells were found in the parenchyma throughout multiple regions of the brain, and we show that
the vast majority of these cells had differentiated into microglia since they were colabeled with
the microglial marker iba1. Most importantly, these donor-derived microglial cells are likely to
be effective antigen-presenting cells (APCs), because they contained high constitutive levels of
the surface molecule CD11c. These data may be of great value for understanding the etiology of
many neurodegenerative diseases and for cancer patients undergoing allogeneic hematopoietic
stem-cell transplantation.
EXPERIMENTAL PROCEDURES
Animals
Adult male C57BL/6J mice (~25 g; Jackson Laboratory, Bar harbor, ME) were acclimated to
standard laboratory conditions (14 h light, 10 h dark cycle; lights on at 0600 and off at 2000 h)
with free access to rodent chow and water. Hemizygous transgenic mice expressing GFP under
control of the chicken β-actin promoter and cytomegalovirus enhancer were initially obtained
from the same vendor (Jackson Laboratory). A colony was then established and maintained in a
C57BL/6J background. GFP mice were used as cell donors at 3-5 months of age. All protocols
were conducted according to the Canadian Council on Animal Care guidelines, as administered
by the Laval University Animal Welfare Committee.
Irradiation and bone marrow transplantation
Mice were exposed to 10 gray total-body irradiation using a cobalt-60 source (Theratron-780
model, MDS Nordion, Ottawa, ON, Canada). A few hours later, the animals were injected via a
tail vein with ~5 x 10
6
bone marrow cells freshly collected from GFP mice. The cells were
aseptically harvested by flushing femurs with Dulbecco’s PBS containing 2% fetal bovine serum
(DPBS-FBS). The samples were combined, centrifuged, and passed through a 25 gauge needle
and then filtered through a 40 µm nylon mesh. Recovered cells were resuspended in DPBS-FBS
at a concentration of 5 x 10
6
viable nucleated cells per 200 µl. Irradiated mice transplanted with
this suspension were housed in autoclaved cages and treated with antibiotics (0.2 mg
trimethorpine and 1 mg sulfamethoxazole/ml of drinking water given for 7 days before and 2 wk
after irradiation).
Seven weeks after transplantation, the mice were deeply anesthetized via an intraperitoneal
injection of a mixture of ketamine hydrochloride and xylazine and then were rapidly perfused
transcardially with 0.9% saline containing 10 U/ml of heparin, followed by 4%
paraformaldehyde in sodium phosphate buffer (pH 7.6 at 4°C). Brains were rapidly removed
from the skulls, postfixed for 2 h, and then placed in a solution containing 20% sucrose diluted in
4% paraformaldehyde buffer overnight at 4°C. The frozen brains were mounted on a microtome
(Reichert-Jung, Cambridge Instruments Company, Deerfield, IL), frozen with dry ice, and cut
into 25 µm coronal sections from the olfactory bulb to the end of the medulla. The slices were
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collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, pH 7.3, 30%
ethylene glycol, 20% glycerol) and stored at -20°C.
Immunohistochemistry
Free-floating sections (25 µm thick) were incubated for 30 min in KPBS containing 4% goat
serum, 1% BSA, and 0.4% Triton X-100. With the use of the same buffer solution, the sections
were then incubated for 90 min in primary Ab (monoclonal rat anti-CD31, 1:1000, PharMingen,
San Diego, CA; polyclonal rabbit anti-ionized calcium binding adaptor molecule 1 (iba1),
1:1000, provided by Dr. Y. Imai, National Institute of Neuroscience, Japan; polyclonal Armenian
hamster anti-CD11c, 1:500, PharMingen) at room temperature. The sections were then rinsed 4 x
5 min in KPBS, followed by a 90 min incubation in fluorochrome-conjugated goat secondary Ab
(anti-rat Rhodamine Red-X, 1:625, Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA; anti-rabbit Alexa 633, 1:625, Molecular Probes, Eugene, OR; anti-Armenian hamster
Rhodamine Red-X, 1:1000). Sections were then rinsed 4 x 5 min in KPBS, mounted onto
gelatin-coated slides and coverslipped with antifade medium composed of 96 mM Tris-Hcl, pH
8.0, 24% glycerol, 9.6% polyvinylalcohol, and 2.5% diazabicyclooctane (Sigma). Images were
obtained using a Fluoview confocal microscope (FV-500, Olympus Optical) with x20 and x60
oil objectives. Confocal images were acquired by sequential scanning using a two-frame Kalman
filter and a z-separation of 0.25 µm. The images were then processed to enhance contrast and
sharpness using Adobe Photoshop 7 (Adobe Systems) and were assembled using Adobe
Illustrator (Adobe Systems).
RESULTS
In this study, irradiated mice were transplanted with stem cells expressing GFP (9). We then
assessed the outcome of the donor-derived cells in the CNS of these lethally irradiated mice. We
found that 7 wk after the bone marrow transplant, many GFP-expressing cells were present in
various regions of the CNS, from the olfactory bulb to the end of the medulla (Fig. 1
). Moreover,
most of the donor-derived cells were not associated with blood vessels, indicating that they had
effectively crossed the BBB and infiltrated into the brain parenchyma. Regions that consistently
showed a great number of infiltrating GFP-positive cells included the anterior olfactory nucleus;
medial part (AOM; Fig. 1a
and b); piriform cortex (Fig. 1c and d); the lateral septal nucleus (Fig.
1e and f); the hypothalamus (Fig. 1g and h); the amygdaloid area (Fig. 1i and j); the S1 layer of
the cerebral cortex, trunk region (SiTr, Fig. 1k
and l); the hippocampus (Fig. 1m and n); the
substantia nigra (Fig. 1o
and p); the midbrain (Fig. 1q and r); the medulla (Fig. 1s and t); the
cerebellum (Fig. 1u
and v); and the area postrema (Fig. 1w and x). Though GFP-positive cells
were also present in other regions of the CNS, they were fewer in number compared with the
regions mentioned above (data not shown). Of interest is the fact that these cells were found
across the whole rostro-caudal brain in both neurogenic and non-neurogenic areas, which
indicates that bone marrow precursors have the ability to populate the cerebral tissue in a
nonspecific manner.
We observed that nearly all of the GFP-positive cells had a highly ramified phenotype,
resembling the morphology of parenchymal microglia. We thus assessed whether the ramified
cells expressed the microglial marker iba1 (10). As expected, all of the GFP-positive cells with a
ramified morphology were immunoreactive to iba1, providing direct evidence that donor stem
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cells had differentiated into parenchymal microglia (Fig. 2). In this regard, both GFP cells and
endogenous microglial cells exhibited similar immunoreactive signal levels for iba1. We also
tested the possibility that irradiation caused a massive neurodegeneration or death of other cell
types in the brain, which could account for the high cellular immigration and transdifferentiation
observed using our protocols. We thus used the fluorochrome Fluoro-Jade B (FJB), which is a
sensitive and reliable marker for the histochemical localization of neuronal degeneration (11,
12), and positive signals were not found in the brain of the animals included in this study (data
not shown). Therefore, neurodegeneration may not account for the phenomenon presented in this
study.
Macrophages are known to be very efficient APCs, and they express high levels of major
histocompatibility complex type II (MHC-II), whereas brain microglia are poor APCs and have
low surface levels of MHC-II. It has been shown that MHC-II expression is correlated with
another membrane-bound protein known as CD11c (13). Therefore, to determine if newly
differentiated microglia of donor stem cell origin are good APCs, we assessed the expression
levels of CD11c in tissues of the irradiated mice by immunohistochemistry (Fig. 3
). We found
that all GFP-positive cells colocalized with CD11c staining, whereas virtually none of the
residential microglia exhibited a positive signal for this protein. These data thus suggest that
donor-derived microglia may be proficient APCs.
DISCUSSION
Taken together, our data clearly show that stem cells originating from the bone marrow have the
capacity to migrate across the BBB and to transdifferentiate into microglia in vivo and that
generation of these cells during adulthood does not solely depend on the proliferation of resident
cells. To this date, evidence suggested that stem cells could not generate parenchymal microglia
(4, 5), except for two papers showing that only a few infiltrating cells displayed this phenotype
(7, 8). One of these papers shows that this phenomenon seems to consistently occur only in the
cerebellum (8). This report showed that only very small amounts of bone marrow-derived cells
(<0.3% of total donor-derived cells) had infiltrated past the BBB and differentiated into
parenchymal microglia (8). To the contrary, our data show that the large majority of GFP cells
were clearly not associated with blood vessels, although a small percentage of cells derived from
the donor had a perivascular nature. It is difficult to explain why bone marrow precursors were
able to fully differentiate into parenchymal microglia throughout the cerebral tissue here,
whereas previously analyzed mice exhibited only perivascular cells except for the cerebellum
(8). Mice of both studies were exposed to the same total radiation (10 gray), although it was
delivered via different protocols. Indeed, the animals in the previous study were irradiated with
two doses of 5 gray separated by a 3 h interval (8) for a total of 10 gray, whereas a single
radiation session was administered to the animals of the present study.
The fact that we now demonstrate that hematopoietic cells are capable of infiltrating and
generating microglia in various regions of the CNS has wide implications. Indeed, MHC-II is
expressed only on the surface of activated APCs, such as macrophages, dendritic cells, and
microglial cells in the brain. Expression of MHC-II is necessary for APCs to present a specific
foreign antigen to T cells. Parenchymal microglial cells display the same characteristics as
macrophages, but they are not recognized as being very potent APCs and their MHC-II levels are
low compared with other APCs. It is also known that effective APCs express high CD11c levels,
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whereas parenchymal microglia contain very small quantities of this protein (13). Our data show
that microglia of bone-marrow stem cell origin express high levels of CD11c, which is a strong
indication that these newly differentiated parenchymal microglia are likely to be effective APCs.
This could have a critical impact on brain disorders that have an immune etiology, such as
multiple sclerosis (MS). Indeed, macrophage-derived IL-12 stimulates the differentiation of a
subset of T lymphocytes (CD4+) into T helper 1 cells (Th-1) that produce interferon gamma
(IFNγ). These activated Th-1 cells are actually believed to play a critical role in MS, especially
during the demyelinating episodes. Immigrating microglia from blood circulation may therefore
contribute to the development of such immune processes in the cerebral environment.
Monocyte chemoattractant protein 1 (MCP-1) is likely to be essential in this recruitment of bone
marrow-derived cells, because the release of this chemokine by endothelial cells is the key
mechanism for the chemoattraction of cells of monocytic lineage. In this regard, MCP-1 and its
receptor CCR2 have been implicated in a number of inflammatory diseases and the report that
mice lacking CCR2 are resistant to experimental autoimmune encephalomyelitis (EAE), the
animal model of MS, provided decisive evidence that this chemokine has a leading role in the
cerebral infiltrating process (14). This concept is further supported by the total absence of
mononulear cell inflammatory infiltrates within the cerebral tissue of CCR2-deficient mice and
immunized with the myelin oligodendrocyte glycoprotein peptide 35-55 (14) and the fact that
monocyte recruitment to the CNS is a prerequisite step for the development of inflammatory
lesions in EAE (15). Differentiated macrophages into the brain parenchyma are believed to be
directly responsible for the demyelination and axonal dysfunction, as a consequence of myelin
sheath destruction by these activated phagocytes. The presence of these cells in the CNS of intact
animals may play a critical role in the fate that the innate immune system may have on
neurodegenerative disorders.
These data may also have a great clinical impact for cancer patients exposed to different levels of
chemotherapy and radiotherapy, particularly for those undergoing allogeneic hematopoietic
stem-cell transplantation. A similar immigration process of bone marrow precursors is expected
to take place in the brain of these patients, but the physiological relevance of such a phenomenon
has yet to be unraveled. Because these cells are more efficient APCs than endogenous microglia,
it is tempting to propose that recruitment of bone marrow precursors may be a critical step in
brain diseases with an immune etiology.
ACKNOWLEDGMENTS
The Canadian Institutes in Health Research (CIHR) supported this research. Alain Simard is
supported by a Ph.D. Studentship from the CIHR. Serge Rivest is a CIHR Scientist and holds a
Canadian Research Chair (Junior) in Neuroimmunology. The authors thank Dr. Y. Imai
(National Institute of Neuroscience, Kodaira, Japan) for the anti-ionized calcium binding adaptor
molecule 1 (iba1) antibody.
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Fig. 1
Figure 1. Widespread distribution of bone marrow-derived cells throughout parenchymal elements of the central
nervous system. A primary antibody directed against CD31 labeled with rhodamine red-X-conjugated secondary antibody
was used to stain the endothelium of brain capillaries (red blood vessels). Several highly ramified GFP-positive cells were
identified in various areas of the brain, namely anterior olfactory nucleus, medial part (AOM; a and b), piriform cortex (c
and d), lateral septal nucleus (e and f), hypothalamus (g and h), amygdala (i and j), S1 cortex, trunk region (SiTr, k and l),
hippocampus (m and n), substantia nigra (o and p), midbrain (q and r), medulla (s and t), cerebellum (u and v), and area
postrema (w and x). Note that nearly all GFP-positive cells (green) in these regions had a ramified microglia-like
morphology and most of these cells were not associated with blood vessels (red). Scale = 50 µm.
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Fig. 2
Figure 2.
Bone marrow-derived cells transdifferentiate into microglial cells. CD31-positive cells were stained with
a rhodamine red-X-conjugated secondary antibody (red blood vessels), whereas a primary antibody directed against
iba1 was used to label microglia. They were then stained with an Alexa 633-conjugated secondary antibody (blue).
Donor-derived cells (green) with a ramified morphology were always colocalized with iba1 staining (blue) and rarely
colocalized with blood vessels (red). These results provide direct anatomical evidence that highly ramified cells are
parenchymal microglia, which originate from circulating donor stem cells. Scale bar = 25 µm.
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Fig. 3
Figure 3.
Microglial cells derived from bone marrow stem cells exhibit characteristics of antigen-presentating
cells (APCs). Effective APCs express high levels of the surface molecule CD11c, which was visualized by
immunohistochemistry using an anti-CD11c primary antibody and a rhodamine red-X-conjugated secondary antibody.
Virtually all GFP-positive cells were immunoreactive to CD11c (red). Arrows indicate highly localized cellular expression
of the surface antigen CD11c. These data suggest that infiltrating microglia derived from bone marrow precursors are
immunologically competent to present antigens to cells of the adaptive immunity. Scale bar = 10 µm.
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