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The Journal of Neuroscience, August 1, 2002, 22(15):6623-6630
Remyelination of the Rat Spinal Cord by Transplantation of
Identified Bone Marrow Stromal Cells
Yukinori
Akiyama1, 2, 3,
Christine
Radtke1, 2, and
Jeffery D.
Kocsis1, 2
1 Department of Neurology, Yale University School of
Medicine, New Haven, Connecticut 06516, 2 Neuroscience
Research Center, Veterans Affairs Medical Center, West Haven,
Connecticut 06516, and 3 Department of Neurosurgery,
Sapporo Medical University School of Medicine, Sapporo, Hokkaido
060-8543, Japan
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ABSTRACT |
Bone marrow contains a population of stem-like cells that can
differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on
plastic that proliferated in culture to confluency with a typical
flattened elongative morphology. The large majority of the isolated
stromal cells were GFP expressing and immunopositive for collagen type
I, fibronectin, and CD44. Transplantation of these cells by direct
microinjection into the demyelinated spinal cord of the
immunosuppressed rat resulted in remyelination. The remyelinated axons
showed characteristics of both central and peripheral myelination as
observed by electron microscopy; conduction velocity of the axons was
improved. GFP-positive cells and myelin profiles were observed in the
remyelinated spinal cord region, indicating that the donor-isolated
stromal cells were responsible for the formation of the new myelin. The
GFP-positive cells were colocalized with myelin basic protein-positive
and P0-positive cellular elements. These findings indicate that cells
contained within the stromal cell fraction of the mononuclear cell
layer of bone marrow can form functional myelin during transplantation into demyelinated spinal cord.
Key words:
stromal cell; remyelination; cell transplantation; nonhematopoietic stem cells; myelin; mesenchymal stem cells
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INTRODUCTION |
Bone marrow provides a source of
circulating erythrocytes, platelets, monocytes, granulocytes, and
lymphocytes, which are derived from a hematopoietic stem cell (Phinney
et al., 1999 ). The marrow stroma is complex tissue that contains cells
that are required for lineage commitment of hematopoietic cells.
Although initially thought to be primarily hematopoietic support cells, the marrow stromal cells (MSCs) also contain nonhematopoietic cells
that can differentiate into a variety of mesenchymal cell types,
including bone (Rickard et al., 1994; Pereira et al., 1995), cartilage
(Ashton et al., 1980), muscle (Ferrari et al., 1998), and glia and
neurons (Azizi et al., 1998; Woodbury et al., 2000). The hypothesis has
been generated that MSCs are a unique cell in marrow that differentiate
along multiple mesenchymal cell lineages that can differentiate into
nonhematopoietic and nonlymphopoietic tissues (Owen, 1988; Caplan,
1991; Prockop, 1997). The stromal cells have a propensity to adhere to
tissue culture plastic, and this property has been used as a means to
isolate them from bone marrow (Friedenstein, 1976; Wang and Wolf, 1990;
Simmons et al., 1991).
Neural stem cells are self-renewing precursors of neurons and glia and
can be derived from the adult brain (Reynolds and Weiss, 1992; Lois and
Alvarez-Buylla, 1993; Gage et al., 1995; Johe et al., 1996; Johansson
et al., 1999), including that of humans (Kukekov et al., 1999; Vescovi
et al., 1999; Akiyama et al., 2001). When expanded and injected into
demyelinated lesions in the CNS, these cells can form myelin
(Brustle et al., 1999; Keirstead et al., 1999; Akiyama et al., 2001)
and improve conduction velocity (Akiyama et al., 2001). Given the
pluripotency of MSCs, the prospect of using them to elicit
remyelination has been explored (Chopp et al., 2000; Sasaki et al.,
2001).
Indeed, direct injection of cells acutely isolated from the mononuclear
cell fraction of adult bone marrow, which contains MSCs, into the
demyelinated spinal cord elicits remyelination (Sasaki et al., 2001),
as does intravenous delivery of these cells (Akiyama et al., 2002).
Systemic injection of bone marrow cells into lethally X-irradiated mice
leads to differentiation of neuronal cells in brain (Brazelton et al.,
2000; Mezey et al., 2000) and enhances functional recovery in rodents
with middle cerebral artery infarction (Chen et al., 2001) and after
contusive spinal cord injury (Chopp et al., 2000). Although acutely
isolated cells from the mononuclear fraction of bone marrow can
remyelinate spinal cord axons (Sasaki et al., 2001), it is important to
isolate the cell type within the mononuclear layer that is responsible
for the remyelination. This would indicate the possibility of
harvesting and expanding a homogeneous population of cells to be used
for cell therapy studies. The goal of the present study was to isolate MSCs from green fluorescent protein (GFP)-expressing mice and determine
their myelinating potential when transplanted into the demyelinated
spinal cord. We observed that isolated and expanded GFP-expressing bone
marrow stromal cells injected into the demyelinated rat spinal cord
resulted in considerable remyelination with some recovery of axonal function.
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MATERIALS AND METHODS |
GFP-expressing mice. GFP expressing mice
[C57BL/6-TgN(ACtbEGFP] (The Jackson Laboratory, Bar Harbor,
ME) were bred and used for collection of bone marrow cells.
GFP-expressing mice were identified by their fluorescence properties,
and 8-week-old animals were used for bone marrow cell preparation.
Isolation of mice bone marrow stromal cells. Bone marrow (10 µl) was obtained from femurs and tibias of GFP mice using a
heparinized 24 gauge needle. The samples were diluted in 5 ml of neural
progenitor cell basal medium (NPBM) (Clonetics, San Diego, CA), which
contains 10% fetal bovine serum (FBS), epidermal growth factor, basic
fibroblast growth factor, and neural survival factors (NSF). The
composition of the NSF (Clonetics) is proprietary. The bone marrow
suspension was loaded on a 5 ml Ficoll solution using a Pasteur
pipette. The cells were collected from the mononuclear cell layer after centrifugation (560 × g, 25 min) and resuspended in 5 ml of NPBM. After a second centrifugation (560 × g, 25 min), the cells were collected in NPBM, were plated on untreated
plastic culture dishes, and incubated for 3 d, and the nonadherent
cells were removed by replacing the medium. The cells were maintained
in culture for 2 weeks and then treated with 0.25% trypsin and 1 mM EDTA for 5 min, followed by removal from the
flask and replating in new culture dishes.
Animal preparation and transplantation. A focal demyelinated
lesion was created in the dorsal funiculi of adult Wistar rats (12 weeks old) of the spinal cord using X-irradiation and ethidium bromide
(EB) injections (EB-X) (Blakemore and Crang, 1985, 1989; Honmou
et al., 1996). Briefly, rats were anesthetized with ketamine (75 mg/kg,
i.p.) and xylazine (10 mg/kg, i.p.), and a 40 Grays surface dose of
X-irradiation was focally delivered to the spinal cord caudal to the
10th thoracic level (T-10) using a Siemens (Erlangen, Germany)
Stabilipan radiotherapy machine (Honmou et al., 1996). Three days after
irradiation, rats were anesthetized, and a laminectomy was performed at
T-11. The demyelinating lesion was induced by the direct injection of
EB into the dorsal column via a drawn glass micropipette. Injections of
0.5 µl of 0.3 mg/ml EB in saline were made at depths of 0.7 and 0.4 mm at three longitudinal distances separated by 2 mm each. A suspension
of cultured bone marrow stromal cells (5.0 × 103 cells/µl) in 1 µl of medium or 1 µl of medium without cells for control were injected into the middle
of the EB-X-induced lesion 3 d after EB injection. Both control
and cell-transplanted rats were immunosuppressed with cyclosporine (10 mg · kg 1 · d1,
s.c.; Sandoz, Basel, Switzerland) 1 d before spinal cord
injection and each day thereafter.
Field potential recording. Three weeks after
transplantation, the rats were deeply anesthetized (60 mg/kg sodium
pentobarbital), and then spinal cords of normal control
(n = 12), demyelinated (n = 12), and
transplanted rats (n = 9) were quickly removed and maintained in an in vitro submersion-type recording chamber
with a modified Krebs' solution containing the following (in
mM): 124 NaCl, 26 NaHCO3,
3.0 KCl, 1.3 NaH2PO4, 2.0 MgCl2, 10 dextrose, and 2.0 CaCl2 (saturated with 95%
O2 and 5% CO2). Field
potential recordings of compound action potentials were obtained at
36°C with glass microelectrodes (1-5 M ; 1 M
NaCl) positioned in the dorsal columns, and signals were amplified with
an high-input impedance amplifier. The axons were activated by
electrical stimulation of the dorsal columns with bipolar Teflon-coated
stainless steel electrodes cut flush and placed lightly on the dorsal
surface of the spinal cord. Constant current stimulation pulses were
delivered through stimulus isolation units and the timing device. The
recorded field potentials were positive-negative-positive waves
corresponding to source-sink-source currents associated with
propagating axonal action potentials; the negativity represents inward
current associated with the depolarizing phase of the action potential.
All variances represent ± SE. Differences among groups were
assessed by unpaired two-tailed t test to identify
individual group differences. Differences were deemed statistically
significant at p < 0.05.
Immunocytochemistry. Cultured bone marrow stromal cells were
rinsed in PBS and fixed for 15 min with a fixative solution containing 4% paraformaldehyde in 0.14 M Sorensen's
phosphate buffer, pH 7.4, at 4°C. Fixed cells were incubated for 15 min in a blocking solution containing 0.2% Triton X-100 and 5% normal
goat serum (NGS) before incubation with the primary antibody. The
primary antibodies used were anti-fibronectin (1:400, mouse monoclonal; Sigma, St. Louis, MO) and anti-collagen type I (1:100, rabbit polyclonal; Chemicon, Temecula, CA). Living cells in culture were immunostained with anti-CD44 antibody (1:100, mouse monoclonal; VMRD,
Pullman, WA) and were then incubated with anti-CD44 antibody in NPBM
containing 10% FBS overnight at 37°C, followed by washing three
times with PBS containing with 5% FBS. The primary antibody was
visualized using goat anti-mouse or goat anti-rabbit IgG antibody (1:200; Molecular Probes, Eugene, OR). After immunostaining, coverslips were mounted cell-side down on microscope slides using mounting medium
(Dako, High Wycombe, UK). Two controls were run for all immunocytochemical experiments. Tissue was processed without incubation in either primary or secondary antibody. No background fluorescence was
observed in either of these controls.
Histological processing. After electrophysiological
recordings were obtained, each spinal cord was prepared for
histological study. The histological analysis was blinded until
completion of the study. The spinal cords were fixed for 24 hr in 4%
paraformaldehyde in 0.14 M Sorensen's phosphate
buffer, pH 7.4. Tissue was washed three times and stored overnight in
buffer. The lesions in the spinal cord were separated into rostral and
caudal halves. One-half of the tissue from the lesion was separated for
cryosections to look for GFP fluorescence and immunohistochemistry, and
the other half was prepared for plastic embedding for light and
electron microscopic analysis, respectively. For immunohistochemistry, the tissue was placed in 30% sucrose solution in PBS until the tissue
sank. Cryosections were cut, rinsed, and blocked with 10% NGS and
0.4% Triton X-100 in Tris-buffered saline (TBS) or PBS for 30 min at
room temperature. Sections were incubated with primary antibodies mouse
anti-myelin basic protein (MBP) (1:1000 dilution; Sternberger
Monoclonals, Exeter, UK) or mouse anti-P0 (1:1000 dilution; obtained
from Dr. Juan J. Archelos, Department of Neurology, University of Graz,
Graz, Austria) overnight at 4°C. MBP was diluted with 2% NGS
and 0.4% Triton X-100 in TBS, and P0 was diluted with PBS. After
washing, the sections were incubated with secondary antibody (Alexa
Fluor 594 goat anti-mouse secondary antibody; 1:1000 dilution;
Molecular Probes) for 1 hr at room temperature in the dark and mounted
with fluorescent mounting medium (Dako). Slides were examined on a
Nikon (Tokyo, Japan) Eclipse 800 epifluorescent microscope. For plastic
sections, the segments were postfixed with 1% osmium tetroxide for 4 hr, dehydrated in graded ethanol solutions, and embedded in Epox-812
(Ernest Fullam, Latham, NY). The tissue was serially sectioned on an
ultramicrotome. Finally, the sections were counterstained with 0.5%
methylene blue-0.5% azure II in 0.5% borax. The sections were
examined with a Nikon Eclipse 800 and photographed with a Spot RT Color
CCD camera (Diagnostic Instruments, Inc.). Thin plastic
sections were counterstained with uranyl and lead salts and examined
with a Zeiss (Oberkochen, Germany) EM902A electron microscope operating
at 80 kV.
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RESULTS |
GFP expression in bone marrow cells from GFP-expressing mice
Bone marrow was examined from a transgenic mouse line with an
enhanced GFP (EGFP) cDNA under the control of chicken  -actin promoter and cytomegalovirus enhancer (C57BL/6-TgN(ACTbEGFP)1Osb; The
Jackson Laboratory). This model is suggested to have GFP expression in
all tissues, except for erythrocytes and hair (Okabe et al., 1997). We
found that in acutely isolated raw bone marrow from this strain that
19.6 ± 4.9% of cells were GFP positive and that, in the
mononuclear layer separated on a density gradient, that 40.9 ± 9.8% of cells were GFP positive. In the adherent cell population of
the bone marrow, i.e., stromal cells, 62.5 ± 5.0% were GFP positive at 7 d in culture, and, at 14 and 21 d in culture,
62.4 ± 12.2 and 66.78 ± 7.9%, respectively, were positive
for GFP. This indicates that a significant proportion of cells in the
stromal cell fraction express GFP in this model system. At 2 weeks in culture, the adherent cells increased in number from 0.42 × 106 to 2.1 × 106 cells/ml, indicating a near fivefold
increase in the number of cultured stromal cells.
Figure 1A is a field of
living adherent cells in culture obtained at 2 weeks showing a number
of GFP-expressing cells. The overlay of the fluorescent image of Figure
1A with a differential interference contrast image
(Fig. 1B) shows that both GFP-positive and -negative
cells are present in this field. A variety of cell morphologies was
present at this time culture. These included flattened process-bearing
cells, fusiform cells, and rounded cells. There was no relationship
between GFP expression and cell morphology, i.e., GFP-positive and
-negative cells were present across the cell population. Figure
1C is a higher-power field of fixed cells showing a cluster
of flattened, process-bearing GFP-positive stromal cells at 2 weeks in
culture. Stromal cells isolated in this manner have been reported to
express collagen type I, fibronectin, and CD44 (Zohar et al., 1997;
Azizi et al., 1998; Conget and Minguell, 1999; Pittenger et al., 1999).
Many of the GFP-expressing cells coexpressed GFP with collagen I (Fig.
2A), fibronectin (Fig.
2B), or CD44 (Fig. 2C). The isolation of
plastic adherent cells plated from the mononuclear layer of bone marrow
and the expression of the above three surface antigens places these
cells as conventionally defined marrow stromal cells. Although we did
not do triple-labeling experiments for these antigens, the large
percentage of cells that coexpressed GFP with each of the antigens
studied suggest that collagen I, fibronectin, and CD44 are coexpressed
on the stromal cells.
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Figure 1.
GFP-expressing mouse bone marrow stromal cells
cultured for 14 d. A, A field of GFP-expressing
cells and superimposition on a DIC image of the same field
(B). The cells vary in morphology from flattened
and fusiform to rounded. White arrows indicate positive
and black arrows indicate negative fluorescent cells. A
higher-power image (C) shows a collection of
GFP-expressing cells, which are flattened and process bearing. Scale
bar: A, B, 50 µm; C, 30 µm.
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Figure 2.
Immunocytochemical characterization of cultured
GFP-expressing mouse bone marrow stromal cells. Green fluorescence
(A1, B1, C1) shows
GFP-expressing bone marrow stromal cells from three different cultures
(14 d in culture). Red fluorescence (A2,
B2, C2) shows immunoreactivity to
collagen type I (Col I), fibronectin
(FN), and CD44, respectively. A3,
B3, and C3 are coregistered images from
their respective panels in 1 and 2. The
large majority of GFP fluorescent cells were positive for collagen type
I, fibronectin, and CD44. Scale bar, 10 µm.
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Remyelination after transplantation of bone marrow
stromal cells
In preparation for injection into the demyelinated spinal cord,
the stromal cells were grown in culture for 2 weeks, trypsinized, washed, and suspended in NPBM at a cell concentration of ~5.0 × 103 cells/µl. Control injections were
with NPBM alone to control for possible effects of the trophic factors
and mitogens in the medium. Cells or control medium solution were
injected into the center of the lesion (see Materials and Methods), and
the immunosuppressed rats were prepared for histology at 3 weeks or for
in vitro electrophysiological recording. Figure
3A1 shows a low-power
micrograph of a plastic-embedded section from the normal dorsal
columns. A higher-power micrograph (Fig. 3A2) from this
section shows the abundance of myelinated axons in the normal dorsal
funiculus. In the EB-X-lesioned dorsal funiculus (Fig. 3A3),
there is virtually no myelin; the field is composed of demyelinated
axons, cellular debris, and phagocytic cells. It is important to note
that, in this model system, virtually no endogenous repair occurs for
up to 6-8 weeks after lesion induction (Blakemore and Crang, 1985,
1989; Honmou et al., 1996). Three weeks after direct microinjection of
bone marrow stromal cells into the demyelinated spinal cord, relatively
extensive remyelination is observed (Fig. 3B). The low-power
micrograph in Figure 3B1 shows the lesion site. The
darker stained area in the central region of the dorsal
funiculus is replete with remyelination profiles. The boxed
areas (2 and 3) in Figure 3B1 are
shown at higher power in Figure 3, B2 and B3,
respectively. Notice the near complete remyelination in the center of
the lesion (B2) and the partial remyelination at the lateral
margin of the lesion (Fig. 3B3). The lighter stained
areas on the lateral margins of the lesion zone showed poor
myelination (Fig. 3B1), and the remyelination was most
competent in the dorsoventral axis of the dorsal funiculus.
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Figure 3.
Morphology of control, demyelinated, and
remyelinated dorsal funiculus. Normal dorsal funiculus (area
outlined by arrowheads in A1) of
the T-11 spinal cord is shown at low (A1) and high
(A2) power. A3, High-power field of
demyelinated axons in control lesion with injection of control vehicle
without cells. B1, A low-power micrograph of the dorsal
funiculus lesion zone 3 weeks after GFP mouse bone marrow stromal cell
transplantation. The high-power fields in B2 and
B3 were obtained from the boxed areas in
B1 marked 2 and 3,
respectively. Note that nearly all of the axons in the central lesion
zone in B2 are remyelinated, but that demyelinated axons
are present at the lateral edge of the lesion (B3).
DF, Dorsal funiculus; DH, dorsal horn.
Scale bar: A1, B1, 100 µm;
A2, A3, B2,
B3, 15 µm. All images were obtained with Nikon Eclipse
800 epifluorescent microscope.
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The electron micrographs in Figure
4 show the structure of the remyelinated
axons in greater detail. A lower-power field shows both demyelinated
and remyelinated axons obtained at the edge of the repair zone (Fig.
4A). Some of the myelinated profiles are associated
with large nuclei characteristic of peripheral myelin
(arrows). One can also see cells with large nuclei that have
cytoplasmic regions surrounding axons but have not as yet formed myelin
(asterisks). A higher magnification of a myelinated axon
associated with a large nucleus (Fig. 4B) indicates
that these myelinated axons are surrounded by a basement membrane
(arrowheads). These morphologies suggest that, whereas some
axons show stable myelin formation, others appear to be in the process
of myelinating. Moreover, a significant number of remyelinated axons
have morphological features characteristic of peripheral myelin, which
include a large cytoplasmic and nuclear surround.
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Figure 4.
Electron micrographs of remyelinated axons in the
dorsal funiculus of the spinal cord after bone marrow stromal cell
transplantation. A, A field of axons showing some with
myelin associated with a large cytoplasmic and nuclear surround,
demyelinated axons, and axons surrounded by cytoplasmic regions but not
yet forming myelin. Some of the myelinated axons did have the large
nuclear and cytoplasmic surrounds and others without. B,
A higher-power electron micrograph illustrating a remyelinated axon
characteristic of a peripheral myelination pattern. Note the large
cytoplasmic and nuclear domains and the basement membrane
(arrowheads) surrounding the entire axon and cellular
complex. Scale bar: A, 5 µm; B, 1 µm.
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The low-power field of the dorsal funiculus in Figure
5A from a transplanted rat
shows an abundance of GFP fluorescence. A higher-power micrograph (Fig.
5B) shows cellular elements with GFP fluorescence that may
be myelinated axons. We parceled tissue into adjacent blocks for frozen
and plastic-embedded sections. Figure 5, C and D,
is from adjacent blocks of the same transplanted animal from a frozen
and plastic section, respectively. In virtually all transplanted
animals in which plastic-embedded sections revealed remyelination,
adjacent frozen sections showed intense GFP fluorescence. In animals in
which no or little remyelination was observed, GFP fluorescence was
weak.
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Figure 5.
Identification of donor bone marrow cells
in vivo. A, Dorsal funiculus (DF)
of coronally cut spinal cord from a rat that was injected with bone
marrow stromal cells from GFP-expressing mice 3 weeks after
transplantation. Arrows indicate the lateral margins of
the dorsal funiculus. Numerous GFP-positive cells are observed in the
remyelinated region. DH, Dorsal horn. B,
Higher-power image of same field showing profiles reminiscent of
myelinated axons. C and D are from frozen
and plastic-embedded sections, respectively, from the same animal
showing colocalization of GFP fluorescence and more clearly defined
myelination in the plastic section. E and
F show an H-E-stained frozen section and a fluorescent
unstained image with GFP fluorescence at the same high power. Note
that, in the frozen H-E section, the axon cylinder is collapsed
(arrows) and the myelin is "puffy," as is typical
with this staining technique. G shows a comparable
semithin plastic section from the same animal showing myelinated axons.
The myelin is better preserved and the tissue more shrunken form
dehydration protocols required for plastic embedding. Scale bar:
A, 250 µm; B, 50 µm;
C, D, 40 µm; E,
F, 12 µm; G, 10 µm.
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Figure 5E is a high-power light micrograph from a frozen
section stained with hematoxylin-eosin (H-E) taken within the
transplant zone. The arrows point to collapsed axon
cylinders surrounded by expanded myelin, which is typical with this
histological technique. Note the eosin-stained nuclei
(asterisks) associated with some of the myelinated axons.
Figure 5F is an unstained frozen section obtained from the
same block as in E, showing a cluster of cells that are GFP
positive. A field of remyelinated axons from a plastic-embedded section
is shown in Figure 5G for comparison.
Immunostaining was also performed for MBP and P0 on sections from
spinal cords that were transplanted with GFP-expressing stromal cells
(Fig. 6). Plastic sections from adjacent
blocks of the same spinal cords indicated an abundance of
remyelination. Colocalization of both MBP (Fig.
6A-C) and P0 (Fig. 6D-F)
with GFP-expressing cells was observed in the transplant zone. Whereas MBP staining was very specific for myelin (both central and peripheral myelin on attached spinal roots), there was considerable MBP staining in the lesion site that was not colocalized with GFP cells. P0 staining
was very specific for peripheral roots and cells within the transplant
zone. The enhanced MBP staining in the lesion zone may reflect MBP
epitope from central myelin debris within the lesion. These results,
although not quantitative, indicate the colocalization of transplanted
GFP-expressing stromal cells with myelin proteins.
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Figure 6.
Colocalization of MPB and P0 with transplanted
GFP-expressing stromal cells. Cryosections from spinal cord blocks
adjacent to plastic-embedded blocks in which remyelination was
confirmed showed colocalization of MBP (A-C) and
P0 (D-F) with GFP-expressing elements. Images in
A-C were obtained on a confocal microscope, and
D-F were obtained on a conventional epifluorescence
microscope. Scale bar: A-C, 70 µm;
D-F, 30 µm.
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Conduction velocity improves after transplantation of
stromal cells
Electrophysiological studies were performed in vitro to
examine the conduction properties of the axons (Fig.
7). Field potential recordings of
compound action potentials were recorded with a glass microelectrode
from the dorsal columns of control, demyelinated, and bone marrow
stromal cell-injected rats, respectively, at sequential longitudinal
distances (Fig. 7A). The early negativity was indicative of
the fastest conducting fiber group and is shown at three distances along the conduction trajectory for normal, demyelinated, and stromal
cell-transplanted dorsal funiculi (Fig. 7B1-3). The
demyelinated axons displayed considerable conduction slowing (0.89 ± 0.11 m/sec; n = 12) compared with control
(10.76 ± 1.24 m/sec; n = 12). The transplant
group had a conduction velocity of 6.03 ± 1.62 m/sec (n = 9), which was significantly faster than the
demyelinated axons (Fig. 7C). These data indicate that at
least a subpopulation of remyelinated axons in the bone marrow stromal
cell-injected rats showed increased conduction velocity.
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Figure 7.
Conduction velocity measurements of the
remyelinated axons. A, Schematic indicating sites of
stimulation (S) and recording
(R) within the dorsal funiculus.
B, Compound action potentials recorded at 1.0 mm
increments longitudinally along the dorsal columns in normal
(1), demyelinated (2), and
after stromal cell transplantation (3).
The arrows in B indicate the peak
negativities of three primary negativities. C,
Histograms of conduction velocity (error bars indicate SEM) of dorsal
column axons obtained from normal (CONT),
demyelinated (DEMYEL), and remyelinated
(REMYEL) axons. At 36°C, conduction velocity of the
early negativity in controls, demyelinated, and bone marrow-delivered
groups were 10.76 ± 1.24 m/sec (n = 12),
0.89 ± 0.11 m/sec (n = 12), and 6.03 ± 1.62 m/sec (n = 9), respectively.
*p < 0.01, demyelinated versus transplanted;
**p < 0.001, control versus demyelinated.
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DISCUSSION |
In the present study, we demonstrated that bone marrow stromal
cells isolated in culture from the mononuclear layer of bone marrow can
remyelinate demyelinated spinal cord axons after direct injection into
the lesion. Our results show that (1) expanded and isolated bone marrow
stromal cells differentiate primarily into myelinating cells when
injected into a demyelinated spinal cord lesion, (2) a large number of
the myelinated axons have characteristics of peripheral-like myelin
with large nuclei and a surrounding basement membrane, and (3)
conduction velocity of the remyelinated axons is improved.
Donor cells as the source of myelin-forming cells
in vivo
The lesion model that we used combines focal X-irradiation of the
lesion zone to delay endogenous repair in combination with microinjection of ethidium bromide, which kills glia within the lesion
zone (Blakemore and Crang, 1985, 1989; Honmou et al., 1996). Although
our lesion model is unique in that it represents an aglial zone and
most naturally occurring demyelinating disorders are associated with
gliosis, this system does provide an important and simple model system
to study the interaction of transplanted precursor cells and
demyelinated axons in vivo. Virtually no myelination is
observed in the lesion zone for 6-8 weeks, at which time, some endogenous myelin repair begins. Our studies were performed at 3 weeks
after lesion induction, which is well within the time at which
persistent demyelination is observed in this model. However, one cannot
completely rule out the possibility that the injected cells recruited
or facilitated an endogenous repair mechanism. To address this issue,
we used GFP-expressing donor mouse MSCs. The majority of the MSCs
cultured from the GFP-expressing mouse strain were GFP positive. It is
also important that, in our control experiments, we did not observe
remyelination. There are a number of trophic and mitogenic agents in
the control media that was injected without cells. This strongly
indicates that the medium alone did not enhance or accelerate an
endogenous repair mechanism.
MSCs in culture were immunopositive for CD44, collagen I, and
fibronectin, which have been shown to be expressed on MSCs (Zohar et
al., 1997; Azizi et al., 1998; Conget and Minguell, 1999; Pittenger et
al., 1999). Although we did not triple label the cells, the large
proportion of cells immunopositive for these three antibodies suggest
colocalization. Our results indicate that, in the spinal cord of rats
showing remyelination after injection of donor GFP-expressing MSCs,
that GFP-expressing cellular profiles were observed in the remyelination zone and were associated with the myelinated axons. In
experiments in which no remyelination was observed, some GFP-like fluorescence was observed, but the intensity was low and no cellular profiles associated with axons were observed. Although we cannot say
conclusively that every myelinated axon was the result of the injected
donor cells, which is a caveat for all such studies, the intense GFP
fluorescence and the cellular morphology of the GFP profiles in areas
of remyelination strongly suggest that the donor cells contributed to
the remyelination.
Morphology of the remyelinated axons
The remyelinated axons were densely packed and tended to cluster
in the area of cell injection. The lateral margins of the dorsal
funiculus remained unmyelinated, suggesting that either cell injection
number was relatively low or the cells had limited ability to migrate.
However, remyelination was observed along the entire dorsoventral
extent of the dorsal funiculus. Interestingly, many of the remyelinated
profiles were associated with a large cytoplasmic and nuclear surround,
which in turn was enshrouded by a basement membrane. These are hallmark
characteristics of peripheral myelin (Berthold, 1978). Neural precursor
cells derived from the subventricular zone of postnatal rat (Keirstead
et al., 1999) and adult human (Akiyama et al., 2001) brain also can
give rise to both central (oligodendrocyte-like) and peripheral
(Schwann cell-like) myelin.
There was no obvious neuronal differentiation within the
transplantation zone, although marrow cells do have the ability to differentiate into neurons and astrocytes when transplanted into other
regions of brain (Azizi et al., 1998; Woodbury et al., 2000). It is not
clear whether there are different populations of cells within these
progenitor groups or whether regions in the host (transplant recipient)
provide signals to direct these cells along different glial or neuronal
lineages. It is also unclear as to why these cells develop an almost
exclusive myelinating phenotype when transplanted into the demyelinated
lesion. One possibility is that the demyelinated white matter in our
lesion model is replete with bare axon membrane and phagocytic cells.
The ethidium bromide treatment kills virtually all endogenous cells,
including astrocytes and oligodendrocytes within the lesion zone, and
no endogenous neurons are present in the dorsal funiculus. Axon
membrane is known to provide important signals to promote
myelin-forming cells to form myelin (Wood and Bunge, 1975; Salzer et
al., 1980; Maurel and Salzer, 2000). It is possible that these local
axonal signals in the demyelinated lesion direct the lineage of the
transplanted MSCs to a myelinating phenotype.
Conduction velocity of the remyelinated axons
In vitro electrophysiological studies were
performed to assess conduction velocity of the remyelinated axons. The
results indicate that conduction velocity was significantly increased in the transplantation group compared with the demyelinated group. The
velocity was not as fast as controls but was several times greater than
the demyelinated axons. However, the increase in conduction velocity as
studied over several millimeters of conduction suggest that functional
and sequential internodal segments are deposited on at least a subset
of the fibers. We limited this study to 3 weeks after transplantation.
It will be important in the future to determine whether continued
maturation of the myelinated axons is accompanied by greater
improvement in conduction velocity. These results do indicate that at
least a level of functional recovery of conduction is elicited by the
MSCs transplantation-induced remyelination.
Oligodendrocytes are the cells that normally myelinate CNS axons, but
peripheral myelin-forming cells, such as Schwann cells (Blakemore,
1977) and olfactory ensheathing cells (Franklin et al., 1996; Imaizumi
et al., 1998), can myelinate CNS axons in vivo and restore
near normal conduction properties (Honmou et al., 1996; Imaizumi et
al., 1998). Peripheral myelin-forming cells may have the advantage, if
used as a cell therapy in multiple sclerosis (MS) patients, of not
having the antigenic properties of oligodendrocytes, which are targets
of an immune response in MS patients. Harvesting sufficient numbers of
Schwann cells from peripheral nerve biopsy and cell expansion is
problematic. However, the development of human clonal neural precursor
cells derived from either embryonic or adult CNS (Keirstead et al.,
1999; Kukekov et al., 1999; Vescovi et al., 1999; Akiyama et al., 2001)
may allow for an abundant source of myelin-forming cells to be used in
transplantation studies. Brustle et al. (1999) have demonstrated that
human embryonic stem cell-derived glial precursors can be used as a
source of myelinating cells in the CNS. Advances in the cell biology of
progenitor cells derived from embryonic, fetal, or adult CNS open the
prospect of developing cell lines as a potential source of a cell
therapy for demyelinating diseases.
Bone marrow stromal cells may offer advantages if developed for cell
therapies because the cells are relatively easy to isolate from small
aspirates of bone marrow that can be obtained under local anesthesia,
and they could provide a vast source of autologous cells for reparative
therapies. Thus, MSCs have the potential to provide an efficient and
renewable source of cells for autologous transplantation studies for
demyelinating diseases. The demonstration that bone marrow stromal
cells can form functional myelin when transplanted into demyelinated
CNS regions suggests a potentially important interaction between bone
marrow cells and CNS white matter repair mechanisms.
| |
FOOTNOTES |
Received Feb. 22, 2002; revised May 9, 2002; accepted May 20, 2002.
This work was supported by National Institutes of Health Grant NS10174,
the Department of Veterans Affairs, and National Multiple Sclerosis Society Grant RG2135.
Correspondence should be addressed to Dr. Jeffrey D. Kocsis, Yale
University School of Medicine, Neuroscience Research Center (127A),
Veterans Affairs Medical Center, West Haven, CT 06516. E-mail:
jeffery.kocsis{at}yale.edu.
| |
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ABSTRACT
INTRODUCTION
