doi:10.1093/brain/awm125
Brain (2007), 130, 2175^2185
Schwann cell precursors: a favourable cell for myelin
repair in the Central Nervous System
A. Woodhoo,1 V. Sahni,1 J. Gilson,2 A. Setzu,2 R.J.M. Franklin,2 W.F. Blakemore,2 R. Mirsky1 and K.R. Jessen1
1
Department of Anatomy and Developmental Biology, University College London, London and
Repair and Department of Veterinary Medicine, University of Cambridge, UK
2
Cambridge Centre for Brain
Correspondence to: Prof. K.R. Jessen, Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
E-mail: [email protected]
Cell transplant therapies are currently under active consideration for a number of degenerative diseases. In the
immune-mediated demyelinating-neurodegenerative disease multiple sclerosis (MS), only the myelin sheaths of
the CNS are lost, while Schwann cell myelin of the PNS remains normal.This, and the finding that Schwann cells
can myelinate CNS axons, has focussed interest on Schwann cell transplants to repair myelin in MS. However,
the experimental use of these cells for myelin repair in animal models has revealed a number of problems
relating to the incompatibility between peripheral glial cells and the CNS glial environment. Here, we have
tested whether these difficulties can be avoided by using an earlier stage of the Schwann cell lineage,
the Schwann cell precursor (SCP).
For direct comparison of these two cell types, we implanted Schwann cells from post-natal rat nerves and SCPs
from embryo day 14 (E14) rat nerves into the CNS under various experimental conditions. Examination 1 and 2
months later showed that in the presence of naked CNS axons, both types of cell form myelin that antigenically
and ultrastructurally resembles that formed by Schwann cells in peripheral nerves. In terms of every other
parameter we studied, however, the cells in these two implants behaved remarkably differently. As expected
from previous work, Schwann cell implants survive poorly unless the cells find axons to myelinate, the cells do
not migrate significantly from the implantation site, fail to integrate with host oligodendrocytes and astrocytes,
and form little myelin when challenged with astrocyte-rich environment in the retina. Following SCP implantation, on the other hand, the cells survive well, migrate through normal CNS tissue, interface smoothly and
intimately with host glial cells and myelinate extensively among the astrocytes of the retina. Furthermore,
when implanted at a distance from a demyelinated lesion, SCPs but not Schwann cells migrate through
normal CNS tissue to reach the lesion and generate new myelin.
These features of SCP implants are all likely to be helpful attributes for a myelin repair cell. Since these cells also
form Schwann cell myelin that is arguably likely to be resistant to MS pathology, they share some of the main
advantages of Schwann cells without suffering from the disadvantages that render Schwann cells less than ideal
candidates for transplantation into MS lesions.
Keywords: multiple sclerosis; Schwann cell precursors; Schwann cells; remyelination
Abbreviations: SCP ¼ Schwann cell precursors; MS ¼ multiple sclerosis
Received March 5, 2007. Revised May 3, 2007. Accepted May 4, 2007. Advance Access publication June 4, 2007
Introduction
Damage and death of oligodendrocytes is a key feature of
multiple sclerosis (MS). This results in the formation of
scattered demyelinated lesions throughout the CNS,
a process that is accompanied by breakdown of the
blood–brain barrier, activation of the immune system
and loss of axons (Lucchinetti and Lassmann, 2001).
Because the causes of MS are essentially unknown,
a number of therapeutic strategies are under consideration.
Broadly they fall into two categories, namely attempts to
block the disease process, and attempts to repair the damage,
notably the demyelination, caused by the disease (Scolding,
1999; Lubetzki et al., 2005). Myelin repair, in turn, can be
approached either through stimulation of the inherent
remyelination capacity of the CNS, or by transplantation of
exogenous cells that can populate the MS lesions, remyelinate
the axons and prevent further axon loss. The work presented
here is relevant for the second of these approaches.
ß The Author (2007). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
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Brain (2007), 130, 2175^2185
A central issue when considering exogenous myelin
repair is the choice of cell type for transplantation. Three
classes of cell have received most attention: cells from
various stages of the oligodendrocyte lineage, olfactory bulb
ensheathing cells and Schwann cells from the post-natal
PNS (Franklin, 2002; Halfpenny et al., 2002; Stangel and
Hartung, 2002; Zhao et al., 2005; Raisman and Li, 2007).
More recently, a number of studies have also been carried
out using neural or non-neural stem cells (Baron-Van
Evercooren and Blakemore, 2004; Pluchino and Martino,
2005). For myelin-generating transplants, each of these cells
have different advantages and drawbacks, and at present no
one cell type has emerged as the undisputed cell of choice.
Transplanted Schwann cells myelinate CNS axons in vivo
in a variety of experimental models, and can restore
function to demyelinated axons. Similarly, in some myelin
mutants, and following injection of toxins that kill
astrocytes and oligodendrocytes, endogenous Schwann
cells provide significant areas with long lasting and
functional myelin (Blakemore, 1977; Duncan et al., 1981;
Itoyama et al., 1983; Baron van Evercooren et al., 1992;
Felts and Smith, 1992; Blakemore et al., 1995; Honmou
et al., 1996; Duncan and Hoffman, 1997; Iwashita et al.,
2000). With respect to the possibility of using Schwann cells
as a therapeutic tool in MS, these observations indicate that
cells of the Schwann cell lineage can in principle, and given
the right conditions, achieve stable and functional myelin
repair within the CNS. In addition, Schwann cells and
oligodendrocytes show considerable molecular differences,
and Schwann cell myelin is unaffected in MS. Schwann cell
myelin within the CNS may therefore not be a target
for ongoing disease activity.
Together, these are some of the main arguments for
Schwann cells as the cell of choice for eventual therapeutic
implants in MS. Indeed Schwann cells were chosen for the
first clinical trial of transplanted cells in MS patients
(Pluchino et al., 2003).
Potential drawbacks to the use of Schwann cells within
the CNS are nevertheless apparent and relate primarily to
three issues: migration, survival and the complex interplay
between astrocytes and Schwann cell myelination.
For practical implantation strategies in MS patients,
an important requirement is that the implanted cells can
migrate in the CNS. It is therefore unfortunate that
according to most studies, implanted Schwann cells
do not migrate significantly through the normal CNS
(Iwashita et al., 2000), a problem also encountered with
oligodendrocyte progenitors (Franklin et al., 1996; O’Leary
and Blakemore, 1997). Even in the case of myelination by
endogenous Schwann cells, mentioned earlier, this is seen
only in relatively restricted areas of the CNS. It is therefore
unlikely that Schwann cells would migrate sufficiently
widely from the implantation site in the human CNS.
This would preclude the use of a single injection of these
cells to populate multiple lesions and reduce the chances
A. Woodhoo et al.
of implanted cells effectively reaching the lesion they were
aimed for.
Survival is, of course, a prerequisite for migration. It is
therefore highly relevant that in normal CNS tissue, Schwann
cells appear to survive very poorly, although in lesions, where
the cells can access naked axons and myelinate, they thrive,
as mentioned earlier (Iwashita et al., 2000).
Interactions between astrocytes and Schwann cells have
been the subject of numerous studies. The majority of these
indicate that co-existence or close interaction between these
cells, in vivo or in vitro, is rarely obtained, and that under
most conditions astrocytes inhibit Schwann cell myelination
in the CNS (Itoyama et al., 1985; Blakemore et al., 1986;
Franklin and Blakemore, 1993; Wilby M et al., 1999;
Lakatos et al., 2003). On the other hand, extracellular
matrix provided by astrocytes can promote myelination by
implanted Schwann cells (Blakemore, 1984; Franklin et al.,
1992). This complex and paradoxical relationship is likely
to pose a significant problem for the use of Schwann cells
to establish myelin within astrocyte-rich environments
such as chronic MS lesions.
In the present paper, we have tested whether the
difficulties outlined above can be avoided using a close
relative of the Schwann cell, the Schwann cell precursor
(SCP). This cell represents a well-characterized developmental stage in the Schwann cell lineage that stands between
migrating neural crest cells and specified Schwann cells
(Jessen et al., 1994; Jessen and Mirsky, 2005). SCPs are
present in embryonic rat and mouse nerves at embryo day
14/15 and 12/13, respectively, and cells with similar
morphology are found in embryonic human nerves (Jessen
et al., 1994; Dong et al., 1999).
Our results reveal striking differences between SCP and
Schwann cell transplants. The behaviour of SCPs in the CNS
indicates that the SCP phenotype retains key advantages of
post-natal Schwann cells for myelin repair. Importantly,
however, the use of SCPs appears to circumvent some of the
main problems associated with the use of Schwann cells as
a myelin repair cell.
Materials and methods
Cell preparation
SCP and Schwann cell culture preparations are described in detail
elsewhere (Jessen et al., 1994) and assessment of the purity of the
cultures by L1 immunolabelling is described elsewhere (Dong
et al., 1995). Purification of Schwann cell cultures by negative
immunopanning using Thy1 has also been described elsewhere
(Dong et al., 1999). The donor cells used in our experiments were
obtained from transgenic rats that expressed the green fluorescent
protein (GFP) under the chicken bactin promoter. Transgenic
males were crossed with normal Sprague-Dawley females and
usually half the offspring obtained carried the transgene and were
used as donors. These animals were generated by Dr Masaru
Okabe (University of Osaka, Osaka, Japan) (Okabe et al., 1997; Ito
et al., 2001) were obtained from Japan SLC, Inc. (Hamamatsu,
Japan).
Schwann cell precursors
Surgery and tissue processing
For cell transplantation, female Sprague-Dawley rats (n ¼ at least 8
for each type of experiment) were used and in each experiment,
1 ml of cell suspension at a density of 10 000 cells/ml was injected.
For spinal cord transplant experiments, cells were implanted into
ethidium bromide-induced focal areas of demyelination or into
the normal CNS and at the relevant time, the spinal cord segments
processed for frozen or resin sections (see Blakemore and Crang,
1992; Iwashita et al., 2000 for detailed descriptions).
For retinal transplant experiments, the cells were injected
(described in detail in Setzu et al., 2004, 2006) and after 4 or
6 weeks, the retinae were dissected out, flat-mounted and
immunolabelled with mouse anti-P0 (1:500; Astexx, Austria)
antibody (see later). All areas of the retinae containing
P0þ myelinating cells were captured using a Multi-Photon UV
confocal microscope (Leica, UK) and the area quantified using
image analysis software (Image J, USA).
Immunohistochemistry
For immunohistochemistry, the spinal cord sections or retinal flatmounts were fixed in 4% paraformaldehyde (PF) in phosphatebuffered saline (PBS), permeablized with ice-cold methanol,
blocked with 0.2% tritonX-100 in ADS (PBS containing 10%
calf serum, 0.1% lysine and 0.02% sodium azide) and primary
(overnight) and relevant secondary antibodies (30 min) were
applied in blocking solution. In addition to the P0 antibody, the
following antibodies were used; rabbit anti-GFAP (1: 500;
Dakopatts, Denmark), rabbit anti-S100b1 : 1000; (Dakopatts,
Denmark), mouse anti-MBP (1 : 200; Sternberger Monoclonals,
USA), mouse anti-nestin (Rat401) (1 : 1000; Developmental
Studies Hybridoma Bank, USA), goat anti-mouse Ig rhodamine
(MP Biomedicals, USA) and goat anti-rabbit Ig Cy3 (Jackson
ImmunoResearch, USA).
Results
The properties of the transplanted cell
populations
All the experiments in the present work were done by
acutely transplanting cells dissociated from dissected nerves,
without an intervening cell-culture period. SCPs were
obtained from the sciatic nerve and brachial plexus of
embryo day 14 (E14) rat embryos and Schwann cells from
the sciatic nerve and brachial plexus of newborn or postnatal day 1 (P1) rats. The molecular expression profile of
SCPs and Schwann cells has been well characterized (Jessen
and Mirsky, 2005). Except for immunopanning, which was
carried out for Schwann cells only, the cells were handled
and injected in an identical way so that a strict comparison
of the transplantation properties of these two cell types
could be made. Prior to injection, both cell types cells were
resuspended in DMEM containing 10 ng/ml neuregulin at
a density of 10 000 cells/ml. One microlitre of this cell
suspension was injected in every case. Fibroblasts can have
deleterious effects on Schwann myelination in the CNS as
their presence in transplanted preparations results in axon
degeneration (Brierley et al., 2001) and so to determine the
Brain (2007), 130, 2175^2185
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purity of the SCPs, samples from the cell suspensions to be
injected were plated on coverslips and immunolabelled 3 h
later with L1 antibodies to identify SCP (Dong et al., 1995).
This showed that 99.1 5.6% of the cells in these cultures
were L1þ. Following purification by immunopanning,
the purity of Schwann cells was determined in a similar
way and found to be 99.8 1.3%.
To enable us to unambiguously identify the transplanted
cells in CNS tissue, all experiments were done with cells
from transgenic rats that express the enhanced GFP under
the chicken bactin promoter. Transgenic males were
crossed with normal Sprague-Dawley females and usually
half the offspring obtained carried the transgene and were
used as donors.
In de-myelinated CNS lesions, Schwann cell
precursors survive and myelinate at least as
efficiently as Schwann cells
Most previous studies on Schwann cell myelination in the
CNS have used cells that were maintained in long/mediumterm culture before transplantation (Blakemore, 1977;
Duncan et al., 1981; Blakemore and Crang, 1985, Iwashita
et al., 2000, Shields et al., 2000; Brierley et al., 2001).
Because the properties of Schwann cells are often determined by their environment, it was necessary to examine
whether the acutely transplanted cells used here behaved
like the cultured cells used previously. Freshly dissociated
Schwann cells were injected into an ethidium bromide
(EB)-induced de-myelinating lesion in rat spinal cords and
1 month later, the spinal cord segments were dissected out
and processed for embedding in resin or for generation of
frozen sections. The implanted Schwann cells were found to
be well distributed within the EB lesions in the dorsal
funiculus of the spinal cord (Fig. 1A). Even after 1 month
following transplantation, the cells showed intense green
fluorescence, and no immunolabelling was required to
detect the GFP protein. Immunolabelling of frozen sections
with P0 antibody, which binds to Schwann cell myelin,
showed that the cells had re-myelinated the de-myelinated
axons. (Fig. 1B–D). Examination of semi-thin resin sections
stained for myelin with 1% toluidine blue solution, revealed
the myelin sheaths formed by the transplanted cells
as circular structures that were usually less heavily
stained than the endogenous myelin sheaths formed by
the oligodendrocytes surrounding the lesions (Fig. 1E).
Ultra-thin sections viewed in a transmission electron
microscope showed that the transplanted GFP–Schwann
cells formed a 1:1 relationship with axons and individually
myelinated single axons (Fig. 1F). Another characteristic
feature was the significant extracellular space present
between axon-Schwann cell units, an architectural
arrangement that replicates that seen in peripheral nerves.
Together these experiments suggest that acutely implanted
Schwann cells are not significantly different from cultured
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Brain (2007), 130, 2175^2185
Fig. 1 Freshly dissociated Schwann cells survive and
myelinate demyelinated axons in an EB lesion. Schwann cells were
transplanted into an EB lesion at thoracic level 13 (T13) and 1 month
later, the spinal cord segment was dissected out and processed for
resin or frozen sections. (A) Frozen transverse section of spinal
cord showing transplanted Schwann cells, identified by the
expression of GFP (green), within the EB lesion in the dorsal
funiculus. The structure of the spinal cord is seen in red from
a brightfield exposure of the same field. The border between
the white matter and grey matter is shown by a white line.
(B, C) Frozen sections immunolabelled with P0 antibody.
P0 immunoreactivity (C) colocalizes with GFP (D), expressed by
the transplanted cells (B). (E) Semi-thin resin section showing part
of an EB lesion containing transplanted Schwann cells. The section
was stained with toluidine blue. Dark circular structures on the
right represent oligodendrocyte (arrows). Light circular structures
on the left represent myelin formed by transplanted Schwann cells
(e.g. arrowheads). (F) Electron micrographs showing transplanted
Schwann cells in EB lesions. The cells are separated by large
extracellular spaces, form typical 1 : 1 relationship with CNS axons
and generate compact myelin sheaths. A, CNS axons; M, myelin.
Schwann cells in terms of their myelinogenic properties
in CNS implants (Shields et al., 2000).
It was uncertain whether SCPs would survive CNS
implantation because they are, unlike Schwann cells, acutely
dependent on extrinsic survival factors. Furthermore, it has
not been demonstrated that after isolation from embryonic
nerves these cells can give rise to cells that myelinate
A. Woodhoo et al.
axons, although it has previously been demonstrated that
a conditionally immortalized cell line derived from these
cells can myelinate axons within the CNS (Lobsiger et al.,
2001). To answer these questions, freshly isolated SCPs
were used in experiments similar to those described earlier
for Schwann cells.
We found that 1 month after SCP transplantation, GFPþ
cells had spread throughout the lesions, much like Schwann
cells (Fig. 2A). Furthermore, they had generated myelinating Schwann cells, in the sense that they had formed P0þ
Schwann cell-like myelin around de-myelinated CNS axons
(Fig. 2B and C), which had the ultrastructural characteristics of peripheral myelin (Fig. 2F and G). Axon-Schwann
cell units were also separated by significant extracellular
space containing collagen fibrils (Shields et al., 2000).
In addition to myelination, another way of following the
maturation of implanted SCPs is to examine expression of
S100b. This protein marks the developmental transition
between SCPs and immature Schwann cells during
embryonic nerve development (E14/15–E17/18), because
S100b is very low or absent in SCPs but high in Schwann
cells (Jessen and Mirsky, 2005). Immunolabelling of frozen
sections from EB lesions with S100b 1 month after
transplantation of SCPs showed that the large majority
of the GFPþ-implanted SCPs now expressed S100b
(Fig. 3A–C). Thus, in terms of S100b protein expression
and ability to myelinate, SCPs develop into Schwann cells
when injected into areas of demyelination in the CNS.
These cells also expressed nestin, a protein expressed by
the Schwann cell lineage but not by astrocytes, mature
oligodendrocytes or fibroblasts (Friedman et al., 1990)
(Fig. 3D–F).
These results show that transplanted SCPs survive in
de-myelinated lesions and progress to form Schwann
cell-like cells in these transplants, although this is achieved
in a CNS environment. Interestingly, however, other
experiments (later) indicate that these cells are not identical
to implanted Schwann cells, but differ from them in ways
that enhance their survival and migration potential. For
ease of description, we will generally refer to GFPþ cells
observed in the CNS 1 month (or later) after transplantation of SCPs as ‘SCPtrans-derived’ Schwann cells, and to
GFPþ cells observed in the CNS after transplantation of
Schwann cells as ‘post-natal’ Schwann cells.
SCPtrans -derived Schwann cells survive better
than post-natal Schwann cells within the
normal CNS
Schwann cells show notoriously poor long-term survival
when transplanted in normal CNS (where denuded axons
are not available for re-myelination), and very few cells
remain 4 weeks after transplantation (Iwashita et al., 2000).
Long-term survival would be a prerequisite for significant
Schwann cell migration through CNS tissue and such
migration, in turn, is likely to be an important attribute
Schwann cell precursors
Fig. 2 Following transplantation of Schwann cell
precursors, SCPtrans-derived Schwann cells survive and remyelinate
CNS axons similar to freshly isolated post-natal Schwann cells.
SCPs were transplanted into an EB lesion at T13 and 1 month later,
the spinal cord segment was dissected out and processed for resin
or frozen sections. (A) Frozen transverse section showing that
CNS-generated Schwann cells, identified by expression of GFP
(green), fill the EB lesion site in the dorsal funiculus. The surrounding tissue is revealed in red (brightfield exposure of image). The
border between the white and grey matter is shown by a white
line. (B ^D) Frozen sections immunolabelled with P0 antibody
show myelin sheaths (C) made by the transplanted GFPþ cells (B).
(D) Represents overlay. (E) A high-power confocal image demonstrating that SCPtrans-derived Schwann cells, identified by green
GFP fluorescence can form P0þ Schwann cell-like myelin in the
CNS (red). Asterisk: two examples of individual myelinated axons;
arrow: a P0þ myelin sheath. (F) Electron micrograph illustrating the
general appearance of SCPtrans-derived myelinating Schwann cells
in EB lesions. They adopt a 1 : 1 relationship with the CNS axons
and elaborate compact myelin wraps around them. A, CNS axon;
M, myelin. (G) High magnification of an ultra-thin section showing
part of the myelin sheath of a SCPtrans-derived myelinating cell
with the characteristic periodicity of peripheral myelin (15 nm).
of an effective myelin repair cell (Franklin, 2002). Inability
to survive in the CNS, except when forming myelin, is
therefore a significant obstacle for the use of Schwann cells
for myelin repair.
For this reason we compared the ability of Schwann cells
and SCPs to survive following injection into normal white
Brain (2007), 130, 2175^2185
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20 µm
5 µm
20 µm
5 µm
20 µm
5 µm
Fig. 3 Antigenic profile of SCPtrans-derived Schwann cells present
in EB lesions. SCPs were transplanted into an EB lesion and 1
month later, the spinal cords were dissected out and
processed for frozen sections. (A^C) Immunolabelling with
antibodies to S100b (red) a marker of mature Schwann cells, reveals
substantial co-localization with GFP expression (A; green) showing
that most of the CNS-generated Schwann cells express this
marker of mature Schwann cells. Overlay is shown in (C). (D ^F)
Immunolabelling with antibodies to nestin (red), which labels cells
of the Schwann cell lineage, co-localizes with GFP (E; green)
showing that the transplanted cells are likely to be still in the
Schwann cell lineage and have not trans-differentiated into other
glial cell-types. Overlay is seen in (F).
matter of the dorsal funiculus at thoracic level 13 (T13) of
the spinal cord. One and two months later, frozen sections
from the injected segments were immunolabelled with
antibodies against myelin basic protein (MBP). MBP
immunohistochemistry labels oligodendrocyte myelin and
reveals the general architecture of the spinal cord, but does
not label Schwann cell myelin, using the present protocol,
due to lower MBP content (not shown).
As expected, post-natal Schwann cells survived poorly
and very few GFPþ cells were present in the white matter at
1 and 2 months (Fig. 4A and B). In contrast, large numbers
of the SCPtrans-derived Schwann cells were present in the
dorsal funiculus 1 month after injection of SCPs and their
number was maintained at 2 months (Fig. 4C–E).
Quantification of the areas containing GFPþ cells showed
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A. Woodhoo et al.
that post-natal Schwann cells occupied only 2–3% of the
area occupied by SCPtrans-derived Schwann cells (Fig. 4E).
The difference between these two cells in their ability to
survive without forming myelin is, however, even more
clear-cut than these experiments suggested. This was shown
by immunolabelling the transplants with P0 antibodies to
reveal Schwann cell myelin. We found that the large
majority of the post-natal Schwann cells that still survived
2 months after transplantation had, in fact, formed myelin
sheaths, presumably around axons that had lost myelin
due to the trauma associated with cell injection (Fig. 4F).
In contrast, in the case of SCPtrans-derived Schwann cells,
areas associated with P0þ myelin sheaths constituted only
a small fraction of the implant area (Fig. 4G).
These results show that post-natal Schwann cells are
unable to survive for significant periods in the uninjured
white matter in agreement with previous reports, a feature
that by itself would be sufficient to significantly limit their
ability to migrate in the CNS. More importantly, the
present study also reveals that SCPtrans-derived Schwann
cells behave quite differently, since these cells thrive for
long periods among white matter oligodendrocytes, without
themselves forming myelin sheaths.
SCPtrans -derived Schwann cells, unlike
post-natal Schwann cells, integrate with
injured CNS tissue
Fig. 4 SCPtrans-derived Schwann cells survive much better than
post-natal Schwann cells in normal CNS tissue. Schwann cells and
SCPs were transplanted into the normal white matter at spinal
cord segment T13 and 1 and 2 months later, the spinal cords were
processed for frozen sections. The sections were immunolabelled
for oligodendrocyte myelin using MBP antibody. (A, B) Post-natal
Schwann cells survive poorly in the normal white matter at 1
month (Açtransverse section of the spinal cord) and 2 months
(Bçparasagittal section of the spinal cord), with only very few
GFPþ cells (green) seen in the white matter. MBP
immunolabelling (red) shows oligodendrocyte myelin. (C, D)
SCPtrans-derived Schwann cells survive well in normal white matter
and at 1 month (Cçtransverse section of the spinal cord) and
2 months (Dçparasagittal section of the spinal cord), with a large
number of GFPþ cells (green) present in the white matter.
MBP immunolabelling (red) shows oligodendrocyte myelin.
(E) Quantification of the number of surviving cells in normal white
matter after 1 and 2 months following transplantation. The areas
containing GFPþ cells were quantified using an image analysis
software (Image J, USA) and compared in animals transplanted
with post-natal Schwann cells and SCPs. At 1 and 2 months,
post-natal Schwann cells occupy an area of about 72.5 32.5 and
108.9 60.7 pixels, respectively and SCPtrans-derived Schwann cells
occupy an area of about 3297.6 226.7 and 2945 767.1 pixels,
respectively (n ¼ 6, P50.001; denotes significant difference).
The difference in areas occupied by SCPtrans-derived Schwann cells
at 1 and 2 months was not statistically significant. (F, G) Frozen
sections of spinal cords 2 months after transplantation were
immunolabelled with P0 antibody. (F) Most of the surviving
The success of myelin repair will be determined not only by
the relationship between implanted cells and oligodendrocytes, but equally by the interactions between such cells and
astrocytes. What we know about the interactions of
Schwann cells and astrocytes is complex and sometimes
contradictory (Franklin and Barnett, 1997). Nevertheless,
a large body of observations shows that these cells intermix
poorly and that their presence in CNS tissue is often
mutually exclusive, indicating that astrocytes, which are
present in many MS lesions, are potentially significant
inhibitors of Schwann cell re-myelination.
To determine whether SCPs interfaced with astrocytes
behave more favourably than Schwann cells, we labelled
astrocytes, using GFAP antibodies and examined the
interface between implanted cells and host tissue,
2 months after injection of SCPs or Schwann cells into
de-myelinating EB lesions (as before). When examining the
Schwann cell implants, we found, as reported previously,
that essentially all the cells remained within the lesion and
that a clearly defined boundary was established between
them and the surrounding astrocytes with minimal
intermixing between the two cell types (Fig. 5A).
The interface between SCPtrans-derived Schwann cells and
post-natal Schwann cells (green) are associated with P0þ myelin
(red). However, only very few of the SCPtrans-derived Schwann cells
(green) are associated with P0þ myelin (red).
Schwann cell precursors
Brain (2007), 130, 2175^2185
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5 µm
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Schwann cells, entered host tissue and spread among the
host myelinated axons forming an extensive area around
the implant where host and transplanted cells associated
closely with each other (Fig. 5D).
The results indicate that the implantation of SCPs results
in the generation of Schwann cell-like cells in the CNS
that show no adverse interactions with the astrocytes or
oligodendrocytes of the host tissue, but migrate freely
among them. In strictly comparable experiments, implanted
post-natal Schwann cells fail to mix with the cells of the
host tissue.
The migration rates of post-natal and
SCPtrans -derived Schwann cells in the CNS
20 µm
20 µm
Fig. 5 SCPtrans-derived Schwann cells intermix with
astrocytes and integrate with host white matter, while post-natal
Schwann cells do not. Schwann cells and SCPs were transplanted
into an EB lesion and 2 months later, the spinal cords were
processed for frozen sections. These were immunolabelled with
either GFAP antibody, which labels astrocytes, but not Schwann
cells using the present protocol, due to the lower GFAP content
of Schwann cells (A, B) or MBP antibody, which, in this assay, labels
oligodendrocyte myelin but not Schwann cells because of the
lower MBP levels in Schwann cell myelin (C, D). (A) Confocal
showing that post-natal Schwann cells (green) remain separated
from the GFAPþ tissue (red) around the lesion. The astrocytes and
Schwann cells exist as separate domains with minimal
intermingling. (B) Confocal picture showing that SCPtrans-derived
Schwann cells (green) intermingle with astrocytes. Between the
area containing only host astrocytes (redçextreme left) and
implanted cells (greençextreme right), there is an extensive region
where the two cell types mix, with many of the implanted cells
appearing to be in direct contact with astrocytic processes.
(C) Confocal picture showing post-natal Schwann cells (green)
within the lesion in the dorsal funiculus. MBP immunolabelling (red)
shows the presence of oligodendrocyte myelin. There is a clear
demarcation between the lesion area (green) and the normal CNS
tissue (red). (D) Confocal picture showing SCPtrans-derived
Schwann cells (green) at the border between the implant and
host tissue (red). Note that many of the transplanted cells are
able to integrate into the normal CNS tissue and there is no
clear demarcation between the lesion area and the normal CNS
tissue.
astrocytes was quite different, since it showed extensive and
intimate intermixing between GFPþ cells and astrocytes
(Fig. 5B).
Close observation of the border between these implants
into EB lesions and myelinated white matter tracts revealed
a similar pattern. Schwann cell implants showed a clear
border, with minimal entry into MBPþ tissue of the
transplanted GFPþ post-natal Schwann cells, all of which
remained within the lesion (Fig. 5C) whilst SCPtrans-derived
The ability to migrate through the normal CNS is likely to
be an important attribute of a myelin repair cell.
We therefore obtained a quantitative measure of cell
migration rate in the spinal cord following implantation
of Schwann cells and SCPs, and compared the two cell
types.
The cells were injected in the normal, unlesioned dorsal
funiculus at T13 and 1 and 2 months later, the spinal cords
were processed for frozen sections. Serial transverse sections
of the cord were collected on coverslips, covering the entire
rostro-caudal stretch containing GFPþ cells. The total
number of sections containing GFPþ cells was counted
and the length of spread calculated by multiplying the
number of sections with a factor of 20 mm (thickness of
sections).
Post-natal Schwann cells remained as a focus of cells that
showed very limited evidence of migration following
transplantation. The length of spread measured 0.7 0.1
and 0.9 0.2 mm at 1 and 2 months post transplantation,
respectively. SCPtrans-derived Schwann cells, on the other
hand, had spread 3.7 0.1 mm at 1 month to 7.7 0.3 mm
at 2 months post transplantation (Fig. 6). An important
aspect of these results is the finding that SCPtrans-derived
Schwann cells retain their migratory potential over time, as
shown by the difference in spread at 1 and 2 months. Using
this parameter to calculate migration rate, SCPtrans-derived
Schwann cells migrated at 2 mm/month while post-natal
Schwann cells migrated at 0.1 mm/month.
SCPtrans -derived Schwann cells readily
migrate and myelinate axons in the
astrocyte-rich environment of the retina
There is substantial evidence that astrocytes have the
potential to compromise the ability of Schwann cells to
myelinate CNS axons (Franklin and Blakemore, 1993;
Blakemore, 2005). This is not a significant factor in the
EB lesions we studied because they do not contain
astrocytes. However, it would be important in MS lesions,
which do contain astrocytes. To determine whether
SCP-derived Schwann cells were beset by the same
2182
Brain (2007), 130, 2175^2185
A. Woodhoo et al.
SCP injection. We therefore used GFP fluorescence to
examine the distribution of implanted cells in the retina,
6 weeks after injection. Although these observations were
not quantified, it was obvious that SCPtrans-derived
Schwann cells were present in larger numbers and covered
substantially larger area of the retina than post-natal
Schwann cells. Representative examples are shown in
Fig. 7D and F.
These experiments suggest that the astrocytic environment of the retina hinders effective migration and
myelination by post-natal Schwann cells, in agreement
with many previous observations of adverse interactions
between these cell types. On the other hand astrocytes do
not appear to hinder migration or myelination by SCPtransderived Schwann cells. This could confer an important
advantage on SCPs as myelin repair cells in the context
of MS.
Fig. 6 Evidence for cell migration following Schwann cell and SCP
transplantation in normal white manner. The length of spread of
SCPtrans-derived and post-natal Schwann cells after 1 and 2 months
following transplantation into the normal dorsal funiculus was
measured. Note that SCPtrans-derived Schwann cells migrate much
further than post-natal Schwann after 1 month and that they
continue a significant migration between the 1 and 2 months
(n ¼ 3, P50.005, denotes level of significance).
problems, we examined myelination in the retina. In this
tissue, an astrocyte-rich matrix envelops a network of
unmyelinated axons, and the preparation has the additional
advantage that myelination can be quantified (Setzu et al.,
2004, 2006).
Schwann cells and SCPs were injected in the retina of
adult rats and after 4 and 6 weeks, the retinae were
dissected out, flat-mounted and immunolabelled with P0
antibody to reveal the extent of myelination. All areas
showing P0 immunoreactivity were acquired by confocal
microscopy and quantified. Although little myelination was
seen at 4 weeks, the SCPtrans-derived Schwann cells had
formed about twice as much myelin as the post-natal
Schwann cells (Fig. 7A). Remarkably, at 6 weeks the
amount of myelin formed by SCPtrans-derived Schwann
cells had increased about 18-fold (Fig. 7A). At the same
time myelin formed by post-natal cells increased by only
about 2-fold. Thus, at 6 weeks, nearly 20 times more
myelin was formed in the retina following injection of
SCPs, compared to that seen when Schwann cells
were used.
One reason for this striking difference might be
differential migration. Even though an equal number of
SCPs and Schwann cells were injected, the results from our
experiments in the spinal cord (above) predicted that the
cells would spread more extensively in the retina following
SCPtrans -derived Schwann cells migrate
through CNS tissue to reach a demyelinated
lesion where they provide new myelin
The experiments above show that following CNS transplantation, SCP-derived cells possess a combination of
features not found in post-natal Schwann cells, namely the
ability to remyelinate CNS axons and an ability to survive
and integrate well in host CNS tissue. An additional and
important feature of an ideal myelin-repair cell would be an
ability to migrate towards demyelinated lesions to populate
and myelinate axons in more than one focus of demyelination in MS. Therefore, we investigated how SCPs and
post-natal Schwann cells compared in their ability to
migrate through normal CNS tissue towards a focal
demyelinated lesion and provide new myelin within the
lesion. An EB lesion was created in the ventral region of the
spinal cord near the ventral roots and Schwann cells and
SCPs were then transplanted at a significant distance from
the lesion in the dorsal funiculcus 10 mm rostral to the
plane of the EB lesion. The animals were sacrificed 1 month
later and GFP fluorescence used to examine the distribution
of the transplanted cells in frozen transverse sections of the
spinal cord. As expected, the post-natal Schwann cells
survived poorly in the spinal cord and no cells were seen
present in the lesion site (Fig. 8A). SCPtrans-derived
Schwann cells, on the other hand, survived well and were
seen apparently migrating towards the lesion site and
populating it (Fig. 8B). The cells that had reached the
lesions were able to remyelinate the injured axons as
shown by P0 immunoreactivity (Fig. 8C).
These experiments, together with the studies described in
previous sections, show that SCPtrans-derived Schwann cells
differ remarkably from post-natal Schwann cells in number
of aspects that are likely to be relevant for defining
the phenotype of a promising myelin-repair cell in the
context of MS.
Schwann cell precursors
Brain (2007), 130, 2175^2185
2183
Discussion
Fig. 7 SCPtrans-derived Schwann cells perform substantially better
than post-natal Schwann cells in (i) myelinating retinal axons and
(ii) migrating in the astrocyte-rich environment of the retina.
Schwann cells and SCPs were injected into the retina and 4 and
6 weeks later, the retinae were dissected out, flat-mounted,
immunolabelled with the Schwann cell myelin marker P0.
(A, B) Myelination: all areas containing P0þ myelinating cells
were acquired using a confocal microscope and the total area of
myelination quantified. (A) P0þ -myelinated segments from a
retina transplanted with SCPs 6 weeks earlier. (B) Graph
showing the total amount of P0þ myelin formed by post-natal
Schwann cells and SCPtrans-derived Schwann cells after 4 and
6 weeks following transplant. At 6 weeks, the area
myelinated by SCPtrans-derived Schwann cells is nearly 20 -fold
larger than that formed by post-natal Schwann cells (n ¼ 5, P50.05,
denotes significant level of difference). (C^F) Migration: picture
montages show the distribution of GFPþ cells in flat-mounts of
radial segments of the retina extending from the injection sites
(white arrows) to the retinal edges (arrowheads). The tissue was
dissected out 6 weeks after transplantation. Post-natal Schwann
cells (C, Brightfield; D, GFP fluorescence) are mostly found near
the point of injection and the cells do not migrate great distances
In the present experiments, we have implanted two stages
of the Schwann cell lineage, SCPs and immature Schwann
cells (Jessen and Mirsky, 2005), into the CNS under various
experimental conditions. Examination 1 and 2 months
later shows that when presented with naked CNS axons,
extensive myelin is formed in both types of implant by cells
that antigenically and ultrastructurally resemble myelinating
Schwann cells. In terms of every other parameter we have
studied, however, the cells behaved remarkably differently.
As expected from previous work (for refs, see ‘Introduction’
section), Schwann cell implants survive poorly unless the
cells find axons to myelinate, the cells do not migrate
significantly from the implantation site, fail to integrate
with host cells and form little myelin when challenged with
astrocyte-rich environment in the retina. Following SCP
implantation, on the other hand, the cells survive well,
migrate through normal CNS tissue, interface smoothly
and intimately with host glial cells and form extensive
myelin among the astrocytes of the retina.
These particular features of SCP implants are all likely to
be helpful attributes for a myelin repair cell. Since these
cells also form Schwann cell myelin that is arguably likely to
be resistant to MS pathology, they share some of the main
advantages of Schwann cells without suffering from
the disadvantages that render Schwann cells unsuitable
candidates for transplantation into MS lesions.
SCPs are the early glial cells of embryo day 14/15
(E14/15) rat nerves, and in vivo they convert to immature
Schwann cells during the next 2–4 days, so that by E17/18
the large majority of the glial cells are Schwann cells.
This transition is characterized by a number of phenotypic
changes including a strong up-regulation of cytoplasmic
S100b (Jessen and Mirsky, 2005). In the present experiments, the bulk of the implanted SCPs express S100b
1 month after the implantation and at that time they have
generated large numbers of P0þ myelinating Schwann cells
in demyelinated EB lesions. These cells also express nestin,
a protein found in the Schwann cell lineage, but not in
mature astrocytes or oligodendrocytes (Friedman et al.,
1990). Clearly, the SCPs survive the implantation, although
in peripheral nerves in vivo they are acutely dependent on
axon-associated neuregulin-1 for survival (Wolpowitz et al.,
2000; Winseck and Oppenheim, 2006). They progress to
generate cells that resemble Schwann cells, rather than CNS
glia. Nevertheless, the behaviour of these cells, referred to
here as SCPtrans-derived Schwann cells, differs from that of
Schwann cells generated in peripheral nerves that we
implanted in to the CNS in identical experiments
(above). To resolve this paradox, it would be of interest
over the retinal surface. CNS-generated Schwann cells
(E, Brightfield; F, GFP fluorescence) are able to migrate
throughout most of the surface of the retina and are present
at edges of the retinal flat-mount.
2184
Brain (2007), 130, 2175^2185
Fig. 8 SCPtrans-derived Schwann cells can, unlike post-natal
Schwann cells, migrate towards an area of focal demyelination in
the spinal cord and myelinate the demyelinated axons. An EB lesion
was created in the ventral region of the spinal cord near the
ventral roots and Schwann cells and Schwann cell precursors were
then transplanted in the dorsal funiculcus 10 mm rostral to the
plane of the EB lesion. One month later the spinal cords were
processed for frozen sections. (A) Transverse section of spinal
cord showing a few surviving post-natal Schwann cells, identified
by the expression of GFP (green), in the dorsal funiculus at
transplantation site (arrow), which do not migrate towards the site
of lesion (white circle). (B, C) Transverse section of spinal cord
showing numerous CNS-generated Schwann cells, identified by
the expression of GFP (green), in the dorsal funiculus at the
transplantation site (arrow). A great number of the cells are
present in the normal grey matter, apparently migrating towards
the site of lesion (white circle) (B). The transplanted GFPþ cells
(green) reach the lesion (C). In this high-power micrograph,
SCPtrans-derived Schwann cells, identified by green GFP
fluorescence, are seen making P0þ myelin sheaths (e.g. arrow)
around individual axons (e.g. asterisk).
in future studies to examine the time course of the
SCP/Schwann cell transition in the transplants and, perhaps
more importantly, to retrieve these Schwann cell-like cells
from the CNS and compare in detail their phenotype
with that of Schwann cells from neonatal nerves.
More generally, these observations suggest that glia,
neurons or stem cells that are implanted into the CNS for
reparative purposes may, in that alien environment, form
cell types that are significantly different from any normal
cells found in developing or adult organisms. The success of
such implants are likely to depend on how much of the
desirable functions of the implanted cells, in the present
case the ability to myelinate, is retained by the modified
‘novel’ cells formed within the CNS.
It has often been pointed out that Schwann cells, if used
for myelin repair, could be autografted, thereby avoiding
A. Woodhoo et al.
immune rejection. It is harder to see how this could apply
to the cells we describe here. While SCPtrans-derived
Schwann cells appear to have many advantages for myelin
repair, the SCPs that give rise to them are obtained
from embryonic nerves. This raises the questions of how
such cells could be obtained for implantation purposes.
At present, the answers to such questions are speculative
although not implausible. Progress is continuously being
made in defining more clearly the phenotypic differences
between SCPs and Schwann cells (Jessen and Mirsky, 2005)
and progress in this area will help identify the features
responsible for the advantageous behaviour of SCPs.
With that knowledge it is possible that nerve-derived
Schwann cells could be genetically modified prior to
implantation to acquire the relevant SCP phenotype.
Another route, perhaps more feasible in the short term, is
to employ cellular systems already developed for generating
Schwann cells in vitro from bone marrow cells (e.g. Keilhoff
et al., 2006). Refining these systems and adapting them
so that the developmental sequence is halted at the SCP,
rather than the Schwann cell, stage, should be possible.
Lastly, many recent demonstrations of unexpected cellular
plasticity (e.g. Collas, 2007) encourage the view that SCPs,
that can already be made to form Schwann cells in vitro,
can be generated in reverse, namely from Schwann cells.
The significance of the present work, in this context, is to
help defining the cellular phenotype that is likely to serve
well for myelin repair. This information, in turn, can form
the basis of efforts aimed at generating such cells in
quantities that make their use in myelin repair a practical
possibility.
Acknowledgements
This work was supported by a grant from the Multiple
Sclerosis Society of Great Britain and Northern Ireland.
We are very grateful to Dr M. Okabe for providing the
GFP-transgenic rats.
Conflict of interest statement. None declared.
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