progenitor cells generate myelinating oligodendrocytes and Schwann cells in spinal cord demyelination and dysmyelination

Experimental Neurology 213 (2008) 176–190

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r

Transplanted neural stem/progenitor cells generate myelinating oligodendrocytes and Schwann cells in spinal cord demyelination and dysmyelination Andrea J. Mothe a,⁎, Charles H. Tator a,b a b

Division of Genetics and Development, Toronto Western Research Institute, Krembil Neuroscience Centre, Toronto, ON, Canada M5T 2S8 Division of Neurosurgery, University of Toronto, ON, Canada M5S 1A8

a r t i c l e

i n f o

Article history: Received 17 January 2008 Revised 12 May 2008 Accepted 23 May 2008 Available online 10 June 2008 Keywords: Neural stem/progenitor cells Transplantation Spinal cord Myelination Focal demyelination lesions Shiverer mouse

a b s t r a c t Stem cell therapy is a promising approach for remyelination strategies in demyelinating and traumatic disorders of the spinal cord. Self-renewing neural stem/progenitor cells (NSPCs) reside in the adult mammalian brain and spinal cord. We transplanted NSPCs derived from the adult spinal cord of transgenic rats into two models of focal demyelination and congenital dysmyelination. Focal demyelination was induced by X-irradiation and ethidium bromide injection (X-EB); and dysmyelination was in adult shiverer mutant mice, which lack compact CNS myelin. We examined the differentiation potential and myelinogenic capacity of NSPCs transplanted into the spinal cord. In X-EB lesions, the transplanted cells primarily differentiated along an oligodendrocyte lineage but only some of the oligodendrocytic progeny remyelinated host axons. In this glial-free lesion, NSPCs also differentiated into cells with Schwann-like features based on ultrastructure, expression of Schwann cell markers, and generation of peripheral myelin. In contrast, after transplantation into the spinal cord of adult shiverer mice, the majority of the NSPCs expressed an oligodendrocytic phenotype which myelinated the dysmyelinated CNS axons forming compact myelin, and none had Schwann cell-like features. This is the first study to examine the differentiation and myelinogenic capacity of adult spinal cord stem/progenitors in focal demyelination and dysmyelination of the adult rodent spinal cord. Our findings demonstrate that these NSPCs have the inherent plasticity to differentiate into oligodendrocytes or Schwann-like cells depending on the host environment, and that both cell types are capable of myelinating axons in the demyelinated and dysmyelinated adult spinal cord. © 2008 Elsevier Inc. All rights reserved.

Introduction There is considerable interest in stem cell therapy for the treatment of the injured or diseased nervous system. The presence of neural stem/progenitor cells in the adult mammalian brain and spinal cord (Reynolds and Weiss, 1992; Weiss et al., 1996) has suggested their potential therapeutic application. Adult neural stem/ progenitor cells (NSPCs) are self-renewing and multipotent, capable of generating both neurons and glia in vitro (Reynolds and Weiss, 1992; Weiss et al., 1996). When cultured in the presence of growth factors they form neurospheres which are free-floating colonies of cells primarily composed of progenitor cells andb1% stem cells (Morshead et al., 1994). Transplantation of NSPCs derived from the adult rodent spinal cord or subventricular zone of the forebrain produced limited functional recovery after spinal cord injury (Vacanti et al., 2001; Hofstetter et al., 2005; Karimi-Abdolrezaee et al., 2006; Pfeifer et al., 2006). In the spinal cord, NSPCs reside close to the ependymal/ periventricular region since multipotential self-renewing neurospheres were generated only when the cultured tissue included ⁎ Corresponding author. E-mail address: [email protected] (A.J. Mothe). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.05.024

parts of the central canal (Martens et al., 2002). In lower vertebrates, ependymal cells rapidly proliferate and differentiate into neurons and glia to regenerate the transected cord (Nordlander and Singer, 1978; Dervan and Roberts, 2003). In adult mammals, ependymal cells proliferate in response to several types of spinal cord trauma (Vaquero et al., 1981; Bruni and Anderson, 1987; Wallace et al., 1987; Beattie et al., 1997; Johansson et al., 1999; Namiki and Tator, 1999; Takahashi et al., 2003; Mothe and Tator, 2005; Horky et al., 2006). However, endogenous NSPCs appear to have only limited regenerative capacity for repair. Increasing cell numbers through transplantation may enhance this regenerative potential. Stem cell therapy may be particularly effective for remyelination in diseases or injuries associated with demyelination. Recently, we reported that NSPCs derived from the periventricular region of the adult spinal cord showed an intrinsic capacity for predominant oligodendrocytic differentiation without targeted manipulation by neurotrophic factors or other agents (Kulbatski et al., 2007). The current study examines the differentiation potential and myelinogenic capacity of adult spinal cord NSPCs transplanted into the spinal cord of two complementary models of demyelination, focal demyelination induced by X-irradiation/ethidium bromide (X-EB) which leaves a population of demyelinated axons in a glial-free environment

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(Blakemore, 1982; Crang et al., 1992), and adult shiverer mutant mice which carry a spontaneous mutation of myelin basic protein (MBP) resulting in a lack of central myelin, a model of congenital dysmyelination with a long-term irreversible myelin deficiency (Privat et al., 1979; Chernoff, 1981). Although the myelination potential of many cell types has been examined in experimental studies (Radtke et al., 2007), to our knowledge, this is the first study to examine the differentiation and myelinogenic capacity of adult spinal cord stem/ progenitors in focal demyelination and dysmyelination of the adult rodent spinal cord. Our findings demonstrate that spinal cord NSPCs differentiate into oligodendrocytes or Schwann-like cells depending on the host environment, and that these cells are capable of myelinating axons in the demyelinated and dysmyelinated adult rodent spinal cord. Materials and methods Isolation and culture of adult periventricular spinal cord NSPCs NSPCs were isolated from the spinal cords of transgenic adult Wistar rats expressing enhanced green fluorescent protein (GFP) (Wistar-TgN(CAG-GFP)184ys) (YS Institute Inc., Utsunomiya, Tochigi, Japan). The GFP transgene is driven by chicken β-actin promoter and cytomegalovirus enhancer (Hakamata et al., 2001). NSPCs isolated from these rats stably express the transgene long term both in vitro and in vivo (Mothe et al., 2005). The isolation and generation of periventricular neurospheres were performed based on methods described previously (Kulbatski et al., 2007). The rat cervical and thoracic spinal cord was excised under sterile conditions and washed in Dulbecco's phosphate-buffered saline supplemented with 30% glucose (Sigma-Aldrich, Oakville, Ontario) and 1% penicillin/streptomycin (Sigma-Aldrich, Oakville, Ontario). The overlying meninges, blood vessels, and white matter were removed so the periventricular region including the ependymal, subependymal, and some gray matter tissue surrounding the central canal were harvested. The dissected tissue was cut into 1 mm3 pieces, enzymatically dissociated in a papain dissociation enzyme solution (Worthington Biochemicals, New Jersey) containing 0.01% papain and 0.01% DNase I for 1 h at 37 °C, and then mechanically dissociated into a cell suspension which was centrifuged using a discontinuous density gradient to remove cell membrane fragments. Cells were resuspended in Neurobasal-A medium (Gibco-Invitrogen, Burlington, Ontario) supplemented with B27 (Gibco-Invitrogen, Burlington, Ontario), L-glutamine (GibcoInvitrogen, Burlington, Ontario), penicillin/streptomycin (Gibco-Invitrogen, Burlington, Ontario), 20 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich, Oakville, Ontario), 20 ng/ml fibroblast growth factor-2 (FGF2) (Sigma-Aldrich, Oakville, Ontario), 2 µg/ml heparin (SigmaAldrich, Oakville, Ontario), and hormone mix consisting of 1:1 DMEM/ F-12, 0.6% glucose, 25 µg/ml insulin, 100 µg/ml transferrin, 5 mM HEPES, 3 mM sodium bicarbonate, 30 nM sodium seleniate, 10 µM putrescine, and 20 nM progesterone (all from Sigma-Aldrich). Cell viability was assessed with trypan blue staining, and cells were seeded at a density of 20 cells/µl in Nunc T25 culture flasks (VWR International, Mississauga, Ontario). The neurospheres generated were passaged weekly by mechanical dissociation in serum-free medium described above. In vitro immunocytochemistry Neurospheres were assessed for self-renewal and multipotentiality according to published protocols (Tropepe et al., 1999). Neurospheres were mechanically dissociated into single cells and plated on Matrigel (BD Biosciences, Mississauga, Ontario) coated glass coverslips in multi-well culture plates (50,000 cells per well) in growth factor free media containing 1% fetal bovine serum (FBS). The cultures were grown for 7 days to induce differentiation and then fixed with 4%

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paraformaldehyde (PF) and washed three times with 0.1 M phosphate-buffered saline (PBS). For characterization of undifferentiated cultures, neurospheres were dissociated into single cells and plated for 2 h in serum-free growth factor medium before fixation as described above. In some cases, undissociated whole neurospheres were plated (Fig. 1 nestin and Ki67 immunostaining). After fixation and washing, the cultures were blocked with 10% normal goat or donkey serum in PBS or 0.3% Triton-X 100, (depending on the antibody) for 1 h at room temperature and then incubated with the primary antibody overnight at 4 °C. The following primary antibodies were used: mouse anti-nestin (1:100; BD Biosciences Pharmingen, Mississauga, ON, Canada) for neural stem/progenitor cells, mouse anti-Ki67/MM1 (1:100; Novocastra Laboratories, Newcastle, UK) for proliferating cells, mouse anti-GFAP (1:200; Chemicon, Temecula, CA) for astrocytes, mouse anti-RIP (1:5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and mouse anti-MBP (1:1000; Sternberger Monoclonals, Lutherville, MD) for oligodendrocytes, mouse anti-βIII tubulin (1:500; Covance Research Products, Berkeley, CA) for neurons, mouse anti-p75 (1:100; Chemicon) for Schwann cells, and mouse anti-P0 (1:300; kind gift from Dr. Juan Archelos, Medical University, Graz, Austria) for myelinating Schwann cells. Cultures were washed three times with 0.1 M PBS and then incubated with fluorescent Alexa 568 goat anti-mouse secondary antibody (1:500; Invitrogen) for 1 h, washed three times with PBS, and coverslipped with Vectashield hardset mounting media containing DAPI (4′, 6-diamidino-2-phenyl-indole) (Vector Laboratories, Burlingame, CA) to counterstain the nuclei. Images were taken using a Zeiss LSM 510 laser confocal microscope. Demyelinating lesion and transplantation of spinal cord NSPCs All animal procedures were approved by the animal care committee of the University Health Network in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care. Focal demyelination was induced chemically in the adult rat spinal cord with ethidium bromide (EB lesion) and X-irradiation to suppress endogenous remyelination (X-EB lesion) (Blakemore, 1978, 1982; Crang et al., 1992), resulting in a population of demyelinated axons in a glial-free environment (Franklin et al., 1991). A total of 39 adult female Wistar rats (200 g; Charles River Laboratories, St. Constant, QC) were used in the focal demyelination study, EB lesions in 23 rats, and X-EB lesions in 16 rats. Rats were anesthetized by inhalation of 5% halothane which was reduced to 2% during surgery, in combination with a mixture of nitrous oxide and oxygen (1:2, v/v). For the EB lesions, the spinal cord was exposed by laminectomy at the T8/9 vertebral level, and a small opening was made in the dura with a sterile 30 gauge needle. A 1.0 µl injection of 0.1% EB was delivered unilaterally at the stereotactic coordinates for the ventrolateral funiculus (VLF) (0.7 mm lateral to midline and depth of 1.5 mm) of the thoracic spinal cord, as described previously (Talbott et al., 2006). With the aid of an operating microscope, the EB was stereotactically injected at a rate of 0.5 µl/ min using a Hamilton syringe with a 32 gauge customized needle and a motorized microinjector (Model 780310; Stoelting, IL). The X-EB rats had focal X-irradiation 3 days prior to the EB lesion to block endogenous remyelination as previously described (Crang et al., 1992; Akiyama et al., 2001). Animals were anesthetized by inhalation of 2% halothane and a 40 Gy surface dose of X-irradiation was focused along the thoracic cord encompassing the T8/9 vertebral level through a 2 cm diameter opening in a 4 mm thick lead shield (100 kV, 10 mA, dose rate 510.2 cGy/min). After irradiation, animals were given 5 ml saline (s.c.) and housed in a 26 °C warm room. All transplantations were performed 3 days after the EB lesion, as previously reported (Liu et al., 2000; Akiyama et al., 2001; Blakemore et al., 2003; Sasaki et al., 2006). Following induction of anesthesia, the

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Fig. 1. Adult rat spinal cord NSPCs are multipotential in vitro. NSPCs isolated from the periventricular region of the spinal cord from an adult GFP transgenic rat were grown as freefloating neurospheres in uncoated tissue culture flasks. The neurospheres were dissociated weekly into single cells and passaged for expansion. Neurospheres from passages 3 or 4 were characterized in vitro and used for transplants into the focal demyelination lesion in the rat and into the shiverer mouse spinal cord. GFP positive neurospheres expressed nestin (A–C), a marker for neural stem/progenitor cells, and Ki67 (D–F), a marker for proliferating cells. Neurospheres were dissociated into single cells and plated onto a Matrigel™ substrate in the presence of 1% FBS and in the absence of EGF and FGF2. Immunocytochemistry on these cultures 1 week after plating, showed differentiation into GFAP positive astrocytes (G–I), βIII tubulin positive neurons (J–L), and RIP positive (M–O) and MBP positive (P–R) oligodendrocytes. Double-labeled cells are shown in the merged panels.

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T8/T9 laminectomy site was re-exposed. NSPCs were transplanted as neurospheres (diluted in growth medium) after 4–6 days in vitro in passages 3 or 4. The NSPCs were transplanted as neurospheres because we found better survival than when transplanted as dissociated cells (Mothe et al., in press). The viability of cells within a sphere in vitro was N95% as determined with propidium iodide staining. Using the motorized microinjector and operating microscope, a 1 µl volume of neurospheres equivalent to 100,000 cells (determined from neurosphere dissociation and trypan blue staining) or a 1 µl volume of media (control) was delivered at a rate of 0.5 µl/min directly into the EB lesion using the same stereotactic coordinates as described above. The needle was left in place for an additional 2 min to prevent back-flow of cells. To aid transplant survival and integration, animals were immunosuppressed daily until sacrifice with 15 mg/kg of cyclosporine (Sandimmune, Novartis, Dorval, QC, Canada) injected subcutaneously. Rats were sacrificed at 1 week (n = 8 EB lesions and n = 4 X-EB lesions) or 3 weeks (n = 10 EB lesions and n = 7 X-EB lesions) for immunohistochemical analysis, and 3 weeks (n = 5 EB lesions and n = 5 X-EB lesions) for ultrastructural analysis. At 3 weeks posttransplantation, GFP+ cells were detected in 10/10 EB rats and 6/7 X-EB rats. Cell survival was quantified in 4 rats which showed the highest number of transplanted cells. Cell survival averaged 6.2% in EB lesions and 4.1% in X-EB lesions. Transplantation into shiverer mice Adult spinal cord NSPCs were also transplanted into the spinal cord of adult shiverer mutant mice which carry a spontaneous mutation of MBP resulting in lack of central myelin (Privat et al., 1979; Chernoff, 1981). A total of 12 young adult (6–8 weeks of age) homozygous shiverer mice (C3Fe.SWV-Mbp-shi/J; Jackson Laboratory, Bar Harbor, ME) were used. Mice were anesthetized by inhalation of 1.5% halothane in combination with a mixture of nitrous oxide and oxygen (1:2, v/v). A T6-T8 laminectomy was performed, and a small opening was made in the dura with a sterile 30 gauge needle. As described above for the transplants into focal demyelination lesions, adult rat spinal cord NSPCs were transplanted as neurospheres at passages 3 or 4 (4–6 days in vitro) into the spinal cord of shiverer mice. With the aid of an operating microscope, a 1 µl volume of neurospheres equivalent to 300,000 cells (determined from neurosphere dissociation and trypan blue staining), was delivered at a rate of 0.5 µl/min using a Hamilton syringe with a 32 gauge customized needle and the motorized microinjector. One injection was made into the dorsal spinal cord, immediately adjacent to the midline to avoid the midline dorsal vein, and at a depth of 1 mm. The needle was left in place for an additional 2 min to prevent back-flow of cells. To aid transplant survival and integration, mice were immunosuppressed daily until sacrifice with 20 mg/kg of cyclosporine (Sandimmune, Novartis, Dorval, QC, Canada) injected subcutaneously. Mice were sacrificed at 1 week (n = 2), 2 weeks (n = 2), 3 weeks (n = 6), and 4 weeks (n = 2). In initial pilot studies with a group of 5 mice following transplantation of 100,000 NSPCs with no immunosuppression, GFP+ cells were detected in only one mouse and cell survival was extremely low (0.09%). Therefore, we increased the number of transplanted cells to 300,000 and administered 20 mg/kg cyclosporine daily since mice metabolize faster than rats and this level of immunosuppression has been used effectively in mice (McKenzie et al., 2006). Under these conditions, GFP+cells were detected in 8/12 mice with an average of 0.3% cell survival. Tissue processing and immunohistochemistry Animals were sacrificed with a lethal dose of sodium pentobarbital, followed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Spinal cords were dissected and the tissue cryoprotected in 30% sucrose. In the rats, a

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0.8 cm segment of spinal cord encompassing the transplant site was embedded in Shandon Cryomatrix compound (VWR Laboratories, Mississauga, ON, Canada), and cryosectioned transversely into 20 µm serial sections collected on Superfrost slides (Fisher Scientific, Ottawa, ON, Canada). Every eighth section was stained with luxol fast blue and hematoxylin and eosin (LFB/H and E) for myelin and general morphology. In the mice, the rostro-caudal segment of the spinal cord 1–1.5 cm in length encompassing the transplant site was embedded and cryosectioned longitudinally in the horizontal plane from the dorsal surface to the ventral surface into 20 µm serial sections. For immunostaining, sections were rehydrated in 0.1 M PBS and permeabilized and blocked with 0.3% Triton-X 100, 10% normal goat or donkey serum in PBS or 0.3% Triton-X 100, 5% nonfat milk, 1% BSA in PBS (depending on the antibody) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C, washed three times, and then incubated with fluorescent-conjugated secondary antibodies for 1 h at room temperature. The following primary antibodies were used: mouse anti-Ki67/MM1 (1:100; Novocastra Laboratories, Newcastle, UK) for proliferating cells, mouse anti-nestin (1:100; BD Biosciences Pharmingen, Mississauga, ON, Canada) for neural stem/progenitor cells, rabbit anti-NG2 (1:800; Chemicon, Temecula, CA) for glial progenitors, mouse anti-GFAP (1:200; Chemicon) for astrocytes, mouse anti-MAP2 (1:500; Chemicon) and mouse anti-NeuN (1:500; Chemicon) for neurons, mouse anti-NF200 (NF) (1:500; Sigma) for axons, mouse anti-CC1/APC (1:1000; Calbiochem, San Diego, CA) and mouse anti-MBP (1:1000; Sternberger Monoclonals, Lutherville, MD) for oligodendrocytes, goat anti-Olig2 (1:500; R and D Systems, Minneapolis, MN) for oligodendrocyte lineage cells, mouse anti-p75 (1:100; Chemicon) and mouse anti-P0 (1:300; kind gift from Dr. Juan Archelos, Medical University, Graz, Austria) for Schwann cells. Secondary antibodies used were as follows: Alexa 568 goat anti-mouse, Alexa 568 donkey anti-goat, Alexa 568 goat anti-rabbit, and Alexa 647 goat anti-mouse (1:500; all from Invitrogen). In triple labeling for GFP/CC1/NF, GFP/MBP/CC1, GFP/MBP/ NF, or GFP/P0/NF, sections were incubated with mouse anti-CC1 antibody, MBP, or P0, followed by Alexa 568 secondary antibody incubation. Sections were then incubated with mouse anti-NF or CC1, followed by Alexa 647 secondary antibody. Slides were washed three

Fig. 2. Quantitation of immunopositive cells from dissociated neurospheres grown in undifferentiated and differentiating culture conditions. Most of the undifferentiated dissociated neurospheres were nestin positive (82.8 ± 3.0%), and very few positive for GFAP (0.4 ± 0.2%), RIP (8.6 ± 1.2%), or βIII tubulin (0.5 ± 0.3%). In contrast, dissociated neurospheres grown in differentiating culture conditions for 1 week showed small numbers of nestin positive (7.9 ± 2.2%), GFAP positive (11.6 ± 3.7%), and βIII tubulin positive cells (13.1 ± 2.2%), but a high incidence of RIP positive cells (60.3 ± 19.1%). Error bars indicate SD.

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Fig. 3. EB-induced demyelination in the lateral funiculus of the spinal cord. Stereotactic microinjection of EB into the lateral funiculus resulted in a discrete focal demyelinated lesion, as shown with luxol fast blue and hematoxylin and eosin (LFB/H and E) staining indicating a loss of myelin throughout the lesion site (A, outlined) after 1 week. EB is toxic to both GFAP positive astrocytes (B) and CC1 positive oligodendrocytes (C) (3 weeks after lesion induction). Ultrastructural analysis shows that after 3 weeks in an X-EB rat that was not transplanted, most of the axons remained demyelinated (D). Low magnification image of DAPI stained spinal cord cross-section showing GFP positivity from the engrafted NSPCs 1 week following transplantation into an EB lesion (E, outlined).

times with PBS and then coverslipped with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) nuclear counterstain. Species specific non-immune IgG was used as negative controls, in addition to tissue controls where available, such as p75/P0 positive roots and negative spinal cord or MBP deficient shiverer spinal cord. Immunofluorescent tissue was examined using a Zeiss LSM 510 confocal microscope. Electron and immunoelectron microscopy Rats were transcardially perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (PB). The spinal cords were rinsed in 0.1 M PB and embedded in 4% agar (agarose A, biotechnology grade, Rose Scientific, Edmonton, AB) for vibratome sectioning. Freefloating sections were obtained at 100 µm thickness and then processed for immunoperoxidase staining. The sections were washed

in PBS three times and then blocked for endogenous peroxidase with 1% hydrogen peroxide for 20 min. After blocking with 2% normal goat serum with 1% bovine serum albumin for 1 h, sections were incubated with rabbit anti-GFP antibody (Chemicon, Temecula, CA) at 1:200 overnight at 4 °C. After three PBS washes, sections were incubated with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) at 1:400 for 2 h, followed by avidin-biotin peroxidase complex (Vectastain Elite ABC Kit Standard, Vector Laboratories) for another 2 h at room temperature. After 3 washes, sections were reacted with 0.04% diaminobenzidine and then washed 3 times. The sections were osmicated with 1% osmium tetroxide in PBS for 1 h at room temperature. Sections were then dehydrated in a graded series of ethanol (50% through 100% ethanol) with 3 changes of 100% ethanol, each for 10 min, followed by 2 changes of propylene oxide, and then progressively infiltrated with Epon-Araldite resin. The sections were embedded and polymerized in a 70 °C vacuum oven

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overnight, and 1 µm thick sections were cut with an ultramicrotome (Reichert-Jung Ultracut E, Vienna, Austria), and stained with 0.5% toluidine blue. Subsequently, 70 nM thin sections were collected and mounted on copper mesh grids, and unstained sections were examined with a transmission electron microscope (JEOL 1230, Akishima, Japan) at 80 kV. Other tissue specimens were processed for immunoelectron microscopy (IEM) with glutaraldehyde postfixation, as described previously with modifications (Wu et al., 2002). Frozen 20 µm cryostat sections mounted on glass slides were thawed and washed three times in PBS and processed as described above. Following the avidin-biotin peroxidase application, sections were washed 3 times and then postfixed with 1% glutaraldehyde for 10 min at room temperature. After 3 washes, the sections were reacted with diaminobenzidine then removed from the glass slides with the aid of a razor blade. The sections were then osmicated and processed as described above. Similar results were obtained with both methods. Negative controls included sections from rats that did not receive cell transplants and sections from rats that received cell transplants with omission of the primary antibody. Spinal cord tissue from shiverer mice was processed for electron microscopy as described above and previously with modifications (Wu et al., 2002). Frozen 20 µm cryostat sections mounted on glass slides were thawed and washed in PBS and postfixed with 1%

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glutaraldehyde for 10 min at room temperature. After washing, the sections were removed from the glass slides with the aid of a razor blade, and osmicated with 1% osmium tetroxide in PBS for 1 h at room temperature. Sections were then dehydrated and then progressively infiltrated with Epon-Araldite resin, as described above. The sections were embedded in Beem capsules and polymerized in a 70 °C vacuum oven overnight. Thin sections were cut with an ultramicrotome and sections were examined with a transmission electron microscope (JEOL 1230, Akishima, Japan) at 80 kV. Quantitative analysis To quantify the differentiation pattern in vitro, the number of immunopositive cells for each antibody was counted as a percentage of GFP/DAPI positive cells in 10 random fields (n = 3). Fluorescent cells were examined and images were taken using a Zeiss LSM 510 laser confocal microscope. To quantify the differentiation pattern of transplanted cells, we used confocal microscopy to count the number of GFP/DAPI positive cells that were double-labeled with different cell markers. All transplanted cells in the EB lesion core and along the lesion border were quantitated. Double-labeled cells were examined in Z-series obtained with the Zeiss LSM 510 confocal microscope using multi-track scanning. We chose the spinal cord sections with the

Fig. 4. Adult spinal cord NSPCs primarily differentiate along an oligodendrocyte lineage. Confocal immunohistochemistry was performed on cross-sections of EB and X-EB lesions transplanted with GFP expressing spinal cord NSPCs. A, Low magnification image, at 1 week following transplantation into an EB lesion, shows astrocytes along the lesion periphery (denoted by line) extending long processes towards the lesion (right side of image). GFP positive transplanted cells express GFAP (A, arrow). B–D, High magnification image at 3 weeks after transplantation shows GFP+/GFAP+ cells (arrows) along the lesion perimeter. E, Low magnification image at 1 week following transplantation into an EB lesion, shows a large proportion of GFP positive cells located near the lesion border expressing CC1 (a marker for mature oligodendrocytes). The lesion area is outlined. F, High magnification of boxed area in E, shows many GFP positive transplanted cells expressing CC1. G, Higher magnification of boxed area in merged image in F showing GFP+/CC1+ cells (arrows indicate colocalization). H, Many GFP+/CC1+ double-labeled cells 3 weeks after grafting in an EB lesion. I–K, Many GFP positive transplanted cells located near the lesion border also express Olig2 (an oligodendroglial transcription factor) at 1 week following transplantation into an EB lesion (inset, higher magnification), and at 3 weeks (L) after transplantation into a X-EB lesion.

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Fig. 5. Oligodendrocytic progeny from GFP expressing spinal cord NSPCs transplanted into EB lesions ensheath demyelinated axons and generate MBP. Confocal images of triplelabeled cross-sections 3 weeks after transplantation, show (A–D), transplanted cells (GFP, green) expressing the oligodendrocyte marker CC1 (red) and ensheathing axons (identified by NF200 (NF) staining, blue). E–H, These images show 3 GFP positive profiles (GFP, green) ensheathing axons (NF, blue), 2 of which are also generating myelin (MBP, red). I–L, A GFP positive cell is shown (I, nuclei counterstained with DAPI, pseudocolored turquoise) ensheathing multiple axons (NF, blue) and generating myelin (MBP, red).

highest number of transplanted cells from four animals (n = 4). For each cell marker, we immunostained 15 sections per animal (140 µm apart). Three of these sections with the highest number of transplanted cells were examined for double-labeled cells (cells positive for both GFP and the specific antibody), and 3 regions per section were counted. The proportion of GFP positive cells that were double-labeled was calculated for each animal. Data are presented as mean ± standard deviation (SD). To quantify the number of transplanted cells, spinal cords (n = 4 rats, n = 3 mice) were cryosectioned at a thickness of 20 µm, and every section was collected. Sections were counterstained with DAPI. For each spinal cord, every eighth section throughout the thickness of the cord was analyzed, and all GFP positive cells containing a DAPI positive nucleus were counted. To determine the total number of surviving transplanted cells per animal, the number of GFP positive cells in all of the counted sections were multiplied by 8 to compensate for the sampling frequency. Results Adult spinal cord periventricular neurospheres are multipotent NSPCs were isolated from the periventricular region of the spinal cord from adult GFP transgenic rats and grown as free-floating neurospheres in uncoated tissue culture flasks. The neurospheres were cultured in the presence of the growth factors EGF and FGF2 and passaged weekly for expansion. Neurospheres from passages 3 or 4 were used in both the in vitro and in vivo experiments.

Figs. 1A–C shows GFP expression of a single sphere with high levels of nestin, a marker for neural/stem progenitor cells, demonstrating that the neurospheres comprised primarily undifferentiated neural stem/progenitors. Under these conditions in the presence of the growth factors, most of the stem/progenitor cells were proliferating, as shown with the proliferating cell marker, Ki67 (Figs. 1D–F). The same immunostaining results were obtained when the neurospheres were dissociated and single cells were plated on Matrigel in growth factor medium (data not shown). These neurospheres when dissociated, but not differentiated, also showed low levels of immunoreactivity for mature neural markers, such as GFAP for astrocytes (0.4 ± 0.2%), RIP for oligodendrocytes (8.6 ± 1.2%), and βIII tubulin for neurons (0.5 ± 0.3%) (Fig. 2). In contrast, when EGF and FGF2 were removed from the culture medium and replaced with 1% fetal bovine serum (FBS), and dissociated neurospheres were plated onto a Matrigel™ substrate for 1 week, the cells displayed a heterogenous morphology suggesting they were undergoing differentiation. Immunocytochemistry demonstrated that some of these cells acquired morphological and antigenic properties of astrocytes, as shown with GFAP (Figs. 1G–I; Fig. 2, GFAP 11.6 ± 3.7%), and neurons, as shown with βIII tubulin (Figs. 1J–L; Fig. 2, βIII tubulin 13.1 ± 2.2%). However, the majority of the cells were immunoreactive for the oligodendrocyte marker RIP (Figs. 1M–O; Fig. 2, RIP 60.3 ± 19.1%), and also expressed the myelin marker, myelin basic protein (MBP) in culture (Figs. 1P–R). Only a small percentage of these cells remained nestin positive (Fig. 2, nestin 7.9 ± 2.2%) in differentiating conditions in culture. The present results confirm previous observations in our laboratory and elsewhere

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Fig. 6. Adult spinal cord NSPCs transplanted into EB lesions express Schwann cell markers. At 1 week (A) and 3 weeks (B) after transplantation into an EB lesion, some GFP positive cells in the lesion core express p75, a marker for nonmyelinating Schwann cells. C, Low magnification image of GFP positive transplanted cells within the core of an EB lesion 3 weeks after transplantation showing many positive P0 profiles (marker for peripheral myelin and myelinating Schwann cells), indicating migration of endogenous Schwann cells into the lesion. D, Higher magnification of boxed area in C, showing some of the transplanted cells expressing P0 (red). E–H, Triple-labeled cross-sections of EB lesions 3 weeks after transplantation show transplanted cells (GFP, green) that are ensheathing axons (NF, blue) and generating peripheral myelin (P0, red).

that adult spinal cord NSPCs are multipotent, having the ability to differentiate into all three neural cell types in vitro (Weiss et al., 1996; Martens et al., 2002; Kulbatski et al., 2007), and also show a greater propensity for oligodendrocytic differentiation in culture. Adult spinal cord NSPCs transplanted into focal demyelination lesions primarily differentiate into oligodendrocytes Adult spinal cord NSPCs were subacutely (3 days post EB) transplanted into the lateral funiculus of the spinal cord in rats after either ethidium bromide (EB) or X-irradiated (40 Gy) EB lesions (XEB). Transplanted rats were sacrificed after 1 and 3 weeks, and tissue was examined histologically and ultrastructurally. Injection of EB into the lateral funiculus of the adult rat spinal cord consistently resulted in a focally demyelinated zone as shown by LFB/H and E staining of frozen sections from the lesion epicenter (Fig. 3A, lesion outlined). We confirmed that the lesion was devoid of astrocytes and oligodendrocytes as shown by a lack of GFAP and CC1 immunoreactivity (Fig. 3B and C, lesion outlined). Oligodendrocytes and reactive astrocytes were present at the perimeter of the lesion and sharply delineated the lesion from the adjacent uninjured white matter (Talbott et al., 2005). Electron microscopy at 3 weeks in a nontransplanted X-EB rat showed many viable demyelinated axons in the lesion (Fig. 3D). Fluorescence microscopy showed GFP positive cells engrafted within the EB lesion 1 week following transplantation (Fig. 3E). Confocal microscopy and immunostaining with cell type-specific markers were used to identify the phenotypes of the spinal cord NSPCs transplanted into EB and X-EB lesions, and cells transplanted into either type of lesion showed no differences in their phenotypic expression pattern. We found that only 1.9 ± 0.8% (Fig. 7 graph) of the GFP positive transplanted cells expressed GFAP, a marker for astrocytes (Figs. 4A–D), and no double-labeled cells were observed for GFP and either of the neuronal markers, NeuN or MAP2 (Fig. 7 graph, immunostaining data not shown). In contrast, 67.4 ± 8.3% of the transplanted cells expressed CC1 (Figs. 4E–H, and Fig. 7), a marker for mature oligodendrocytes (Bhat et al., 1996). Most of these cells were found near the lesion border (Fig. 4E). Furthermore, many of these transplanted cells also expressed the Olig2 protein (Figs. 4I–L) which is

a bHLH transcription factor important for oligodendrocyte development and is expressed by oligodendrocytes at all developmental stages (Lu et al., 2000; Zhou et al., 2000). Olig2 is also found prominently in neurons, but with cytoplasmic localization. However, Olig2 staining in GFP positive cells was nuclear indicating that the Olig2 expression was in oligodendrocytes. Before transplantation, the vast majority of spinal cord NSPCs were nestin positive and proliferating (Fig. 1). However, we did not observe any double-labeled GFP+/nestin+ or GFP+/Ki67+ cells at 1 or 3 weeks following transplantation (data not shown), suggesting the engrafted cells had acquired a more differentiated phenotype and did not continue to proliferate in vivo.

Fig. 7. Differentiation profile of adult spinal cord periventricular NSPCs after transplantation into a focal demyelination lesion. Three weeks after transplantation into an EB lesion, GFP positive cells expressed CC1 (67.4 ± 8.3%), MBP (6.9 ± 3.8%), P0 (28.5 ± 2.4%), and GFAP (1.9 ± 0.8%). There was no co-localization of GFP with the neuronal marker NeuN. Error bars indicate SD.

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Some oligodendrocytic progeny of transplanted NSPCs remyelinate axons in focal demyelination lesions We next examined whether the oligodendrocytic progeny of transplanted spinal cord NSPCs can functionally remyelinate the demyelinated axons in the EB and X-EB lesions. GFP was highly expressed in both the cell body and processes of the transplanted adult spinal cord NSPCs. Thus, with high magnification confocal microscopy, we were able to examine the association of donor cell processes with host axons in triple-labeled cross-sections. We found transplanted GFP positive, CC1 expressing oligodendrocytes ensheathing NF200 immunopositive (NF) axons at 3 weeks after grafting into an EB lesion (Figs. 5A–D). Most of the GFP positive, CC1 expressing, transplant-derived cells did not express the myelin marker MBP, suggesting that most of the cells transplanted into the EB or X-EB lesion differentiated into nonmyelinating ensheathing oligodendrocytes. However, some of the oligodendrocytic progeny of transplanted cells (GFP+/CC1+) expressed MBP, with multiple processes extending to MBP positive myelin rings, indicating that GFP positive processes

had generated myelin around NF positive axons (Figs. 5E–L). Quantitative confocal analysis revealed that 6.9 ± 3.8% of GFP positive cells expressed MBP (Fig. 7). Figs. 5I–L show a GFP positive oligodendrocyte with many MBP positive processes ensheathing several neighboring axons. Spinal cord NSPCs transplanted into focal demyelination lesions also express Schwann cell markers and remyelinate axons EB lesions will spontaneously remyelinate without X-irradiation (Crang et al., 1992; Franklin et al., 1996). We immunostained with Schwann cell markers the EB spinal cord after transplantation to assess the infiltration of host Schwann cells into the lesion site. Many p75 profiles were apparent within the lesion at 1 week posttransplantation (Fig. 6A), suggesting that Schwann cells were invading the lesion site. However, some GFP positive cells in the lesion core also expressed p75, a marker for nonmyelinating Schwann cells, at 1 week (Fig. 6A) and 3 weeks (Fig. 6B) posttransplantation. In addition, at 3 weeks, numerous P0 positive

Fig. 8. Adult spinal cord NSPC cultures are not contaminated with Schwann cells but have the potential to express Schwann cell markers in differentiating conditions. Neurospheres at passages 3 or 4 were dissociated and plated onto a Matrigel™ substrate in undifferentiated (growth factors and no serum) and differentiating (no growth factors and 1% FBS for 1 week) conditions, and cultures were immunostained with the Schwann cell markers p75 and P0. Dissociated spinal cord-derived neurospheres do not express p75 (A–C) or P0 (G–I) in undifferentiated culture conditions. When cultured in differentiating conditions in the presence of serum, dissociated neurospheres derived from the spinal cord show immunoreactivity for p75 (D–F) and P0 (J–L).

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profiles were apparent within the EB lesion core (Fig. 6C), which were not apparent at 1 week (data not shown). Protein P0 is the major protein of peripheral myelin which is made by Schwann cells and is absent from CNS myelin. This suggests that the nonmyelinating Schwann cells detected at 1 week have matured into myelinating Schwann cells by 3 weeks. Importantly, at 3 weeks, some GFP positive transplanted cells also co-expressed P0 (Figs. 6C–D). Clusters of engrafted GFP positive cells were observed ensheathing NF positive axons with P0 positive myelin rings (Figs. 6E–H), indicating that GFP positive cells had generated peripheral myelin around axons. Quantitative confocal analysis revealed that 28.5 ± 2.4% of GFP positive cells expressed P0 (Fig. 7) in the EB lesion. GFP+/P0+ transplanted cells were observed in both EB and X-EB lesions, indicating that irradiation was not responsible for the observed Schwann cell-like differentiation. Since the grafted cells specifically express GFP, transplanted cells can be easily distinguished from endogenous host cells. Also, we did not observe remyelination in nontransplanted X-EB lesions, consistent with previous studies (Blakemore and Crang, 1989; Keirstead et al., 1999; Talbott et al., 2006), where endogenous remyelination was suppressed, suggesting

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that the Schwann cells present within the remyelinated X-EB lesion were generated from the transplanted cells. Neurospheres derived from the adult spinal cord are not contaminated with Schwann cells We then asked whether the neurosphere cultures were contaminated with Schwann cells. This possibility was unlikely since the periventricular region of the spinal cord was isolated, and Schwann cells from any attached roots would have been removed. Nevertheless, we dissociated neurospheres at passages 3 or 4 and plated cells in undifferentiated conditions exactly as before, and found no expression of Schwann cell markers, p75 (Figs. 8A–C) or P0 (Figs. 8G–I), demonstrating that the neurospheres used in the transplantation experiments were not contaminated with Schwann cells or cells expressing Schwann cell markers. However, when NSPCs were cultured in the presence of serum for 1 week, dissociated neurospheres expressed p75 (Figs. 8D–F) and P0 (Figs. 8J–L), showing that adult spinal cord NSPCs have the potential to express Schwann cell markers under differentiating conditions in culture.

Fig. 9. Immunoelectron microscopy of GFP expressing spinal cord NSPCs 3 weeks after transplantation into X-EB lesions. A, GFP positive cells along the lesion border with cytoplasmic reaction product (arrows) showing characteristic oligodendrocyte or Schwann cell morphology. B, Higher magnification of the boxed area in A showing labeled processes (arrows) surrounding axons (a) with no myelin formation. C, Cytoplasmic peroxidase reaction product is apparent in this cell with oligodendrocyte morphology (n, nucleus) whose processes ensheath several axons. D, Higher magnification of the boxed area in C showing labeled process (arrows) ensheathing an axon and forming myelin (arrowhead). E, Another GFP labeled cell along the lesion border (arrows denote reaction product) with oligodendrocyte morphology ensheathing several axons (a) and forming thin myelin (arrowheads). F, High magnification image of a cross-section of another axon (a) showing cytoplasmic reaction product (arrows) and remyelination (arrowheads). G, These labeled processes (arrows) are myelinating several axons (a). H, Higher magnification of the boxed area in G showing labeled processes (arrows) ensheathing an axon (a) and forming thin myelin (arrowheads). I, Cytoplasmic reaction product (arrows) is apparent in the soma and processes ensheathing and myelinating an axon (a). This cell ultrastructurally resembles a Schwann cell, with a 1:1 cell-to-axon ratio, the nucleus (n) closely apposed to myelin sheaths (arrowhead), and the presence of basal lamina (bl). J, Cross-sections of myelinated axons from the lesion core surrounded by cytoplasm containing reaction product and a basal lamina surrounding the cell membrane (bl). K, High magnification of another labeled cell (arrows) ultrastructurally resembling a Schwann cell ensheathing and remyelinating (arrowhead) an axon (a). L, Higher magnification of the boxed area in K showing the presence of peroxidase reaction product associated with the myelin membrane around the axon.

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Ultrastructural evidence of remyelination We further examined the remyelinating ability of transplanted adult spinal cord NSPCs using IEM and to better characterize the engrafted cells ultrastructurally. IEM for the GFP reaction product was performed on tissue 3 week after transplantation, to allow correlation with the immunohistochemical data described above. The IEM data is from X-EB rats where spontaneous remyelination has been suppressed. Thus, any evidence of remyelination in the X-EB lesion can be attributed to the grafted NSPCs identified by the GFP immunoperoxidase reaction product. Ultrastructurally, labeled cells with morpho-

logical characteristics of either oligodendrocytes or Schwann cells were identified. Labeled cells resembling oligodendrocytes with a large nucleus, scant cytoplasm, and processes ensheathing several axons were observed (Figs. 9A, C, E, G) near the lesion border. Some of the processes ensheathed axons with thin myelin sheaths and showed varying degrees of remyelination (Figs. 9D, F, H). However, there were many labeled processes that ensheathed axons with no evidence of remyelination (Fig. 9B). Numerous labeled cells in the lesion core were also observed that ultrastructurally resembled myelinating Schwann cells, with a 1:1 cell-to-axon ratio, the nucleus closely apposed to myelin sheaths, and the presence of a basal lamina (Figs. 9I–L).

Fig. 10. Transplanted spinal cord NSPCs express MBP and form compact myelin in the adult shiverer spinal cord. Low magnification images of longitudinal sections of MPB deficient shiverer mouse spinal cord transplanted with GFP positive spinal cord NSPCs (green) showing expression of MBP (red) as early as 1 week (A, merged) after grafting, and robust generation of myelin 3 weeks (B–D) after transplantation. Sections were counterstained with DAPI (blue) to show all the cells. Note the complete absence of MBP in regions that do not contain the transplanted NSPCs. E–H, High magnification confocal images of a transplanted longitudinal spinal cord section triple-labeled for GFP (E, green), MBP (F, red), and NF (G, NF200, blue; H, merge), showing GFP+/MBP+ cells associating with NF positive axons, 3 weeks after grafting. I–L, shows one GFP positive oligodendrocyte with numerous processes associated with NF positive axons and generating MBP. Electron micrographs from M–N, nontransplanted shiverer spinal cord showing axons lacking myelin or surrounded by 2–3 layers of noncompacted membrane. O, Three weeks after transplantation, myelinated axons were observed in transplanted segments of spinal cord containing multilayered compact myelin. P, Photomicrograph at higher magnification shows myelinated axon and compact myelin with major dense lines.

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Adult spinal cord NSPCs remyelinate the dysmyelinated shiverer adult spinal cord The EB model is a chemically induced model of focal demyelination that lacks endogenous astrocytes and oligodendrocytes. We then asked whether adult periventricular spinal cord NSPCs could myelinate dysmyelinated CNS axons in the myelin deficient adult shiverer mouse spinal cord. The shiverer mouse CNS is characterized by the lack of MBP and extensive dysmyelination, so that the presence of myelin can be attributed to the transplanted cells (Chernoff, 1981). As early as 1 week after transplantation, adult spinal cord NSPCs expressed MBP in the shiverer mouse spinal cord (Fig. 10A). Three weeks after transplantation, there was a more robust expression of MBP by the GFP positive grafted cells which had migrated 2–3 mm along the rostro-caudal axis from the single injection site (Figs. 10B– D). Immunohistochemistry revealed that MBP expression was limited to the region of the transplanted NSPCs: MBP was not found in adjacent regions that did not contain grafted cells (Figs. 10A–D) or in shiverer spinal cord that was not transplanted (not shown). MBP positive, GFP negative cells were never observed, confirming that MBP (in addition to the GFP marker) could be used as a reliable genetic marker for myelinating transplanted cells within the shiverer nervous system (McKenzie et al., 2006). High magnification confocal images of immunostained sections showed a close association of the GFP+/MBP+ expressing NSPC derived oligodendrocytes with host NF positive axons (Figs. 10E–L). Transplanted cells showed an oligodendrocyte morphology (Figs. 10E–L), and a normal myelination pattern for oligodendrocytes was apparent because a single GFP positive oligodendrocyte could be observed elaborating MBP positive processes around several neighbouring axons (Figs. 10I–L). Electron microscopy of the nontransplanted shiverer spinal cord showed axons with 2–3 layers of noncompacted myelin membrane (Figs. 10M–N). At 3 weeks after transplantation, we observed newly myelinated axons in the adult shiverer spinal cord identified by their multilayered myelin figures (Fig. 10O). Multilayered compact myelin sheaths were approximately 100 nm thick (Fig. 10P), which is similar

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to the amount of myelination observed at 6 weeks after transplantation with brain derived precursors (Eftekharpour et al., 2007). Confocal immunofluorescence microscopy with cell type-specific markers was used to identify double-labeled cells at 1 to 4 weeks after transplantation. Virtually all of the NSPCs transplanted into the adult shiverer spinal cord differentiated along an oligodendroglial lineage with 99.1 ± 2% of the GFP positive cells staining for the oligodendrocyte transcription factor Olig2 (Figs. 11A–D). Moreover, 90.6 ± 8% of the transplanted cells expressed the mature oligodendrocyte marker CC1 (Figs. 11E–F). In contrast, none of the transplanted NSPCs expressed the astrocytic marker GFAP (Fig. 11G), or the neuronal markers, MAP2 or NeuN (data not shown). Also, transplanted NSPCs did not express the Schwann cell markers p75 or P0 in the shiverer spinal cord (Fig. 11H; P0 shown) as they did in the EB or X-EB lesions in the spinal cord. Similar to the focal demyelination lesion, the transplanted cells were also negative for nestin and Ki67 (data not shown), suggesting they had acquired a more differentiated phenotype after transplantation into the shiverer spinal cord. Discussion In the present study, we show that transplanted adult spinal cord NSPCs preferentially differentiate along an oligodendroglial lineage in both the focal demyelination lesion and the dysmyelinated shiverer spinal cord. However, in both EB and X-EB lesions, some of the oligodendrocytic progeny remyelinate axons, yet a significant proportion of the transplanted cells express a Schwann cell phenotype and generate peripheral myelin. In culture, we show that spinal cord NSPCs do not express a Schwann cell phenotype in proliferating undifferentiated conditions. Thus, the transplanted neurospheres were not contaminated with Schwann cells or included cells with a Schwann cell-like phenotype. In addition, we have shown that adult spinal cord NSPCs have the in vitro potential to express a Schwann cell phenotype in differentiating culture conditions in the presence of serum, and in vivo in the glial-free environment of the EB lesion. In contrast to the focal demyelination lesion, virtually all transplanted

Fig. 11. GFP positive NSPCs express markers for oligodendrocytes but not Schwann cells in the shiverer spinal cord. A, Low magnification confocal merged image showing most of the GFP positive transplanted cells expressing Olig2 1 week after transplantation. B–D, Higher magnification images showing GFP positive (B, green), Olig2 positive (C, red) cells (D, merge; 2 weeks). E, Low magnification images showing most of the transplanted NSPCs expressing CC1 at 1 week after transplantation. F, Higher magnification image showing GFP+/ CC1+ cells (merge; 2 weeks). G, GFP positive transplanted cells did not express the astrocyte marker, GFAP (merge; 1 week) H, Transplanted cells also did not express the Schwann cell marker, P0 (3 weeks). Inset in H shows positive control staining for P0 in a peripheral root adjacent to the spinal cord of a shiverer mouse.

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NSPCs differentiated along an oligodendroglial lineage in the adult shiverer spinal cord and robustly myelinated dysmyelinated axons. There were no Schwann-like transplanted cells in the dysmyelinated mice. These results demonstrate inherent plasticity of adult NSPCs derived from the periventricular region of the spinal cord to respond to environmental cues resulting in the preferential differentiation of these multipotent cells along an oligodendrocytic lineage in both models, and in addition to differentiate into Schwann-like cells in the focal demyelination model. Furthermore, these lineage propensities were expressed in the absence of pre-differentiation or neurotrophic stimulation to enhance oligodendrogenesis. In the present study, we examined the remyelinating capacity of the NSPCs generated from the periventricular region of the spinal cord because we have found that these cells have an intrinsic capacity for oligodendrocytic differentiation in vitro (Kulbatski et al., 2007). This propensity for oligodendrocytic differentiation is a unique feature of these cells, and relative to other reports on differentiated spinal cord cultures of stem/progenitors (Shihabuddin et al., 2000; Cao et al., 2001; Vroemen et al., 2003; Hofstetter et al., 2005), may reflect the periventricular source of cultured tissue surrounding the central canal, and/or specific culture conditions enhancing oligodendrogenesis. We also find that these cells maintain the capacity for oligodendrocytic differentiation following transplantation into the intact and traumatically injured spinal cord (Parr et al., 2007; Mothe et al., in press). Also, as shown here in models of demyelination and dysmyelination, adult spinal cord NSPCs show minimal neuronal or astrocytic fate in the spinal cord. Other groups have shown that endogenous ependymal cells have the ability to regenerate the cord in lower species (Dervan and Roberts, 2003), and recently, we demonstrated significant improvement in functional recovery after delayed transplantation of spinal cord NSPCs into the traumatically injured spinal cord of adult rats (Parr et al., unpublished data). A potential source of adult human spinal cord stem/progenitor cells are organ transplant donors (Dromard et al., 2008). Here, we now show that adult spinal cord NSPCs are capable of remyelinating the demyelinated axons in EB lesions in rats and myelinating the myelin deficient spinal cord of the shiverer mouse. In both models, there was significant graft rejection, and rejection of the xenograft in the shiverer mouse was substantial even with immunosuppression. Rejection of allografts into shiverer mice has also been reported in other studies (Eftekharpour et al., 2007). In the EB and X-EB lesions, cell survival was consistent with other studies of cell transplantation into the traumatically injured spinal cord (Vroemen et al., 2003; Hofstetter et al., 2005; Parr et al., 2007). Recently, we have found that some of the grafted NSPCs undergo apoptosis soon after transplantation into the intact rat spinal cord (Mothe et al., in press), although we cannot rule out the possibility of partial silencing of the GFP transgene. Strategies such as intrathecal infusion of mitogenic growth factors may enhance cell survival, as shown with adult mouse brain derived precursor cell transplants into the injured spinal cord (Karimi-Abdolrezaee et al., 2006). The differentiation of transplanted multipotent cells is strongly influenced by the environmental signals present at the site of transplantation (Keirstead et al., 1999; Shihabuddin et al., 2000; Cao et al., 2001; Akiyama et al., 2002; Talbott et al., 2006). Keirstead et al. (1999), showed that neural precursors positive for the polysialated form of the neural cell adhesion molecule (PSA-NCAM) and derived from the postnatal rat brain, differentiated into Schwann cells after transplantation into X-EB lesions. Clonal analysis of multipotential cells from the rat spinal cord suggests that a common CNS precursor can generate both CNS and PNS phenotypes (Mujtaba et al., 1998). Multipotent progenitors from both CNS (adult human brain) and bone marrow lineages also differentiated into Schwann cells following transplantation into X-EB lesions (Akiyama et al., 2001; Sasaki et al., 2001; Akiyama et al., 2002). It has been proposed that the glial-free environment of the EB lesion is responsible for promoting Schwann

cell-like differentiation of transplanted cells (Blakemore, 2005). Recently, lineage restricted oligodendrocyte progenitor cells (OPCs) isolated from the adult spinal cord were shown to give rise to Schwann cell myelination following transplantation into X-EB lesions (Talbott et al., 2006). This was shown to be mediated by bone morphogenetic proteins (BMPs), as Schwann cell differentiation was inhibited by the BMP-antagonist noggin (Talbott et al., 2006). This group elegantly demonstrated the lack of noggin in EB lesions due to the lack of astrocytes which normally secrete noggin, resulting in unopposed signalling of BMPs which have been shown to instruct neural stem cells to differentiate along a neural crest lineage (Mujtaba et al., 1998; Gajavelli et al., 2004). Thus, there is a potential mechanism for OPCs or other CNS derived cells to be reprogrammed to differentiate into remyelinating Schwann cells (Talbott et al., 2006). Consistent with this report, is our finding that adult spinal cord NSPCs differentiated into cells expressing a Schwann cell phenotype, but only in the glial-free environment of the EB lesion core. We have not observed differentiation of spinal cord NSPCs into Schwann cells after transplantation into either the intact or injured cord (Mothe et al., in press) or in the dysmyelinated shiverer spinal cord as shown here. Thus, the environment of the EB lesion is conducive to Schwann cell differentiation but relatively inhibitory to oligodendrocyte remyelination. The difference in either the numbers of cells transplanted or the level of immunosuppression in the two models is unlikely to account for the apparent differences in the phenotypic fate of the transplanted cells. In other studies in our laboratory, we found similar phenotypic fates after transplantation of different numbers of NSPCs with varying cyclosporine dosages into either intact (Mothe et al., in press) or injured rat spinal cord (Parr et al., 2007). It is conceivable that a longer survival period could result in a greater number of CC1+/MBP+ transplanted cells. The quantitation of GFP+/MBP+ cells was performed on EB animals only. However, there were no differences between MBP+ transplanted cells in EB or X-EB models. It is possible that host competition for the remyelination of demyelinated axons could have limited the potential of transplanted cells to remyelinate demyelinated axons. Although only a small percentage of oligodendrocyte progeny remyelinated axons after transplantation into the EB lesion, the majority of the transplanted NSPCs expressed CC1, a marker for mature oligodendrocytes. Transplanted cells expressing oligodendrocyte markers were found near the lesion border rather than the lesion core where the majority of the cells expressing Schwann cell markers were found. Thus, the environment of the EB lesion did not appear to inhibit oligodendrocyte differentiation of NSPCs. In contrast, following OPC transplantation into the EB lesion, grafted cells did not express CC1 even at 4 weeks post-transplantation (Talbott et al., 2006). In the present study, we transplanted NSPCs that are multipotent 3 days after EB induction, compared to transplanting a more lineage restricted population of cells such as OPCs, 4 days after EB induction (Talbott et al., 2006). Collectively, these data suggest that multipotent NSPCs may be more responsive to environmental cues for differentiation, or alternatively, NSPCs may be less responsive to inhibitory factors for differentiation, relative to more restricted phenotypes such as OPCs. In either case, NSPCs derived from the periventricular region of the adult spinal cord maintain an intrinsic capacity for oligodendrocytic differentiation, even in the glial-free environment of the EB lesion. Another important factor is the expression of inhibitory molecules in the EB lesion that could inhibit remyelination. For example, PSANCAM is normally expressed in the CNS during development and down-regulated in the adult. However, PSA-NCAM has been shown to be up-regulated in focal demyelination lesions induced by lysolecithin (Oumesmar et al., 1995), and recently, in non-remyelinating multiple sclerosis lesions (Charles et al., 2002). PSA-NCAM expression by axons was also shown to negatively regulate CNS myelination (Charles et al., 2000). Thus, it is possible that up-regulation of PSA-NCAM in the EB

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lesion inhibited the remyelination of demyelinated axons by the oligodendrocyte progeny of transplanted NSPCs. Shiverer mice have been used extensively to study myelination by several other types of transplanted cells (Kohsaka et al., 1986; Lubetzki et al., 1988; Gumpel et al., 1989; Lachapelle et al., 1994; Yandava et al., 1999; Liu et al., 2000; Mitome et al., 2001; Windrem et al., 2004; Cummings et al., 2005; Nistor et al., 2005; McKenzie et al., 2006; Eftekharpour et al., 2007). However, most of these studies involve transplantation into neonatal mice rather than dysmyelinated adults that have a long-term irreversible myelin deficiency. Shiverer mice do not produce MBP which is essential for myelin compaction, and contain axons that are devoid of myelin or are surrounded by one or two noncompacted wraps of myelin membrane (Yandava et al., 1999). Therefore, detection of multilayered compact myelin and MBP immunostaining following transplantation into shiverer mice has been used to demonstrate myelination from transplanted oligodendrocytes (Kohsaka et al., 1986; Lubetzki et al., 1988; Lachapelle et al., 1994; Yandava et al., 1999; Windrem et al., 2004; Nistor et al., 2005). In the present study, NSPCs derived from the periventricular region of the spinal cord robustly myelinated the dysmyelinated axons and formed multilayered compact myelin 3 weeks after transplantation into the adult shiverer spinal cord. It is interesting that nearly all the transplanted adult spinal cord NSPCs differentiated into myelinating oligodendrocytes in the shiverer spinal cord. Such a high percentage of oligodendrocytic progeny after transplantation of multipotent NSPCs into the shiverer CNS has not been previously reported. Moreover, strategies requiring pre-differentiation of cells in culture or in vivo infusion of growth factors in order to achieve a high proportion of oligodendrocytic progeny was not required in the present study. Thus, we show for the first time that adult spinal cord NSPCs demonstrate remarkable plasticity in lineage fate both in vitro and in vivo, and an inherent capacity for oligodendrocytic differentiation, and that these cells are capable of myelinating both demyelinated and dysmyelinated axons. Acknowledgments This work was supported by operating grants to C.H.T from the Christopher Reeve Paralysis Foundation, MS Society of Canada, International Foundation of Research in Paraplegia, and in part by CIHR NET team grant. A. J. M. was supported by fellowships from the Ontario Neurotrauma Foundation and CIHR. We thank Linda Lee and Rita van Bendegem for the technical assistance and Iris Kulbatski for culturing some of the cells used for the focal demyelination transplantation experiments. We thank Dr. Armand Keating and Dr. Xinghua Wang of the Cell Therapy Program, Princess Margaret Hospital and Ontario Cancer Institute, for maintaining the transgenic rat colony. We thank Bob Kuba and Dr. Richard Hill of the Division of Applied Molecular Oncology, Princess Margaret Hospital and Ontario Cancer Institute, for their help with the X-irradiation of rats. We thank Sheer Ramjohn and Dr. Patrick Shannon of the Department of Cellular and Molecular Pathology, University of Toronto, for their help with the ultrastructural analysis. We thank Stephanie Tung for her assistance with cell counts and Dr. Juan Archelos for the gift of P0 antibody. References Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J.D., 2002. Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 39, 229–236. Akiyama, Y., Honmou, O., Kato, T., Uede, T., Hashi, K., Kocsis, J.D., 2001. Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol 167, 27–39. Beattie, M.S., Bresnahan, J.C., Komon, J., Tovar, C.A., Van Meter, M., Anderson, D.K., Faden, A.I., Hsu, C.Y., Noble, L.J., Salzman, S., Young, W., 1997. Endogenous repair after spinal cord contusion injuries in the rat. Exp Neurol 148, 453–463. Bhat, R.V., Axt, K.J., Fosnaugh, J.S., Smith, K.J., Johnson, K.A., Hill, D.E., Kinzler, K.W., Baraban, J.M., 1996. Expression of the APC tumor suppressor protein in oligodendroglia. Glia 17, 169–174.

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Blakemore, W.F., 1978. Observations on remyelination in the rabbit spinal cord following demyelination induced by lysolecithin. Neuropathol Appl Neurobiol 4, 47–59. Blakemore, W.F., 1982. Ethidium bromide induced demyelination in the spinal cord of the cat. Neuropathol Appl Neurobiol 8, 365–375. Blakemore, W.F., 2005. The case for a central nervous system (CNS) origin for the Schwann cells that remyelinate CNS axons following concurrent loss of oligodendrocytes and astrocytes. Neuropathol Appl Neurobiol 31, 1–10. Blakemore, W.F., Crang, A.J., 1989. The relationship between type-1 astrocytes, Schwann cells and oligodendrocytes following transplantation of glial cell cultures into demyelinating lesions in the adult rat spinal cord. J Neurocytol 18, 519–528. Blakemore, W.F., Gilson, J.M., Crang, A.J., 2003. The presence of astrocytes in areas of demyelination influences remyelination following transplantation of oligodendrocyte progenitors. Exp Neurol 184, 955–963. Bruni, J.E., Anderson, W.A., 1987. Ependyma of the rat fourth ventricle and central canal: response to injury. Acta Anat (Basel) 128, 265–273. Cao, Q.L., Zhang, Y.P., Howard, R.M., Walters, W.M., Tsoulfas, P., Whittemore, S.R., 2001. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 167, 48–58. Charles, P., Hernandez, M.P., Stankoff, B., Aigrot, M.S., Colin, C., Rougon, G., Zalc, B., Lubetzki, C., 2000. Negative regulation of central nervous system myelination by polysialylated-neural cell adhesion molecule. Proc Natl Acad Sci U S A 97, 7585–7590. Charles, P., Reynolds, R., Seilhean, D., Rougon, G., Aigrot, M.S., Niezgoda, A., Zalc, B., Lubetzki, C., 2002. Re-expression of PSA-NCAM by demyelinated axons: an inhibitor of remyelination in multiple sclerosis? Brain 125, 1972–1979. Chernoff, G.F., 1981. Shiverer: an autosomal recessive mutant mouse with myelin deficiency. J Hered 72, 128. Crang, A.J., Franklin, R.J., Blakemore, W.F., Noble, M., Barnett, S.C., Groves, A., Trotter, J., Schachner, M., 1992. The differentiation of glial cell progenitor populations following transplantation into non-repairing central nervous system glial lesions in adult animals. J Neuroimmunol 40, 243–253. Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M., Summers, R., Gage, F.H., Anderson, A.J., 2005. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A 102, 14069–14074. Dervan, A.G., Roberts, B.L., 2003. Reaction of spinal cord central canal cells to cord transection and their contribution to cord regeneration. J Comp Neurol 458, 293–306. Dromard, C., Guillon, H., Rigau, V., Ripoll, C., Sabourin, J.C., Perrin, F.E., Scamps, F., Bozza, S., Sabatier, P., Lonjon, N., Duffau, H., Vachiery-Lahaye, F., Prieto, M., Tran Van Ba, C., Deleyrolle, L., Boularan, A., Langley, K., Gaviria, M., Privat, A., Hugnot, J.P., Bauchet, L., 2008. Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro. J Neurosci Res. Eftekharpour, E., Karimi-Abdolrezaee, S., Wang, J., El Beheiry, H., Morshead, C., Fehlings, M.G., 2007. Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27, 3416–3428. Franklin, R.J., Crang, A.J., Blakemore, W.F., 1991. Transplanted type-1 astrocytes facilitate repair of demyelinating lesions by host oligodendrocytes in adult rat spinal cord. J Neurocytol 20, 420–430. Franklin, R.J., Bayley, S.A., Blakemore, W.F., 1996. Transplanted CG4 cells (an oligodendrocyte progenitor cell line) survive, migrate, and contribute to repair of areas of demyelination in X-irradiated and damaged spinal cord but not in normal spinal cord. Exp Neurol 137, 263–276. Gajavelli, S., Wood, P.M., Pennica, D., Whittemore, S.R., Tsoulfas, P., 2004. BMP signaling initiates a neural crest differentiation program in embryonic rat CNS stem cells. Exp Neurol 188, 205–223. Gumpel, M., Gout, O., Lubetzki, C., Gansmuller, A., Baumann, N., 1989. Myelination and remyelination in the central nervous system by transplanted oligodendrocytes using the shiverer model. Discussion on the remyelinating cell population in adult mammals. Dev Neurosci 11, 132–139. Hakamata, Y., Tahara, K., Uchida, H., Sakuma, Y., Nakamura, M., Kume, A., Murakami, T., Takahashi, M., Takahashi, R., Hirabayashi, M., Ueda, M., Miyoshi, I., Kasai, N., Kobayashi, E., 2001. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 286, 779–785. Hofstetter, C.P., Holmstrom, N.A., Lilja, J.A., Schweinhardt, P., Hao, J., Spenger, C., Wiesenfeld-Hallin, Z., Kurpad, S.N., Frisen, J., Olson, L., 2005. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci 8, 346–353. Horky, L.L., Galimi, F., Gage, F.H., Horner, P.J., 2006. Fate of endogenous stem/progenitor cells following spinal cord injury. J Comp Neurol 498, 525–538. Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U., Frisen, J., 1999. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25–34. Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Morshead, C.M., Fehlings, M.G., 2006. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci 26, 3377–3389. Keirstead, H.S., Ben-Hur, T., Rogister, B., O'Leary, M.T., Dubois-Dalcq, M., Blakemore, W.F., 1999. Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation. J Neurosci 19, 7529–7536. Kohsaka, S., Yoshida, K., Inoue, Y., Shinozaki, T., Takayama, H., Inoue, H., Mikoshiba, K., Takamatsu, K., Otani, M., Toya, S., et al., 1986. Transplantation of bulk-separated oligodendrocytes into the brains of shiverer mutant mice: immunohistochemical and electron microscopic studies on the myelination. Brain Res 372, 137–142.

190

A.J. Mothe, C.H. Tator / Experimental Neurology 213 (2008) 176–190

Kulbatski, I., Mothe, A.J., Keating, A., Hakamata, Y., Kobayashi, E., Tator, C.H., 2007. Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization. J Histochem Cytochem 55, 209–222. Lachapelle, F., Duhamel-Clerin, E., Gansmuller, A., Baron-Van Evercooren, A., Villarroya, H., Gumpel, M., 1994. Transplanted transgenically marked oligodendrocytes survive, migrate and myelinate in the normal mouse brain as they do in the shiverer mouse brain. Eur J Neurosci 6, 814–824. Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F., McDonald, J.W., 2000. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci U S A 97, 6126–6131. Lu, Q.R., Yuk, D., Alberta, J.A., Zhu, Z., Pawlitzky, I., Chan, J., McMahon, A.P., Stiles, C.D., Rowitch, D.H., 2000. Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system. Neuron 25, 317–329. Lubetzki, C., Gansmuller, A., Lachapelle, F., Lombrail, P., Gumpel, M., 1988. Myelination by oligodendrocytes isolated from 4–6-week-old rat central nervous system and transplanted into newborn shiverer brain. J Neurol Sci 88, 161–175. Martens, D.J., Seaberg, R.M., van der Kooy, D., 2002. In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur J Neurosci 16, 1045–1057. McKenzie, I.A., Biernaskie, J., Toma, J.G., Midha, R., Miller, F.D., 2006. Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system. J Neurosci 26, 6651–6660. Mitome, M., Low, H.P., van den Pol, A., Nunnari, J.J., Wolf, M.K., Billings-Gagliardi, S., Schwartz, W.J., 2001. Towards the reconstruction of central nervous system white matter using neural precursor cells. Brain 124, 2147–2161. Morshead, C.M., Reynolds, B.A., Craig, C.G., McBurney, M.W., Staines, W.A., Morassutti, D., Weiss, S., van der Kooy, D.,1994. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071–1082. Mothe, A.J., Tator, C.H., 2005. Proliferation, migration, and differentiation of ependymal region stem/progenitor cells following minimal injury in the adult rat spinal cord. Neuroscience 131, 177–187. Mothe A.J., Kulbatski I., Parr A., Mohareb M., Tator C.H. (in press). Adult spinal cord stem/ progenitor cells transplanted as neurospheres preferentially differentiate into oligodendrocytes in the adult rat spinal cord. Cell Transplant. Mothe, A.J., Kulbatski, I., van Bendegem, R.L., Lee, L., Kobayashi, E., Keating, A., Tator, C.H., 2005. Analysis of green fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells. J Histochem Cytochem 53, 1215–1226. Mujtaba, T., Mayer-Proschel, M., Rao, M.S., 1998. A common neural progenitor for the CNS and PNS. Dev Biol 200, 1–15. Namiki, J., Tator, C.H., 1999. Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol 58, 489–498. Nistor, G.I., Totoiu, M.O., Haque, N., Carpenter, M.K., Keirstead, H.S., 2005. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49, 385–396. Nordlander, R.H., Singer, M., 1978. The role of ependyma in regeneration of the spinal cord in the urodele amphibian tail. J Comp Neurol 180, 349–374. Oumesmar, B.N., Vignais, L., Duhamel-Clerin, E., Avellana-Adalid, V., Rougon, G., BaronVan Evercooren, A., 1995. Expression of the highly polysialylated neural cell adhesion molecule during postnatal myelination and following chemically induced demyelination of the adult mouse spinal cord. Eur J Neurosci 7, 480–491. Parr, A.M., Kulbatski, I., Tator, C.H., 2007. Transplantation of adult rat spinal cord stem/ progenitor cells for spinal cord injury. J Neurotrauma 24, 835–845. Pfeifer, K., Vroemen, M., Caioni, M., Aigner, L., Bogdahn, U., Weidner, N., 2006. Autologous adult rodent neural progenitor cell transplantation represents a feasible strategy to promote structural repair in the chronically injured spinal cord. Regen Med 1, 255–266.

Privat, A., Jacque, C., Bourre, J.M., Dupouey, P., Baumann, N., 1979. Absence of the major dense line in myelin of the mutant mouse “shiverer”. Neurosci Lett 12, 107–112. Radtke, C., Spies, M., Sasaki, M., Vogt, P.M., Kocsis, J.D., 2007. Demyelinating diseases and potential repair strategies. Int J Dev Neurosci 25, 149–153. Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710. Sasaki, M., Honmou, O., Akiyama, Y., Uede, T., Hashi, K., Kocsis, J.D., 2001. Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 35, 26–34. Sasaki, M., Black, J.A., Lankford, K.L., Tokuno, H.A., Waxman, S.G., Kocsis, J.D., 2006. Molecular reconstruction of nodes of Ranvier after remyelination by transplanted olfactory ensheathing cells in the demyelinated spinal cord. J Neurosci 26, 1803–1812. Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H., 2000. Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 20, 8727–8735. Takahashi, M., Arai, Y., Kurosawa, H., Sueyoshi, N., Shirai, S., 2003. Ependymal cell reactions in spinal cord segments after compression injury in adult rat. J Neuropathol Exp Neurol 62, 185–194. Talbott, J.F., Loy, D.N., Liu, Y., Qiu, M.S., Bunge, M.B., Rao, M.S., Whittemore, S.R., 2005. Endogenous Nkx2.2+/Olig2+ oligodendrocyte precursor cells fail to remyelinate the demyelinated adult rat spinal cord in the absence of astrocytes. Exp Neurol 192, 11–24. Talbott, J.F., Cao, Q., Enzmann, G.U., Benton, R.L., Achim, V., Cheng, X.X., Mills, M.D., Rao, M.S., Whittemore, S.R., 2006. Schwann cell-like differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent. Glia 54, 147–159. Tropepe, V., Sibilia, M., Ciruna, B.G., Rossant, J., Wagner, E.F., van der Kooy, D., 1999. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208, 166–188. Vacanti, M.P., Leonard, J.L., Dore, B., Bonassar, L.J., Cao, Y., Stachelek, S.J., Vacanti, J.P., O'Connell, F., Yu, C.S., Farwell, A.P., Vacanti, C.A., 2001. Tissue-engineered spinal cord. Transplant Proc 33, 592–598. Vaquero, J., Ramiro, M.J., Oya, S., Cabezudo, J.M., 1981. Ependymal reaction after experimental spinal cord injury. Acta Neurochir (Wien) 55, 295–302. Vroemen, M., Aigner, L., Winkler, J., Weidner, N., 2003. Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways. Eur J Neurosci 18, 743–751. Wallace, M.C., Tator, C.H., Lewis, A.J., 1987. Chronic regenerative changes in the spinal cord after cord compression injury in rats. Surg Neurol 27, 209–219. Weiss, S., Dunne, C., Hewson, J., Wohl, C., Wheatley, M., Peterson, A.C., Reynolds, B.A., 1996. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16, 7599–7609. Windrem, M.S., Nunes, M.C., Rashbaum, W.K., Schwartz, T.H., Goodman, R.A., McKhann 2nd, G., Roy, N.S., Goldman, S.A., 2004. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat Med 10, 93–97. Wu, S., Suzuki, Y., Noda, T., Bai, H., Kitada, M., Kataoka, K., Nishimura, Y., Ide, C., 2002. Immunohistochemical and electron microscopic study of invasion and differentiation in spinal cord lesion of neural stem cells grafted through cerebrospinal fluid in rat. J Neurosci Res 69, 940–945. Yandava, B.D., Billinghurst, L.L., Snyder, E.Y., 1999. “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci U S A 96, 7029–7034. Zhou, Q., Wang, S., Anderson, D.J., 2000. Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25, 331–343.