Multiple sclerosis (MS) is a chronic demyelinating disease characterized by inflammation in the central nervous system (CNS) leading to demyelination, axonal loss, and disability (Sospedra and Martin, 2005) . Many treatments are geared towards reducing neuroinflammation with the ultimate goal of reducing progression of disease (Rizvi and Agius, 2004) . However, these treatments often do not address the neurodegenerative phase of disease. Remyelination can occur spontaneously in the adult human brain as evidenced by shadow plaques, in which large regions of white matter undergo remyelination with characteristically thin myelin sheaths (Halfpenny et al., 2002) . However, remyelination does not occur uniformly within a lesion or across many lesions. Two major processes have been proposed to explain the failure of remyelination in MS, notably: (i) difficulties in recruiting oligodendrocyte precursor cells (OPCs) to areas of active demyelination and (ii) inhibition in differentiation of OPCs into oligodendrocytes capable of remyelination (Franklin, 2002) . In each of these two processes signaling by growth factors, cytokines/chemokines, and extracellular matrix molecules are thought to contribute to an environment that is not conducive for promoting remyelination.
Studies in animal models have proven invaluable for identifying new methods for inducing remyelination in animals with established demyelination. For example, transplantation of stem cells into animal models of acute demyelination results in remyelination (Cao et al., 2002) . Transplant of rodent embryonic stem cells into myelindeficient shiverer mice results in cellular migration in the spinal cord, differentiation into oligodendrocytes and astrocytes, and myelination of axons (Brustle et al., 1999) . Similarly, transplant of human embryonic stem cell-derived oligodendrocyte progenitor cells into myelin-deficient shiverer mice results in oligodendrocyte differentiation and remyelination (Nistor et al., 2005) . Other models of demyelination have also reported reduced demyelination following transplantation of stem cells. Injection of adult neuronal precursors into mice with experimental autoimmune encephalomyelitis (EAE) resulted in recovery from disease and a significant decrease in the level of demyelination (Pluchino et al., 2003) . Similarly, transplantation of stromal bone marrow cells into demyelinated rat spinal cord resulted in remyelination (Akiyama et al., 2002) .
This study utilized mouse hepatitis virus (MHV, a murine coronavirus) as a model for MS to define the impact of transplantation on the inflammatory response associated with disease in the CNS. Infection of susceptible mice with MHV results in an acute encephalomyelitis followed by an immunemediated demyelinating disease. Similar to MS, demyelination in MHV-infected mice is associated with impaired motor skills that often are associated with immune cell infiltration in white matter tracts (Lane and Buchmeier, 1997) . Due to the similarities in both clinical and histologic disease between MS and MHV-induced demyelination, the MHV model is considered a relevant model for studying the underlying immunopathological mechanisms contributing to ongoing myelin destruction as well as clinical disease. Recently, a study by Totoiu et al. (2004) used the MHV model of chronic demyelination to determine if cellular transplantation modulated disease severity. Transplantation of glial-committed progenitor cells into MHV-infected mice with on-going demyelination and established clinical disease resulted in remyelination and improved locomotion. These studies highlight the efficacy of cellular transplantation therapy in promoting remyelination in a model in which there is extensive cellular infiltration and demyelination and provide us with the opportunity to investigate the role that inflammation plays in this regenerative process. Here, we report that cellular transplantation resulting in remyelination does not alter inflammation in the CNS nor modulate the ongoing immune response to MHV. These studies imply that modulation of the inflammatory environment is not required for remyelination, a finding that has profound implications for the development of cellular replacement strategies to treat demyelinating diseases.
Age-matched (5-7 weeks) C57BL/6 male mice (H-2 b background) were used for all studies (National Cancer Institute, Bethesda, MD). Following anesthetization with ketamine (Phoenix, St. Joseph, MO), mice were injected intracranially (i.c.) with 500 PFU of MHV strain J2.2-V.1 suspended in 30 μl of sterile Hank's balanced salt solution (HBSS). Control animals (sham) were injected with 30 μl HBSS alone. Animals were euthanized at 22 and 33 days postinfection (p.i.) (corresponding to 10 or 21 days after transplantation) at which point brains, spinal cords, and spleens were removed for analysis.
Cells for transplant were cultured from the striata of postnatal day 1 C57BL/6 mice as previously described and cultured for 6 days before transplantation (Totoiu et al., 2004) . The striata region of the brain was chosen because it contains periventricular areas which are rich in neurogenic stem cells. Twelve days after i.c. infection with MHV, 2.4 × 10 5 cells were injected in 2 μl of DMEM (Gibco-Invitrogen) at T8 of the spinal cord (Totoiu et al., 2004) . Control animals received i.c. infection with MHV and injection of DMEM alone at T8 of the spinal cord (Totoiu et al., 2004) .
Animals were euthanized by inhalation of halothane (Sigma, St. Louis, MO) 10 or 21 days following transplantation and fixed by cardiac perfusion with 4% paraformaldehyde (Fisher Scientific) or 4% glutaraldehyde (Fisher Scientific, Pittsburgh, PA) in 0.1 M PBS, pH 7.4. The length of spinal cord extending 6 mm cranial and 6 mm caudal to the site of implantation was extracted and processed for resin (glutaraldehyde fixed samples) or cryostat sectioning (paraformaldehyde fixed samples) as previously described (Totoiu et al., 2004) . Briefly, 2-mm blocks were cut from 6 mm spinal cord sections both cranial and caudal to the site of transplantation, and even blocks were processed for immunohistochemistry, and odd numbered blocks were processed for resin embedding and toludine blue staining for a total of 3 blocks cut on either side of the transplantation site. For resin sectioning, blocks were exposed to 1% OsO4 (Electron Microscopy Sciences, Fort Washington, PA), dehydrated in ascending alcohols and embedded in Spurr resin (Electron Microscopy Sciences) according to standard protocols. Transverse 1-mm sections were cut from the cranial face of each block, stained with alkaline toluidine blue, cover slipped, and examined by light microscopy on an Olympus A×-80 microscope using OLYMPUS MicroSuite B3SV software. The state of myelination was determined by assessing the thickness of the myelin sheath in relation to the axon diameter (Guy et al., 1989; Hildebrand and Hahn, 1978) . Demyelinated, remyelinated, and normally myelinated axons were counted within 4 × 3750 μm 2 areas, totaling 15,000 μm 2 , on each tissue section using the 100× objective with a 2× optical zoom representing approximately 10% of the total area of remyelination within transplanted animals, which was determined by measuring the total area of remyelination sections from each block in each animal using the 40× objective and averaging areas from all animals in each group. These quantitative assessments were conducted throughout the region extending 6 mm cranial and 6 mm caudal to the transplantation site. The number of demyelinated axons, remyelinated axons, the total number of axons, and the percentage of remyelinated axons were determined for each of the four regions on each tissue block, averaged, then averaged across animals within each group for each tissue block as previously described (Totoiu et al., 2004) . Statistical analyses were conducted using SPSS software (Chicago, IL). For cryostat sectioning, blocks were cryoprotected in 30% sucrose solution in PBS, embedded in OCT (Fisher Scientific) and frozen sectioned in the transverse plane at 20 μm on a JUNG CM3000 Leica cryostat for anti-BrdU staining as previously described (Totoiu et al., 2004) .
Cells were obtained from pooled brains and spinal cords of mice based on a described protocol (Pewe et al., 1998) . A single cell suspension was obtained by grinding the tissue in sterile tissue culture plates on ice in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Cell suspensions were transferred to 15-ml conical tubes and Percoll (Amersham, Uppsala, Sweden) was added for a final concentration of 30%. One ml of 70% Percoll was underlaid, and the cells were spun at 1300 × g for 30 min at 4°C. Cells were removed from the interface, washed, and used for flow cytometry. FC receptors were blocked by incubating cells with a 1:200 dilution of rat anti-mouse CD16/32 (Fcγ III/II receptor) (Pharmingen, San Diego, CA) for 15 min at 4°C then subjected to staining. Activated macrophages/microglia were identified by dual staining with FITC-conjugated rat anti-mouse F4/80 (C1:A3-1; Serotec, Oxford, England) and PE-conjugated CD45 (Pharmingen). The presence of CD8 + T cells recognizing the immunodominant CD8 epitope located within the surface (S) glycoprotein within residues 510-518 (S510-518) was determined using PE-conjugated D b /S510-518 major histocompatibility class I tetramer (Trifilo et al., 2003) . Cell surface expression of CD8 was determined using APC-conjugated rat anti-mouse CD8 (Pharmingen). Cells were incubated with antibodies for 30 min at 4°C, washed, and analyzed on a FACScaliber (Becton Dickinson, Mountain View, CA). The presence of virus-specific CD4 + T cells was determined by intracellular cytokine staining for IFN-γ (PEconjugated anti-IFN-γ, XMG1.2, Pharmingen) following 5h stimulation with the immunodominant CD4 epitope present within the matrix (M) glycoprotein spanning residues 133-147 (M133-147) in medium containing GolgiStop using the Cytofix/Cytoperm kit (Pharmingen) according to the manufactures protocol (Glass et al., 2004) . Cells were washed and blocked with PBS containing 10% FBS and a 1:200 dilution of CD16/32. Cells were then stained with PE-conjugated CD4 for 30 min at 4°C, followed by fixing and staining for intracellular IFN-γ using for 30 min (Glass et al., 2004) . Cells were washed and analyzed on a FACScaliber. Data are presented as the percent positive within the gated population and then numerated based on the number of total live cells per tissue. Total numbers of cells were calculated by multiplying the percentage of positive live cells by the total number of isolated cells. The frequency of virus-specific cells was determined by dividing the total number of antigen-specific cells by the total number of gated cells.
Total RNA was extracted from brains and spinal cords of MHV-infected mice 33 days p.i., 21 days post-transplant. Cytokine, chemokine, and chemokine receptor transcripts were analyzed using custom probesets (Pharmingen). RPA analysis was performed with 12 μg of total RNA using a previously described protocol (Lane et al., 1998 . Probes for L32 and GAPDH were included to verify consistency in RNA loading and assay performance. For quantification of signal intensity, autoradiographs were scanned, and individual transcript bands were normalized as the ratio of band intensity to L32 (Lane et al., 1998 Liu et al., 2000) . Analysis was performed using NIH Image 1.61 software.
Splenocytes were obtained from MHV-infected mice 21 days post-transplant and stimulated with the MHV epitopes specific for either CD4 + T cells (M133-147) or CD8 epitopes (S510-518 and S598-605) (Held et al., 2004) . As control, an ovalbumin (OVA) peptide spanning residues 323-339 (OVA323-339) was used. After 48 h, supernatants were collected, and production of IFN-γ and IL-10 was determined using Quantikine M Mouse IFN-γ Immunoassay kit (R&D Systems, Minneapolis, MN) and mouse IL-10 DuoSet (R&D Systems). Samples were measured in duplicate and cytokine concentrations presented as picograms per milliliters.
Statistical differences between groups of mice were determined by Student's t test, and values of P ≤ 0.05 were considered significant.
Glial-committed progenitor cells were dissected from the striatal region of the brains of day 1 postnatal C57BL/6 mice and cultured for 6 days before use in transplant, resulting in the growth of numerous neurospheres containing progenitor cells (Figs. 1A, B) . These cells were characterized by immunostaining for NG2 (data not shown), confirming that the majority of cells are oligodendrocyte progenitor cells as previously reported (Totoiu et al., 2004) . Differentiation of these cells in vitro resulted in approximately 60-70% oligodendrocytes, 20-30% astrocytes, and 5% other cell types in agreement with previous studies (Totoiu et al., 2004) . Our results agree with previous findings that transplantation of glial-committed progenitor cells to mice chronically infected with MHV (day 12 p.i.) resulted in substantial remyelination 21 days post-transplant as evident by 
Given the importance of the inflammatory response in initiating and maintaining disease in MHV-infected mice Wu et al., 2000) , we sought to determine if one mechanism by which transplanted progenitor cells promoted remyelination was through modifying the severity of inflammation. To address this question, we first evaluated leukocyte infiltration into the CNS by flow cytometry at 10 days post-transplantation of glial-committed progenitor cells or DMEM as a control (22 days p.i.) (Fig. 2) . The overall frequency of infiltrating of CD4 + T cells, CD8 + T cells, and macrophages (F4/80 + CD45 high ) was not altered in mice receiving progenitor cells as compared to control mice (Fig. 2J ). Infiltration of virusspecific T cells was also investigated. The frequency of CD4 + T cells specific for the immunodominant epitope present within the matrix (M) protein at residues 133-147 (M133-147) was assessed by intracellular cytokine staining for IFN-γ after stimulation with the M33-147 peptide (Xue et al., 1995) . The frequency of M133-147-specific-CD4 + T cells was not modulated following transplant of glial-committed progenitor cells as compared to control mice (Fig. 2K) . Similarly, infiltration of virus-specific CD8 + T cells was determined using MHC tetramers containing the immunodominant CD8 epitope present within the surface (S) glycoprotein at residues 510-518 (S510-518) (Castro and Perlman, 1995) . The results of this analysis demonstrated that transplantation of cells did not alter the frequency of S510-518-specific CD8 + T cells in the CNS when compared to control mice (Fig. 2L) .
To confirm the lack of immunomodulation following stem cell transplant, we also looked at a later time point, 21 days following transplant (33 days p.i.). Analysis of inflammatory infiltrates into the CNS at 21 days post-transplant yielded similar results to that seen at 10 days post-transplant (Fig. 3) . Indeed, the percentage of infiltrating CD4 + T cells, CD8 + T cells, and CD45 high F4/80 + macrophages were equivalent in mice receiving cells or DMEM (Fig. 3A) . Further, the frequency of virus-specific CD4 + T cells, as assessed by intracellular cytokine staining for IFN-γ following stimulation with peptide M133-147, was similar in mice receiving cells or control DMEM (Fig. 3B ). In addition, the frequency of virus-specific CD8 + T cells, assessed by staining with the S510-518 tetramer, was identical in mice receiving cells or DMEM (Fig. 3C) .
To further characterize the impact of transplantation of glialcommitted progenitor cells on inflammation, we evaluated the expression of mRNA transcripts for chemokines, chemokine receptors, and cytokines within the CNS of experimental groups of mice. Total RNA was isolated from spinal cords and brains of experimental groups of mice at 21 days posttransplant (33 days p.i.) and expression of inflammatory factors and receptors were analyzed by RNase protection assay (RPA) (Fig. 4) . Numerous chemokines are expressed in the spinal cord at 33 days p.i., with CCL5, CCL2, CCL3, CCL4, and CXCL10 being readily detected in the spinal cord while only CCL5 and CXCL10 were detected in the brain. Transplantation of glial-committed progenitor cells did not alter chemokines expressed in either the spinal cord (Fig. 4B ) or the brain (C). Similar levels of mRNA transcripts for chemokine receptors CXCR4, CCR5, and CCR2 were detected within both the spinal cord (E) and brain (F) of mice receiving cells or DMEM. Additionally, expression of cytokines within the CNS was not modulated following cell transplant (Figs. 4H, I) with low level expression of both TNF-α and IL-1α observed in both groups of experimental animals.
Lastly, we sought to investigate any effect transplantation of glial-committed progenitor cells to MHV-infected mice may have on T H 1/T H 2 response to viral antigens and cytokine production. Spleens were removed from mice 21 days posttransplant (33 days p.i.) and pulsed with MHV CD8 epitopes S510-518 and S598-605, MHV CD4 epitope M133-147, or control peptide for 48 h following which secretion of IFN-γ (T H 1 associated) and IL-10 (T H 2 associated) were measured in cell supernatants by ELISA (Fig. 5) . Responses to CD8 and CD4 epitopes were not altered in splenocytes from mice receiving cell transplant, nor was there any skewing of the T H 1 response in favor of increased T H 2 cytokine production. One out of five mice receiving cells did produce a low amount of IL-10 in response to MHV CD8 and CD4 epitopes, however, this difference was not significant.
Therapeutic targets in MS include inflammation, demyelination, and axonal loss in chronic stages. Transplantation of gliogenic cell populations has the potential of addressing the latter two targets. Several reports have demonstrated the utility of transplantation therapies in experimental models of demyelination. Early studies by Brustle et al. (1999) showed that injection of embryonic stem cells into the spinal cord of myelindeficient rats results in numerous myelinated axons 2 weeks post-transplant. Similarly, transplantation of clonal mouse neural stem cells (NSCs) into mice after spinal cord injury has been shown to support axonal regeneration (Lu et al., 2003) . Successful transplants have been demonstrated using cells from a variety of sources. Bone marrow stromal cells were cultured in vitro and used for intrathecal transplantation into rats with gamma-irradiation or ethidium bromide lesions, resulting in remyelination and increased conduction velocity 3 weeks following transplant (Akiyama et al., 2002) . Further, transplantation of NSCs into various myelin deficient animals, such as the myelin-deficient rat (Hammang et al., 1997) , shiverer mouse (Mitome et al., 2001; Yandava et al., 1999) , and the MAG/Fyn- Fig. 3 . Flow analysis of leukocyte infiltration into the CNS 21 days posttransplant shows that there is no modulation of inflammatory populations within the CNS following transplant when compared to control mice. Leukocytes were isolated from the CNS of sham and infected mice which received a transplant of DMEM or cells, and flow cytometry was performed to determine infiltration of CD4 + T cells, CD8 + T cells, and F4/80 + CD45 high (macrophages) (A). Intracellular cytokine staining for IFN-γ was performed to determine the frequency of M133-147-specific CD4 + T cells (B); and tetramer staining for S510-518 was used to identify antigen-specific CD8 + T cells (C). deficient mouse (Ader et al., 2001 ) results in remyelination. Additional studies using NSCs isolated from mouse spinal cord showed that these cells have long-term survival in vivo, surviving up to 15 weeks following transplantation into hypomyelinated MAG/Fyn-deficient mice (Ader et al., 2004) . Collectively, these studies clearly demonstrate the efficacy of transplant-mediated CNS remyelination in animal models of dysmyelination and hypomyelination. Nonetheless, it is important to note that many animal models do not contain critical features of MS, including astrogliosis and the presence of inflammatory infiltrates.
Given the inflammatory nature of MS and the fact that current human therapies for MS are immunosuppressive in nature (Rizvi and Agius, 2004) , it is critical to understand the influence, if any, that inflammation has on the ability of a transplanted cell population to affect remyelination. Studies using EAE-induced demyelination have evaluated the efficacy of transplant-mediated remyelination in an inflammatory environment. Transplantation of clonal oligodendrocyte progenitor cells (OPCs) into rats prior to induction of EAE results in cell migration within the CNS localizing to inflammatory lesions and surviving up to 50 days post transplant, weeks after recovery from disease (Tourbah et al., 1997) . Likewise, transplantation of adult neural stem cells to mice after onset of EAE resulted in targeted migration of cells to areas of damage, improved clinical disease, as well as reduced demyelination and axonal loss (Pluchino et al., 2003) . Following remyelination of the rat spinal cord by mouse oligodendroglial cells, and subsequent rejection of those cells by removal of immunosuppression, host oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair (Blakemore et al., 1995) . These studies demonstrate that remyelination is possible during inflammation. Indeed, several studies indicate that inflammation enhances survival of transplanted cells, migration of transplanted cells, and remyelination (Foote and Blakemore, 2005 ; O'Leary and Blakemore, 1997; Tourbah et al., 1997) , and it has been suggested that oligodendrocyte progenitors within chronic multiple sclerosis lesions may require induction of inflammation to enable their proliferation and differentiation into myelinogenic cells (Foote and Blakemore, 2005) . Thus, it is important to understand the immune response during instances of successful remyelination in our effort to develop strategies to overcome remyelination failure.
Mechanisms by which transplanted cells facilitate remyelination include replenishing the OPC pool which ultimately enhances remyelination; suppressing local inflammatory gene expression thereby limiting access of inflammatory cells into the CNS; or through amplification of growth factors associated with stimulating endogenous cells to initiate repair. The timing of remyelination observed in previous studies suggests that transplanted cells may directly remyelinate damaged axons (Totoiu et al., 2004) . Furthermore, transplantation of OPCs derived from human embryonic stem cells into the shiverer model of dysmyelination results in differentiation of these cells into oligodendrocytes and results in compact myelin formation providing evidence that transplanted progenitors can differentiate and myelinate naked axons (Nistor et al., 2005) .
The present study examined the possibility that progenitor cell-induced remyelination was associated with attenuation of CNS inflammation which is strongly associated with ongoing myelin destruction in mice persistently infected with MHV. Transplant-mediated attenuation of inflammation would improve clinical score, might alter the capability of endogenous cells to affect remyelination, and is an important consideration in designing therapeutic approaches to treat human disease. A comprehensive overview of infiltration of leukocytes into the CNS was performed by FACS analysis, in which we found that intrathecal transplantation of progenitor cells into mice chronically infected with MHV did not modulate T cell or macrophage infiltration into the CNS at either 10 or 21 days following transplant (Figs. 2 and 3) . Moreover, a systematic assessment of expression of chemokines, chemokine receptors, and cytokines expressed in the CNS by RPA support the finding that inflammation is not modulated following progenitor cell transplantation (Fig. 4) . Further, MHV-specific immunity was not modulated following transplantation. Infiltration of antigenspecific CD4 + and CD8 + was similar in mice receiving transplant of progenitor cells or DMEM (Figs. 2 and 3) . Additionally, T H 1-cytokine expression by mouse splenocytes stimulated with MHV epitopes was not skewed in mice following transplantation (Fig. 5) .
These studies provide a detailed analysis of the state of the immune response following transplant and provide strong evidence that transplantation of glial-committed progenitor cells does not result in immune suppression. This is in contrast to a study in which prophylactic transplant of neural precursor cells (NPCs) into rats with acute EAE had immunosuppressive effects (Einstein et al., 2003) . This study reported reduced perivascular infiltrates in mice receiving NPCs as well as decreased proliferation of lymph node cultures when cocultured with NPCs. It is noteworthy that our studies differ from this report in that we evaluated the therapeutic effects of transplantation for treating demyelinating disease induced by a virus (MHV) in which the transplant was performed after disease onset. The timing of transplant is likely to be critical in influencing the development of an immune response or an ongoing immune response. Our finding that transplantation of glial-committed progenitor cells does not ameliorate inflammation are novel in that other therapies both clinical and experimental target inflammation to improve clinical disease. Specifically, experimental neutralization of chemokine CCL5 in mice chronically infected with MHV results in improved clinical and histological disease and reduces infiltration of leukocytes into the CNS as well as reduces infiltration of antigen-specific CD4 + and CD8 + T cells into the CNS (Glass et al., 2004) . It is possible that combining therapeutic neutralization of CCL5 with transplantation of glial-committed progenitor cells would synergize resulting in a greater degree of remyelination. Studies are underway to evaluate the benefit of such a combined therapy.
Our findings indicate that transplant-mediated remyelination is not mediated or accompanied by a reduced inflammatory response in the CNS. This is the first time in which repair and remyelination in mice chronically infected with MHV has been documented in the absence of reduced inflammation. These data suggest that remyelination can occur within an inflammatory environment and is not dependent upon modulation of inflammation. Other factors that may influence remyelination by transplanted glial-committed progenitor cells include expression of growth factors and extracellular matrix molecules. Further, these studies characterize the effect of transplantation on the immune response in the CNS, an important step in developing transplant-mediated therapies to treat demyelinating disorders. Importantly, transplantation does not reactivate the inflammatory process within the CNS. Additional studies are underway to understand mechanisms by which transplanted cells promote remyelination.