Pluripotent Stem Cells Engrafted into the Normal or Lesioned Adult Rat Spinal Cord Are Restricted to a Glial Lineage

Experimental Neurology 167, 48 –58 (2001) doi:10.1006/exnr.2000.7536, available online at http://www.idealibrary.com on

Pluripotent Stem Cells Engrafted into the Normal or Lesioned Adult Rat Spinal Cord Are Restricted to a Glial Lineage Qi-lin Cao,* Y. Ping Zhang,* Russell M. Howard,* Winston M. Walters,† Pantelis Tsoulfas,† and Scott R. Whittemore* ,‡ *Department of Neurological Surgery and ‡Department of Anatomical Sciences & Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky 40202; and †The Miami Project and Department of Neurological Surgery, University of Miami School of Medicine, Miami, Florida 33136 Received June 12, 2000; accepted July 31, 2000

spinal cord, transplantation of allogeneic embryonic spinal cord or brain tissues has resulted in partial functional recovery (5, 20). However, the clinical use of fetal tissue for transplantation is limited by logistical, immunological (67), and ethical (25) considerations. Potential alternatives to primary CNS grafts for use in replacing lost endogenous neurons include immortalized neural precursor cells and CNS-derived stem cells (18, 65). Both cell types can be expanded through multiple passages, yet retain pluripotentiality, both in vitro and in vivo, to differentiate into neurons, astrocytes, or oligodendrocytes. Numerous investigators have used immortalized neural precursor cell lines in a variety of transplantation paradigms (64). These cell lines undergo ectopic, site-specific differentiation after transplantation into the developing brain and some differentiate into neurons in the adult CNS. However, the fact that they are immortalized with an oncogene may restrict the clinical utility of similarly derived human cell lines. Neural stem cells have been isolated from the adult (21, 27, 46, 49, 62) and embryonic (11, 12, 27, 28, 50) rodent CNS, as well as the fetal (6, 15, 47, 58) and adult (32) human CNS. These cells proliferate in response to mitogens and express the neural precursor intermediate filament nestin, but not more mature markers for differentiated neurons and glia. They are pluripotent in vitro, having the potential to differentiate into neurons, astrocytes, and oligodendrocytes with the absolute numbers of each differentiated cell type altered by specific growth factors (27, 63). After engraftment into the developing mouse CNS (6, 15) or into areas in the adult rat CNS undergoing neurogenesis, hippocampus, dentate gyrus, or subventricular zone (SVZ) (16, 18), these cells differentiate into neurons and glia with precise regional specificity. Stem cell grafts have partially remyelinated the myelin-deficient rodent spinal cord (7, 23, 68). These data support the potential therapeutic use of stem cells in spinal cord injury, where remyelination, neuronal replacement,

Proliferating populations of undifferentiated neural stem cells were isolated from the embryonic day 14 rat cerebral cortex or the adult rat subventricular zone. These cells were pluripotent through multiple passages, retaining the ability to differentiate in vitro into neurons, astrocytes, and oligodendrocytes. Two weeks to 2 months after engraftment of undifferentiated, BrdU-labeled stem cells into the normal adult spinal cord, large numbers of surviving cells were seen. The majority of the cells differentiated with astrocytic phenotype, although some oligodendrocytes and undifferentiated, nestin-positive cells were detected; NeuN-positive neurons were not seen. Labeled cells were also engrafted into the contused adult rat spinal cord (moderate NYU Impactor injury), either into the lesion cavity or into the white or gray matter both rostral and caudal to the injury epicenter. Up to 2 months postgrafting, the majority of cells either differentiated into GFAP-positive astrocytes or remained nestin positive. No BrdU-positive neurons or oligodendrocytes were observed. These results show robust survival of engrafted stem cells, but a differentiated phenotype restricted to glial lineages. We suggest that in vitro induction prior to transplantation will be necessary for these cells to differentiate into neurons or large numbers of oligodendrocytes. © 2001 Academic Press Key Words: stem cells; astrocytes; oligodendrocytes; transplantation; spinal cord; rat.

INTRODUCTION

The capacity for intrinsic repair after mammalian CNS injury is limited because the mature CNS can neither generate new neurons nor initiate substantive functional axonal regenerative responses to damage. At present, transplantation is the only means by which CNS neurons can be replaced, although the potential for inducing endogenous CNS stem cells to neuronally differentiate may prove feasible (10, 31). In the injured 0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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and regeneration of severed ascending and descending axons are necessary for functional repair. Here, we address the lineage restriction of stem cells engrafted into the normal and contused adult rat spinal cord. MATERIALS AND METHODS

Preparation of stem cells in vitro. Stem cells were prepared essentially as described by Johe et al. (27). The cerebral cortices of E14 Fischer 344 rats were carefully dissected in L15 medium and triturated with a P1000 pipette. The viability, assessed by trypan blue exclusion, averaged 70% with a range of 50 – 80%. Cells were plated at 10 4 viable cells/cm 2 on tissue culture dishes precoated with 15 ␮g/ml poly-L-ornithine and 1 ␮g/ml fibronectin (PO-FBN) and grown at 37°C in a 5% CO 2 water-jacketed incubator in DMEM/F12 medium containing N2 supplement and 10 ng/ml FGF2. FGF2 was added daily and the growth medium was changed every other day. After 4 days, the cells were passaged at 60 –70% confluence by brief incubation in HBSS (Ca 2⫹ and Mg 2⫹ free) and dislodging with a cell lifter. For differentiation in vitro, passage 2 (P2) cells were plated onto a PO-FBN-coated 12-well dish at density of 3 ⫻ 10 4/well. The day after plating, the FGF2 was removed from the medium, and 0.5% BSA was added. The cells were differentiated for 5 days and then fixed in 4% paraformaldehyde in PB buffer for immunocytochemical analysis. For grafting, undifferentiated P2 stem cells were labeled by incubation in 10 ␮M bromodeoxyuridine (BrdU) overnight. This resulted in over 90% labeling efficiency. Two hours before transplantation, the labeled cells were detached from the dishes as described above, collected by centrifugation at 1000xg for 5 min, and resuspended in 1 ml culture medium. After the cell count and viability assessment, the cell suspension was centrifuged a second time and resuspended in a smaller volume to give a density of 5 ⫻ 10 4 cells/␮l. The stem cells from the adult SVZ were dissociated as described previously (63). The cells were expanded, passaged, and labeled as described for the embryonic stem cells. Prior to initiation of the transplantation experiments, experiments were done to determine whether BrdU labeling affected differentiation and/or survival. Proliferating cells were labeled by incubation in 10 ␮M BrdU overnight. After washing three times in D/F, the cells were differentiated as described above. Transplantation procedures. The care and treatment of all animals were in strict accordance with NIH and institutional IACUC guidelines. Thirty-six adult female Fischer 344 rats (170 –200 g) were divided into three groups: cortical stem cell grafts into normal spinal cord, grafts into the cavity of injured spinal cord, and grafts one segment rostral and caudal to the lesion epicenter. Twelve more rats were grafted with the

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adult SVZ cells one segment rostral to the lesion epicenter. To graft stem cells in the normal spinal cord, rats were anesthetized with ketamine (0.8 mg/kg, ip) and xylazine (0.5 mg/kg, ip), a T 8 dorsal laminectomy was performed, and the T 7 and T 9 spinous processes were rigidly fixed in a spinal frame. A small parasagittal incision was made in the exposed dura overlying the dorsal surface of the spinal cord. Three microliters of labeled stem cells (1.5 ⫻ 10 5 cells) was injected unilaterally into the white matter in the ventral column and the gray matter in the ventral horn through a glass micropipette with an outer diameter of 50 –70 ␮m and the tip was sharp beveled to 30 –50 o. The microinjection system is powered by compressed 90% O 2:5% CO 2 (38). The muscle and skin were sutured and lactated Ringer’s solution and antibiotics administered to prevent dehydration and infection. The animals survived for 1, 2, 4, or 8 weeks. For stem cell grafts into the injured spinal cord, rats were prepared as described above, and 12.5-g/cm NYU Impactor contusion lesions were performed (22, 38, 41). Ten days postinjury, the spinal cord was reexposed and a small window was opened in the dura. Cells to be grafted into the lesion cavity were resuspended at 5 ⫻ 10 4/␮l in 1% fibrinogen, 8 mM CaCl 2, 0.02% gentamicin, and 0.015% aprotinin in DMEM and drawn into the glass micropipette. The micropipette was inserted through the hole in the dura into the center of lesion cavity and 10 ␮l of cell suspension was injected. For animals in which cells were grafted one segment rostral (T 7) and caudal (T 9) to the lesion epicenter (T 8), cells were suspended at 5 ⫻ 10 4/␮l in D/F and 3 ␮l was stereotactically injected unilaterally into the white matter in the ventral column and into the gray matter as described above. The animals were sacrificed at 1, 2, 4, or 8 weeks posttransplantation. Immunohistochemistry. Immunostaining of cell differentiation in vitro was performed as described previously (66) using Map2a,b (1:400, Sigma, St. Louis, MO), Rip (1:10, DSHB, Iowa City, IA), GFAP (1:400, Boehringer Mannheim, Indianapolis, IN), or nestin (1: 10, DSHB) antibodies. The specificity of staining was evaluated by substituting primary antibodies with normal mouse or sheep serum. No positive labeling was observed in the negative controls. For the grafting experiments, rats were anesthetized with 80 mg/kg Nembutal and perfused transcardially with 0.1 M PBS (pH 7.4), followed by 4% paraformaldehyde in 0.1 M PB. The spinal cord segments that received the grafts were removed, cryoprotected in 30% sucrose buffer overnight at 4°C, embedded in OCT compound, and transversely sectioned at 10 ␮m on a cryostat. Sections were mounted on gelatin-coated slides and stored at ⫺80°C. For staining, the slides were warmed for 20 min, treated in 2 N HCl for 30 min

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TABLE 1 Stem Cell Differentiation in Normal and Injured Adult Spinal Cord

Nestin-positive cells (%)

GFAP-positive cells (%)

mAb328positive cells (%)

Time postgraft

Normal cord

Injured cord

Normal cord

Injured cord

Normal cord

1 week 2 weeks 1 month 2 months

48.86 ⫾ 0.09 29.63 ⫾ 0.01 17.65 ⫾ 0.03 14.05 ⫾ 0.07

56.26 ⫾ 0.35 37.45 ⫾ 0.18 31.05 ⫾ 0.02 20.12 ⫾ 0.03

14.89 ⫾ 0.01 35.88 ⫾ 0.07 47.13 ⫾ 0.11 50.62 ⫾ 0.01

9.71 ⫾ 0.04 23.77 ⫾ 0.25 31.29 ⫾ 0.11 34.96 ⫾ 0.13

4.84 ⫾ 0.01 5.71 ⫾ 0.01 6.77 ⫾ 0.02 7.28 ⫾ 0.02

Note. Data represent the mean ⫾ SEM of the percentage of BrdU-labeled cells in a given microscopic field that express the indicated antigens. Twenty-four fields from 24 randomly chosen sections from two to three independent animals were used to calculate the percentages. The number of total cells/field averaged 94 ⫾ 11. The percentage of nestin-positive cells changed significantly along different postgraft time in both normal and injured spinal cord (ANOVA single factor, normal cord, F ⫽ 663, df ⫽ 3,80, P ⬍ 0.001; injured cord, F ⫽ 317, df ⫽ 3,102, P ⬍ 0.001), as well as the percentage of GFAP-positive cells (normal cord, F ⫽ 883, df ⫽ 3,80, P ⬍ 0.001; injured cord, F ⫽ 289, df ⫽ 3,102, P ⬍ 0.001), and the percentage of mAb328-positive cells in the normal cord (F ⫽ 20, df ⫽ 3,79, P ⬍ 0.01). Student–Newman–Keuls post hoc t test revealed that the percentage of nestin-positive cells in the normal cord is significantly lower than that in injured spinal cord at each postgraft time point (all P ⬍ 0.001). Moreover, the percentage of GFAP-positive cells in normal cord is significantly higher than that in injured cord at each postgraft time point (all P ⬍ 0.001).

at room temperature (RT), and rinsed in borate buffer and 0.1 M Tris-buffered saline (TBS), each for 10 min. After blocking with 10% donkey serum in TBS containing 0.3% Triton X-100 (TBST) for 1 h at RT, the sections were incubated in TBST containing 5% donkey serum, sheep anti-BrdU (1:100, Biodesign International; Kennebunk, ME) and either mouse anti-NeuN (1:200), mouse anti-GFAP (1:200), mouse anti-nestin (1:10), or mouse anti-myelin oligodendrocyte, mAb328 (1:50, Chemicon, Temecula, CA), overnight at 4°C. After three washes of 10 min in TBS, the sections were incubated in TBST containing 5% donkey serum, donkey anti-sheep FITC-conjugated Fab⬘ fragments (1:100, Jackson ImmunoResearch Lab, Baltimore, MD), and donkey anti-mouse Texas red-conjugated Fab⬘ fragments (1:200, Jackson ImmunoResearch Lab) for 1 h at RT. The sections were rinsed in TBS and coverslipped with antifade mounting medium (Molecular Probes, Eugene, OR). Quantification of stem cell differentiation. Cultured cells were immunostained exactly as described previously (63). Images of Hoechst dye staining (to identify the total number of cells in the field) and the respective antigens were captured with an Nikon TE300 inverted microscope equipped with an Optronix three-chip CCD camera and connected to a PowerMac 9600/300 equipped with Image ProPlus (Media Cybernetics, MO) software. The respective images were overlaid and the percentage of positive cells was calculated. Five random fields/well from three replicate wells/experiment were counted. For the grafted animals, sets of eight serial sections were placed on consecutive slides so that each slide contained every eighth section. The slides were immunohistochemically double stained for BrdU and each of

the following antibodies: nestin, mAb328, GFAP, or NeuN. The remaining four sets of slides were kept in case of technical difficulties. The double-stained sections were examined by confocal microscopy. In each animal, images obtained with a Leica TCS 4D confocal microscope were captured from 15 individual sections and two fields in each section. The number of BrdUlabeled nuclei and of double stained cells was counted by a blinded observer. The ratio of differentiated astrocytes, oligodendrocytes, and neurons relative to the total number of engrafted stem cells in each field was calculated. Statistical analyses. The percentages of GFAP and nestin cells in the normal and injured spinal cord were compared at each postgraft time point using Student’s t test (for equal or unequal variances, as applicable). Changes in the number of GFAP- and nestin-positive cells over different postgraft time points in the normal cord or injured cord were analyzed with repeated-measures ANOVA, followed by Student–Newman–Keuls post hoc t test. Differences were considered significant at the 0.05 level. RESULTS

Differentiation of embryonic neural stem cells in vitro. Similar to previous reports (30, 66), proliferating stem cells grown under these conditions have rounded, phase bright cell bodies with thin processes; an occasional cell displayed a flat appearance (Fig. 1A). All of these cells expressed nestin, an intermediate filament protein found in CNS precursors (35) (Fig. 1B). Withdrawal of FGF2 initiated differentiation within 24 h. After culture in D/F-N2 containing 0.5% BSA for 7 days, 32% of the cells differentiated into

GLIAL-RESTRICTED SPINAL CORD STEM CELL GRAFTS

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FIG. 1. Characterization of embryonic cortical neural stem cells in vitro. (A) Proliferating neural stem cells have round and phase bright cell bodies with thin process; an occasional cell will display a flat appearance (arrow). (B) Over 95% of proliferating stem cells express nestin. (C) The majority of proliferating stem cells are labeled after incubation in 10 mM BrdU overnight. (D–F) Removal of FGF2 induces these stem cells to differentiate into astrocytes (D), oligodendrocytes (E), and neurons (F), as shown by GFAP, RIP, and MAP2a,b immunohistochemistry, respectively. BrdU labeling did not alter the differentiation of embryonic stem cells into astrocytes (G), oligodendrocyte (H), or neurons (I). Data in (G–I) represent the mean ⫾ SEM of the percentage of total cells that express the indicated antigens from five fields per well and three replicate wells per experiment from three independent experiments. Analysis of variance revealed no differences between the control and BrdU-labeled cells for any of the indicated cell types (GFAP, t ⫽ 0.25, df ⫽ 15, P ⫽ 0.81; RIP, t ⫽ ⫺0.49, df ⫽ 15, P ⫽ 0.63; Map2a,b, t ⫽ ⫺0.69, df ⫽ 15, P ⫽ 50). Bar, 100 ␮m. FIG. 2. Survival of embryonic neural stem cell 2 weeks after engraftment into the normal adult spinal cord. (A) Surviving BrdU-stained cells in the ventral column white matter. The box in (A) is seen at higher magnification in (B). Bar, 400 ␮m.

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FIG. 3. Differentiation of embryonic cortical stem cells 1 week after engraftment into the normal spinal cord. (A) The engrafted cells, revealed by BrdU immunohistochemistry, were in the ventral column white matter and ventral horn gray matter. The photomicrograph encompasses the entire ventral half of the spinal cord and the base of the ventral median fissure is indicated by the arrowhead. (B–E) Double-labeled confocal images revealed that many of the engrafted cells still expressed nestin (B, arrowheads), some were GFAP-positive astrocytes (C, arrowheads), and few differentiated into mAb328-positive oligodendrocytes (D, arrowheads). Colocalization of BrdU and NeuN was not detected (E). BrdU is visualized with FITC and the indicated antigens with Texas red. Bar, 200 mm. FIG. 4. Differentiation of embryonic cortical stem cells 2 months after engraftment into the normal adult spinal cord. (A) BrdU immunohistochemistry showed that the cells survived in the ventral column white matter and ventral horn gray matter. The central canal is marked with an asterisk. Confocal images of sections double labeled for BrdU (FITC) and the indicated antigens (Texas red) revealed that although some stem cells still expressed nestin (B, arrowheads), most of them were GFAP positive (C, arrowheads). Some cells also expressed mAb328 (D, arrowheads), but no double staining for BrdU and NeuN was observed (E). Bar, 200 ␮m.

GFAP-expressing astrocytes (Figs. 1D and 1G), 15% differentiated into oligodendrocytes expressing Rip (Figs. 1E and 1H), and 13% differentiated into neurons expressing Map2a,b (Figs. 1F and 1I). When incubated in 10 ␮M BrdU overnight, ⬎95% of cells were labeled (Fig. 1C). To test if BrdU labeling affects stem cell differentiation or survival, sister cultures of unlabeled and BrdU-labeled cells were differentiated for 7 days as described above and subse-

quently immunostained for the expression of GFAP, Rip, and Map2a,b. As seen in Fig. 1, labeling with BrdU did not alter the number of differentiated astrocytes (Fig. 1G), oligodendrocytes (Fig. 1H), or neurons (Fig. 1I). Differentiation of embryonic neural stem cells in the normal spinal cord. Grafted stem cells, detected by BrdU immunohistochemistry, survived in all rats at each different time point and were observed 1.5–2 mm

FIG. 5. Differentiation of embryonic cortical stem cells 1 week after engraftment into the injured spinal cord. (A) BrdU immunostaining revealed that engrafted stem cells were distributed from the ventral to dorsal column in the ipsilateral injection site with some scattered in the contralateral side. The photomicrograph encompasses the entire ventral half of the spinal cord and the base of the ventral median fissure is indicated by the arrow. The boxed region in (A) is shown at higher magnification in (B–D). Confocal images of sections double stained for BrdU (FITC) and the indicated antigens (Texas red) revealed that most of the stem cells were nestin positive (B), while a few expressed GFAP (C). Expression of mAb328 was not observed (D). Bar, 200 ␮m. FIG. 6. Differentiation of embryonic cortical stem cells 2 months after engraftment into the injured spinal cord. (A) BrdU immunostaining revealed that engrafted stem cells were distributed around the cavity. The base of the ventral median fissure is indicated by the arrow. The boxed region in (A) is shown at higher magnification in (B–D). Confocal images of sections double stained for BrdU (FITC) and the indicated antigens (Texas red) revealed that some cells expressed nestin (B), but many were now GFAP positive (C). However, expression of mAb328 was still not observed (D). Bar, 200 ␮m. FIG. 7. Differentiation of embryonic cortical stem cells 1 month after engraftment into the cavity of injured spinal cord. (A) BrdU immunostaining revealed that many cells survived in the cavity. The base of the ventral median fissure is indicated by the arrow. The boxed region in (A) is shown at higher magnification in (B–D). Confocal images of sections double stained for BrdU (FITC) and the indicated antigens (Texas red) revealed that many of the engrafted stem cells expressed nestin (B), while fewer numbers of cells were GFAP positive (C). No expression of mAb328 was detected (D). Bar, 200 ␮m. FIG. 8. Differentiation of adult SVZ neural stem cells 2 weeks postengraftment in the injured spinal cord. (A) BrdU immunostaining revealed that engrafted cells were distributed in both sides of the ventral spinal cord. The base of the ventral median fissure is indicated by the arrow. The boxed region in (A) is shown at higher magnification in (B–D). Confocal images of sections double stained for BrdU (FITC) and the indicated antigens (Texas red) revealed that many cells were nestin positive (B), while some were GFAP positive (C). No evidence of oligodendrocyte differentiation was observed (D). Bar, 200 ␮m.

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rostal– caudally from the injection site (Figs. 2A and 2B). In the first week after transplantation, 49% of the engrafted BrdU-positive cells remained undifferentiated, still expressing nestin (Fig. 3B, Table 1), and fewer cells (15%) differentiated into GFAP-expressing astrocytes (Fig. 3C, Table 1). As nestin can be expressed in reactive astrocytes (9, 17), some graft-derived astrocytes may also be nestin positive. Small numbers (5%) of mAb328-positive oligodendrocytes were observed (Fig. 3D, Table 1). The mAb328 antibody was used to identify oligodendrocytes because the harsh acid treatment needed for BrdU detection is not compatible with the Rip antibody, which was used in the in vitro studies. No BrdU-positive cells expressed NeuN (Fig. 3E). The grafted stem cells also did not express the earlier neuronal marker, Map2a,b (data not shown), indicating that the failure to detect neurons in vivo was not because the stem cells initiated neuronal differentiation, but not to the more mature NeuN-positive stage. As shown in Table 1, by 2 and 4 weeks posttransplantation, the proportion of nestinpositive cells progressively decreased to 30 and 18%, respectively, while the number of differentiating astrocytes increased dramatically to 36 and 47%, respectively. A small percentage of BrdU-positive cells differentiated into mAb328-expressing oligodendrocytes at 2 (6%) and 4 (7%) weeks postgrafting. By 2 months postgrafting, the longest time postgraft examined, fewer cells (14%) still expressed nestin (Fig. 4B, Table 1). Most of the grafted stem cells (51%) were GFAP positive (Fig. 4C, Table 1), while 7% of the cells differentiated into oligodendrocytes expressing mAb328 (Fig. 4D, Table 1). No BrdU cells were found to express NeuN at any time point (Fig. 4E). Statistical analyses revealed that the temporal changes in nestin-positive precursor cells, GFAP-positive astrocytes, and mAb328-positive oligodendrocytes were highly significant at each time point (see legend to Table 1). Differentiation of embryonic neural stem cells in the lesioned spinal cord. The engrafted stem cells survived in all of the injured animals. In most animals, the cells remained in the ventral part of the cord at the injection site. In the longest surviving animals, many cells were distributed around the cavity. Compared to grafts in the normal spinal cord, the differentiation of cells engrafted one segment rostral and caudal to the injury site and the lesion cavity was delayed. In the first week, over 56% of the cells still expressed nestin (Fig. 5B, Table 1). Although very strong GFAP immunoreactivity was found in the remaining spinal cord around the cavity, a low percentage of BrdU-positive cells (10%) expressed GFAP (Fig. 5C, Table 1). In both the epicenter grafts and the grafts one segment rostral or caudal to the center, no BrdU-positive cells were found that expressed either mAb328 (Fig. 5D) or NeuN (data not shown).

At 2 and 4 weeks posttransplantation, the proportion of cells expressing GFAP increased to 24 and 31%, respectively, but 37 and 31%, respectively, of the BrdU-positive cells still expressed nestin. At 2 months posttransplantation, the percentage of cells expressing nestin further decreased to 20% (Fig. 6B, Table 1), while the proportion of cells that expressed GFAP increased to 35% (Fig. 6C, Table 1). However, BrdU-positive cells that expressed NeuN or mAb328 (Fig. 6D) were still not detected. Statistical analyses revealed that the temporal changes in nestin-positive precursor cells, GFAP-positive astrocytes, and mAb328-positive oligodendrocytes were highly significant at each time point. Moreover, the differences in the number of nestin-positive precursor cells, GFAP-positive astrocytes, and mAb328-positive oligodendrocytes at each time point were significantly different in the grafts of normal and lesioned spinal cord (see legend to Table 1). Differentiation of adult SVZ neural stem cells in the lesioned spinal cord. Previous studies in our (63) and other laboratories (21, 27) have shown that stem cells from the adult SVZ are pluripotent in vitro, retaining the ability to differentiate into neurons, astrocytes, and oligodendrocytes. To compare the in vivo differentiation of adult SVZ and embryonic cortical neural stem cells, we transplanted the adult SVZ cells into the lesioned spinal cord. The differentiation of adult SVZ neuronal stem cells in the lesioned spinal cord was similar to the cells from embryonic cortex. Two weeks after transplantation, most cells expressed nestin (Fig. 8B), while only a few GFAP-immunoreactive cells were observed (Fig. 8C). No BrdU-positive cells expressed mAb328 (Fig. 8D) or NeuN (data not shown). At longer survival times (1 or 2 months; Fig. 7), the percentage of cells that expressed nestin decreased, while the proportion of cells that expressed GFAP increased. No BrdU cells were double stained with mAb328 or NeuN (data not shown). DISCUSSION

After engraftment into normal spinal cord white and gray matter, all embryonic cortical and adult SVZ stem cells differentiated into glia. Most were astrocytes, but small numbers of oligodendrocytes were observed. Neuronal differentiation was not detected. The lack of stem-cell-derived neurons in vivo is unlikely to result from sensitivity of differentiating neuroblasts to BrdU labeling as similar numbers of differentiated neurons, oligodendrocytes, and astrocytes were observed in the absence and presence of BrdU labeling in vitro. Thus, the differentiation of neural stem cells in the normal adult spinal cord is restricted to the glial lineage. This result is in agreement with grafting experiments in adult brain. When neural stem cells isolated from rodent (18) or human (16) embryonic cortex were en-

GLIAL-RESTRICTED SPINAL CORD STEM CELL GRAFTS

grafted into the adult rat striatum, the majority of cells became astrocytes and oligodendrocytes. Studies engrafting pluripotential immortalized neural precursor cells into the normal adult CNS have shown that neurons are only seen in regions where neurogenesis is still ongoing (16, 18, 56). These results suggest that the developmentally relevant neurogenic signals for grafted undifferentiated stem cells may be absent or down-regulated with progressive maturation of the CNS. Neuronal development in the spinal cord follows a precise temporal sequence of gene expression resulting in competence to respond to the local developmental cues (26). The immature neural precursor cells may lack the appropriate receptor(s) or signaling pathway(s) necessary to respond to local developmental cues. Early defining developmental steps that occur during neurogenesis may be required before later differentiative sequences can be initiated. Clearly the intrinsic state of the engrafted neural stem cells is also important in determining their differentiating phenotype. For example, undifferentiated neural precursor cells engrafted into the adult neocortex where pyramidal neurons were induced to degenerate failed to undergo neuronal differentiation; some astrocytes differentiation was observed (52). However, if the later stage precursors and immature neurons were transplanted into such area, the engrafted cells were found to follow pyramidal neuron differentiation (24, 37, 57). Similarly, when undifferentiated stem cells were engrafted into the lesioned striatum, no neuronal differentiation was observed (59). But, if stem cells were differentiated 5 days in vitro prior to transplantation, the engrafted cells survived and both neuronal and astrocytic differentiation was seen (61). Thus, the ability of donor cells to respond to extracellular signals may depend critically on their level of maturation and state of differentiation, suggesting the possibility that manipulation of more restricted progenitors could result in increased efficiency of complex circuit reconstruction in the damaged CNS. Large numbers of neurons were observed after engrafting the raphe-derived RN33B cell line into the uninjured adult rat brain and spinal cord (43, 44, 53–55), but this is a neuronally restricted precursor cell line (66). The small number of oligodendrocytes observed after stem cell grafts in the normal spinal cord was unexpected, as oligodendrocytes and astrocytes derive from a common glial precursor (48). It is likely that the intact, fully myelinated spinal cord does not provide the epigenetic cues necessary for oligodendrocyte differentiation. Consistent with this suggestion, Hammang et al. (23) did demonstrate some stem cell remyelination following engraftment into the early postnatal myelin-deficient rat. Thus, either the early postnatal rat spinal cord expresses signals instructive for oligodendrocyte differentiation that are not expressed in the adult spinal cord or such signals are

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expressed on the demyelinated axons. Both explanations may account for these latter results, as axons in the corticospinal tracts do not become myelinated until approximately postnatal days 25–30 (13) and unmyelinated axons induce oligodendrocytes to myelinate them (1, 8). Engraftment of undifferentiated neural stem cells into the injured spinal cord showed that a larger proportion of cells remained undifferentiated, that astrocytic differentiation had been delayed, and that oligodendrocyte differentiation was inhibited. These results suggest that the injured spinal cord is a more restrictive environment for the differentiation of neural stem cells. These data are also consistent with previous studies. For example, transplantation of the immortalized neuronal precursor cell line RN33B into the intact spinal cord resulted in differentiated neurons with a complex multipolar morphologies. In the lesioned spinal cord, the engrafted RN33B cells were relatively undifferentiated or differentiated with bipolar morphologies (43). The neuronal differentiation of RN33B cells in the injured hippocampus (55) and striatum (36) was also inhibited. The oligodendrocyte differentiation observed in the normal spinal cord was not found in the injured spinal cord. These results were counter-intuitive as contusive SCI results in demyelination (4) and stem cells can partially remyelinate the early postnatal myelin-deficient spinal cord (23). However, the competence of engrafted stem cells to remyelinate adult axons should be examined in a lesion model in which all axons are intact, rather than the severed axons that result from the contusion injury. Importantly, traumatic SCI cause changes in the activation state of microglia or macrophages (29, 30) and the expression of various neurotrophic factors (42), cytokines (3), and neurite outgrowth inhibitory molecules (51). All of these influence neuronal and/or glial differentiation and/or regeneration and likely have complicated, synergistic, and/or antagonistic effects on neural stem cell differentiation in vivo. Importantly, both ciliary neurotrophic factor (34, 45) and leukemia inhibitory factor (2, 33) increase following CNS injury. These factors induce astrocytic differentiation of stem cells (27, 63) and this induction is dominant over signals from other growth factors that induce neuronal or oligodendrocytic differentiation (P. Tsoulfax and S. R. Whittemore, unpublished observations). This may explain the observation that only astrocyte differentiation was seen in the injured spinal cord. In contrast to this study, McDonald et al. (40) recently reported that retinoic acid-treated mouse embryonic stem (ES) cells grafted into the contused adult rat spinal cord differentiated predominantly into oligodendrocytes as well as small percentage of astrocytes and neurons. Differences between rat and mouse stem cells have been reported (60). While the blastocyst-

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derived mouse ES cells and the embryonic rat cortical stem cells similarly differentiate into neurons, astrocytes, and oligodendrocytes in vitro, there may be species-specific differences in their competence to respond to epigenetic signals in the adult rat spinal cord. The precise explanation for these different results remains to be elucidated. After engraftment of adult SVZ stem cells into the injured spinal cord, only astrocytic differentiation was observed. No oligodendrocytes or neurons were detected. The adult SVZ stem cells followed the same lineage restriction in the injured spinal cord as the embryonic cortical neural stem cells. These results are consistent with in vitro studies from our (64) and other (27) laboratories that showed that the in vitro proliferation and differentiation are identical for the embryonic and adult SVZ neural stem cells. As the environment in adult spinal cord restricted neural stem cell differentiation along a glial lineage, it is likely that manipulation of both embryonic and adult stem cells in vitro prior to transplantation will be needed to induce neuronal differentiation, as well as increase the number of differentiating oligodendrocytes. Alternatively, neuronal-restricted (39) or glialrestricted (48) precursors may enable neuronal replacement or remyelination, respectively, in the injured spinal cord. Additional injury-induced signals clearly also have the potential to influence the ultimate differentiated phenotype. Understanding the molecular mechanisms that control the fate of the neural stem cells after transplantation into specific regions of the injured CNS under defined experimental conditions must be carefully delineated before their clinical use can be contemplated.

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ACKNOWLEDGMENTS 14. The Rip hybridoma and nestin antibody developed by Susan Hockfield were obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA, under Contract NO1-HD-7-3263 from the NICHD. This project has been funded in whole or in part with Federal Funds from the National Institute of Neurological Disorders and Stroke, National Institutes of Health, under Contract N01-NS6-2349. Additional support was obtained from NS26887 (S.R.W), the Paralysis Project of America (Q.L.C. and P.T.), the Miami Project to Cure Paralysis (P.T.), The Lucille P. Markey Charitable Trust, and The FaBene Foundation (S.R.W., P.T.). The assistance of Ms. Darlene Burke with the statistical analyses and Drs. Alan Dozier and Fred Roisen with the confocal imaging and the critical comments of Drs. Stephen M. Onifer and David S. K. Magnuson are greatly appreciated.

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