Combined Immunosuppressive Agents or CD4 Antibodies Prolong Survival of Human Ne - 《stemcells》干细胞学杂志干细胞之家



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作者：Jun Yana, Leyan Xua, Annie M. Welsha, David Chena, Thomas Hazele, Karl Johee, Vassilis E. Koliatsosa,b,c,d作者单位：aDepartment of Pathology, Division of Neuropathology, and Departments of & n" R- N: C$ M; @8 `/ |

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      【摘要】6 L3 z( B7 D" m8 F7 b& t1 `
   Amyotrophic lateral sclerosis (ALS) is a target for cell-replacement therapies, including therapies based on human neural stem cells (NSCs). These therapies must be first tested in the appropriate animal models, including transgenic rodents harboring superoxide dismutase (SOD1) mutations linked to familial ALS. However, these rodent subjects reject discordant xenografts. In the present investigation, we grafted NSCs from human embryonic spinal cord into the ventral lumbar cord of 2-month-old SOD1-G93A transgenic mice. Animals were immunosuppressed with FK506, FK506 plus rapamycin, FK506 plus rapamycin plus mycophenolate mofetil, or CD4 antibodies. With FK506 monotherapy, human NSC grafts were rejected within 1 week, whereas combinations of FK506 with one or two of the other agents or CD4 antibodies protected grafts into end-stage illness (i.e., more than 2 months after grafting). The combination of FK506 with rapamycin appeared to be optimal with respect to efficacy and simplicity of administration. Graft protection was achieved via the blockade of CD4- and CD8-cell infiltration and attenuation of the microglial phagocytic response from the host. Surviving NSCs differentiated extensively into neurons that began to establish networks with host nerve cells, including -motor neurons. Immunosuppressed animals with live cells showed later onset and a slower progression of motor neuron disease and lived longer compared with immunosuppressed control animals with dead NSC grafts. Our findings indicate that combined immunosuppression promotes the survival of human NSCs grafted in the spinal cord of SOD1-G93A mice and, in doing so, allows the differentiation of NSCs into neurons and leads to the improvement of key parameters of motor neuron disease. 9 r) P" [% S" D3 E
      【关键词】 Differentiation Motor neuron disease Motor neurons Regeneration Superoxide dismutase
   INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of motor neurons in the spinal cord and brain stem, as well as some pyramidal neurons in motor cortex, leading to muscle atrophy and eventual paralysis and death. Approximately 10%¨C13% of cases of ALS are familial, and 20% of these cases harbor mutations in the Cu/Zn superoxide dismutase (SOD1) gene .
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Human NSCs (i.e., cells that may eventually be used in clinical applications) are different from their rodent counterparts in important ways. For example, human NSCs proliferate and differentiate more slowly than rodent cells, and this property may alter the extent and/or rate of migration, axonal elongation, and synapse formation . In the present study, we aimed at optimizing human NSC xenograft survival in SOD1-G93A Tg mice with several combinations of immunosuppressive agents. Our results indicate that it is possible to achieve good survival and differentiation of human NSCs in the spinal cord of SOD1-G93A Tg mice and, in doing so, to improve key clinical parameters of rodent ALS.: E6 F8 f' i  T# I+ j( F# Q
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MATERIALS AND METHODS! }9 m4 p/ P# i2 |" w/ K
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Experimental Subjects and Design
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Heterozygous male B6SJL-TgN (SOD1-G93A) 1Gur mice were purchased from The Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and mated with wild-type females. Animal care and surgical procedures were carried out according to protocols approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions (JHMI). Heterozygous offspring of mixed gender were selected by genotyping at approximately 1 month of age.

Because, in pilot studies, we had failed to achieve acceptable human NSC graft survival with routine cyclosporin immunosuppression, the present study was designed with emphasis on alternative immunosuppressive regimens. Based on evidence for a strong T-cell-mediated rejection in pilot studies, our goal here was to use a potent combination of T-cell activation and proliferation blockers, capitalizing on commercially available calcineurin-dependent inhibitors (FK506, tacrolimus), non-calcineurin-mediated inhibitors (rapamycin, sirolimus), and inosine monophosphate dehydrogenase inhibitors (mycophenolate esters such as mycophenolate mofetil ). Thus, five experimental groups were given different immunosuppressive treatments: the first group was treated with FK506 monotherapy (Prograf; Fujisawa Healthcare, Inc., Deerfield, IL, http://www.us.astellas.com) (2 µg/g per day, n = 23); a second group was treated with a double immunosuppressive regimen combining FK506 and rapamycin (Calbiochem, San Diego, http://www.embbiosciences.com) (1 µg/g per day each, n = 21); a third group received triple immunosuppression with FK506, rapamycin, and MMF (CellCept; Roche, Nutley, NJ, http://www.rocheusa.com) (1 µg/g per day each for FK506 and rapamycin, 100 µg/g per day for MMF, n = 14); a fourth group was treated with CD4 antibodies (clone GK1.5, 20 µg/g per day, n = 12); and a fifth group (n = 10) was treated with FK506 plus rapamycin exactly as the second group, except that subjects were grafted with dead cells. Dead-cell grafts used cells that were exposed to repeated freezing-thawing and served to control for the potential therapeutic effects of immunosuppressants themselves on motor neuron disease. The FK506-rapamycin combination was the only treatment given to animals with dead-cell grafts because it is the simplest immunosuppressant regimen that protects grafts from rejection (see Results). Males and females were randomly admixed in the various experimental groups to minimize a systemic effect of gender on disease progression and treatment response.

A group of animals was euthanized at 1 week (FK506 monotherapy, n = 6; FK506 plus rapamycin treatment, n = 5; FK506 plus rapamycin plus MMF, n = 5; CD4 antibodies, n = 4), and another group was euthanized at 1 month (FK506, n = 5; FK506 plus rapamycin, n = 5; FK506 plus rapamycin plus MMF, n = 4; CD4 antibodies, n = 3) after grafting in order to monitor host immune response to grafted cells and graft survival. All others (FK506, n = 12; FK506 plus rapamycin, n = 11; FK506 plus rapamycin plus MMF, n = 5; CD4 antibodies, n = 5; FK506 plus rapamycin in animals grafted with dead cells, n = 10) were allowed to survive to end-stage illness (as defined in the "Motor Scoring and Other Tests for Clinical Outcomes" section of Materials and Methods; typically >2 months after grafting) in order for us to assess clinical outcomes as well as long-term graft survival and host responses.7 K2 J6 m1 Y8 z: {
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Derivation and Culture of Human NSCs5 e- u! Y$ a  [. ^) ?; q

Human NSCs were prepared from the cervical spinal cord of a single 8-week human fetus donated by the mother in a manner compliant with the guidelines of the National Institutes of Health and the U.S. Food and Drug Administration and approved by an outside independent review board. All JHMI institutional guidelines were followed in obtaining and using these cells in our laboratory. The initial culture was expanded as monolayer in poly-D-lysine and fibronectin-coated dishes using serum-free medium containing fibroblast growth factor (FGF)-2 as described . The resulting cell line, termed "566RSC," was passaged 10 to 12 times prior to grafting. Five to 7 days prior to surgery, one cryopreserved vial of cells was thawed, washed, and cultured again as monolayer. Cultures were seeded so as to reach confluence on the day of surgery. Cells were subsequently harvested by brief enzymatic treatment that deactivated FGF-2, washed in buffered saline, and used within 24 hours. As verified by trypan blue exclusion, viability of cells on ice was typically greater than 80% within this 24-hour period.

Preparation of Monoclonal Antibody Against Mouse CD4

The monoclonal antibody GK1.5, a rat immunoglobulin G (IgG) 2b directed against a surface epitope of mouse CD4 cells, has excellent in vivo efficacy in multiple models of T-cell-mediated rejection . The antibody was prepared from GK1.5 hybridoma cells (a gift from Dr. William Baldwin, Department of Pathology, JHMI) and purified from cell culture supernatant or ascites fluid with a protein G column. For cell culture, cells were plated in plastic flasks containing hybridoma-SFM (serum-free medium) medium with 1% ultra-low IgG fetal bovine serum and penicillin/streptomycin (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in a humidified cell culture incubator with CO2: ambient air = 5:95 (37¡ãC). For ascites preparation, 2-month-old male nude mice with the same strain background as SOD1-G93A (B6.Cg-Foxn 1nu; The Jackson Laboratory) were primed by injecting 0.5 ml of Freund¡¯s incomplete adjuvant 1 week before the injection of 1 x 106 hybridoma cells in 0.5 ml phosphate-buffered saline (PBS) i.p. Ascites fluid was collected 2 weeks later with multiple taps.

Conditioned medium or ascites fluid were centrifuged at 2,000g. Undiluted medium supernatant or ascites supernatant diluted 1:10 in 20 mM phosphate buffer (PB; pH 7.4) were loaded onto a 5-ml Hi-trap protein G column (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com). The column was washed with 20 mM PB (pH 7.4), and bound antibody was eluted with 0.1 M glycine-HCl (pH 2.9) and immediately neutralized with 1 M Tris-HCl (pH 9).

Surgical Procedures

Surgeries were carried out under gas anesthesia (enflurane/oxygen/nitrous oxide = 1:33:66) and aseptic conditions, with the aid of a Zeiss surgical microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) and a Kopf spinal stereotaxic unit (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com) fitted with a mouse mouth-and-nose device. NSCs were grafted via a dorsal laminectomy window into the ventral gray matter of the lower lumbar protuberance of 8-week-old SOD1 G93A mice. To achieve a good correspondence with L4-L5, injections were targeted to the portion of spinal cord immediately underneath the T12 vertebra. Cell suspensions were delivered under aseptic conditions via four injections aimed at ventral horn (2 x 104 NSCs in 0.5 µl per injection site, two injection sites on each side of the spinal cord 1 mm apart) with pulled, beveled glass micro-pipettes connected to 10-µl Hamilton microsyringes (Hamilton Company, Reno, NV, http://www.hamiltoncomp.com) via silastic tubing.
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Animals were treated with four different immunosuppressive regimens as outlined in the "Experimental Subjects and Design" section of Materials and Methods. Immunosuppressant compounds were given with daily i.p. injections beginning 1 day prior to grafting and ending on the day prior to euthanasia. To prepare FK506 for monotherapy, the commercially available FK506 solution (5 mg/ml) was diluted to 1 mg/ml with sterile distilled water. When FK506 was combined with rapamycin, 1 mg of commercially available rapamycin powder was first dissolved in 200 µl of dimethyl sulfoxide and then combined with 200 µl of the commercial FK506 solution (5 mg/ml); the mixture was then diluted to a final 2-ml volume with the addition of 1.6 ml of sterile distilled water. For triple immunosuppression with FK506, rapamycin, and MMF, rapamycin was mixed with FK506 first, and the dilution step was done with 1.6 ml of sterile distilled water in which 100 mg of MMF had been previously dissolved. GK1.5 antibodies (diluted 10 mg/ml in sterile PBS) were injected for a total of 9 days beginning 1 day prior to grafting and then for 5 consecutive days every 4 weeks until sacrifice .8 B: w) K$ {/ Q% p
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Motor Scoring and Other Tests for Clinical Outcomes
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Animals were weighed twice a week, and disease onset was defined as the time point when body weight started to decrease consecutively , primarily in order to distinguish between hindlimb and forelimb strength. The modified Wrathall scale scores were defined as follows: 5, normal gait; 4, mild deficits: hindlimbs lose some weight-bearing; 3, moderate deficits: hindlimbs lose most weight-bearing and toe clearance; 2, severe deficits: hindlimbs lose weight-bearing almost completely, and forelimbs begin to show signs of weakness; and 1, end-stage disease: hindlimbs are completely paralyzed, forelimbs are affected to various degrees, and the mouse cannot right itself when laid on its side. All animals were euthanized at stage 1.

Statistical Analysis of Clinical Outcomes

Clinical outcomes were studied on the same set of data originating from five concurrent treatment groups allowed to survive to terminal illness. These outcomes included disease onset and life span as well as disease progression based on consecutive motor scores (motor score curves) and Kaplan-Meier survival. In one type of analysis, these outcomes were compared among the four groups of subjects treated with live cells but with variable immunosuppression (i.e., FK506 ). Variance in disease onset and life span was analyzed with one-way analysis of variance (ANOVA), followed by a Fisher least significant difference (LSD) post hoc test. Variance in motor score curves was analyzed by repeated-measures ANOVA, followed by Fisher LSD testing. Statistical analysis of Kaplan-Meier survival was based on Log-rank testing.% k7 K# [, I2 X+ D! b( _
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In a second type of analysis, clinical outcomes were compared between two groups of animals, both of which were immunosuppressed with FK506 rapamycin, but one had received live cells whereas the other was treated with dead cells. Disease onset and life span between live- and dead-cell groups were compared with a Student¡¯s t test. Disease progression was compared in the two groups by analyzing differences in motor score and Kaplan-Meier survival curves as described above.
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Histology, Immunocytochemistry, and Microscopy

At end-stage disease and at 1 week and 1 month after grafting, mice were euthanized with an overdose of sodium pentobarbital (5 mg/100 g i.p.) followed by intracardiac perfusion with 4% freshly depolymerized, neutral-buffered paraformaldehyde. Spinal cord tissue blocks containing the entire lumbar region were dissected and immersion-fixed in paraformaldehyde for an additional 4 hours at room temperature after removing the dura. Tissues were then equilibrated in 30% sucrose and sectioned at the transverse plane (30 µm) on a freezing microtome.

Immunocytochemistry (ICC) studies focused on the differentiation of human NSCs, the structural integration of NSCs, and the characterization of type and intensity of immune response to the graft. Many of these experiments required dual-label immunofluorescence. After permeabilization with 0.1% Triton X-100 and nonspecific site blocking with 5% normal serum from the same species as the secondary antibodies, sections were incubated in primary antibodies in 1 mg/ml bovine serum albumin with 0.1% Triton X-100 (4¡ãC, overnight). Primary antibodies were used to address human (graft) versus mouse (host) cell identity, neuronal, astrocytic, and oligodendrocytic phenotype specification, and the type and intensity of host-versus-graft cellular response (supplemental online Table 1). The presence of human cells in mouse tissues can be reliably traced with antibodies against human nuclear antigen (HNu) . Control sections were stained by replacing the primary antibodies with pre-immune IgG from the same species of origin.
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Antigen-antibody binding sites were revealed with Cy2- or Cy3-conjugated secondary goat or donkey IgGs directed against the species of origin of the corresponding primary antibodies (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com). In most cases, Cy3-conjugated goat or donkey anti-mouse IgG was used to trace the human cell marker HNu, and Cy2-conjugated goat or donkey IgG was used for various cellular markers. Secondary antibody incubations were performed for 2¨C4 hours at room temperature. All sections were counterstained with the DNA dye 4,6-diamidino-2-phenylindole (DAPI) (blue) and then dehydrated and coverslipped with DPX (a mixture of distyrene, tricresyl phosphate, and xylene). In the few cases in which our experimental design required triple labeling, the third secondary antibody was coupled with the blue fluorescence dye AMCA (7-amino-4-methyl-coumarin-3-acetic acid; 1:200; Jackson ImmunoResearch Laboratories, Inc.) and DAPI counterstain was omitted. Sections were studied with a Zeiss Axiophot microscope equipped for epifluorescence, and images were captured with a Spot RT Slider digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, http://www.diaginc.com). Confocal microscopic images were captured and optically resectioned in the x- and y-axes using a Zeiss LSM 410 unit.) c# V" L" @, T6 Q8 h1 c; h3 z; ~

RESULTS
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Combined Immunosuppressive Agents or CD4 Antibodies Ameliorate Cell-Mediated Rejection and Improve Human NSC Graft Survival in SOD1-G93A Mice, a  a; r7 _: s# P8 ^

Graft rejection was studied with ICC for HNu (to mark graft-derived cells) and protein epitopes marking blood-borne immune cells (i.e., lymphocytes and natural killer  (Fig. 3). Cytological features and anatomical relationships with blood vessels also helped in the identification of immune cells. For example, blood-borne cells cluster primarily around blood vessels, although this pattern is less distinct with advanced rejection. Under all circumstances, blood-borne cells are rare in host tissues surrounding the grafts.

Figure 1. Effects of various immunosuppressive regimens on graft survival versus CD8 cell infiltration. Spinal cord sections were taken through the graft site and dually labeled with antibodies against human nuclear antigen (HNu) (red) and CD8 (green). Images were captured under epifluorescence, except in top inset of (A), which is a confocal image taken from the same section as the main panel. All insets, except the top one in (A), represent magnifications of the framed areas in main panels. Arrows in insets point to representative CD8  T cells. Arrowheads point to CD8  cellular debris. Cross-sections of some blood vessels are labeled with asterisks. (A, B): Representative sections of mice treated with FK506, surviving for 1 week (A) or 1 month (B) after grafting. Note many intact CD8  cells in (A) with their typical thin cytoplasm. Many CD8  cells are in close proximity to diffuse HNu immunoreactivity that gives the impression of scaffolding around green CD8  profiles (arrows in insets in  top inset). Abbreviations: Ab, antibody; FK, FK506; MMF, mycophenolate mofetil; mo, month; R, rapamycin; wk, week.

Figure 2. Effects of FK506 (A) and combined FK506 rapamycin treatment (B) on graft survival versus CD4 cell infiltration 1 week after grafting. Spinal cord sections were taken through the graft site and dually stained with antibodies against human nuclear antigen (HNu) (red) and CD4 (green). All images were captured under epifluorescence, and insets represent magnifications of framed areas in main panels, except the confocal image in top inset in (A). Note the sharp difference between non-nuclear HNu immunoreactivity in (A) and intense HNu immunoreactivity of densely packed nuclei in (B). (A): Arrows in insets point to representative CD4  cells. In contrast to CD8  cells in Figure 1A, most of these cells are not in close proximity to HNu-immunoreactive material. Cross-section of a blood vessel is labeled with an asterisk. (B): Arrowheads in inset point to CD4  cellular debris, which is the predominant CD4-immunoreactive structure in these preparations. Scale bars = 100 µm (main panels), 20 µm (all insets except confocal in ). Abbreviations: FK, FK506; R, rapamycin; wk, week.

Figure 3. Effects of various immunosuppressive regimens on graft survival versus microglia/macrophage infiltration. Spinal cord sections were dually stained with antibodies against human nuclear antigen (HNu) (red) and Iba-1 (green). Images were captured under epifluorescence, except in top inset of (A), which is a confocal image. All insets except the top one in (A) represent magnifications of framed areas in main panels. Arrows in insets point to representative microglial cells/macrophages. (A, B): Representative sections of mice treated with FK506 and surviving for 1 week (A) or 1 month (B) after grafting. Most Iba-1  cells in (A) show pyramidal shapes with substantial cytoplasm and short processes (i.e., cytological features of macrophages) (arrows in insets). Colocalization of HNu  material internal to the Iba-1  cell surface is evident in the confocal inset (arrow, top inset in ). Abbreviations: Ab, antibody; FK, FK506; MMF, mycophenolate mofetil; mo, month; R, rapamycin; wk, week.2 L$ L) f0 b2 }* m3 \
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CD4  and CD8  cell recruitment to grafting sites was similar in terms of cytology and perivascular clustering with all treatments and at all time points, but the CD4 response was predominant (Figs. 1 and 2). NK cells were not seen frequently in our preparations. A moderately intense NK cell response was seen in subjects treated with FK506 alone, primarily in perivascular locations with little parenchymal invasion (supplemental online Fig. 1A). The cytology of these DX-5  NK cells resembled that of CD4  and CD8  cells. In animals treated with combined immunosuppressive regimens, DX-5 immunoreactivity appeared primarily as debris without obvious cellular localization (supplemental online Fig. 1B, inset). Central nervous system (CNS) microglia was typically seen to invade the graft from surrounding tissues replete with microglial cells at various stages of transformation (Fig. 3). As we established , reactive microglia is featured by a scant cytoplasm and long, highly ramified processes, whereas phagocytic glia has retracted, thick processes and a more substantial cytoplasm volume.' q4 }+ d, q* X5 q8 X

With FK506 treatment alone, HNu staining fails to reveal graft-derived nuclei as early as 1 week after grafting (Figs. 1A, 2A, 3A; supplemental online Fig. 1A). Grafting sites are featured by a diffuse, non-nuclear HNu immunoreactivity often seen in close proximity to CD8  lymphocytes (Fig. 1A, insets) and, to a lesser degree, to NK cells (supplemental online Fig. 1A, insets), but not CD4  cells (Fig. 2A, insets). HNu immunoreactivity is often localized within the cytoplasm of microglial phagocytes (Fig. 3A, insets). One month after grafting, CD8 (Fig. 1B) and CD4 (data not shown) immunoreactivities remain strong in the graft region, but they are localized within small round structures without cellular organization that probably represent cellular debris. Very few live CD4 or CD8  cells are visible at the graft sites (Fig. 1B), a pattern indicating that active T-cell rejection is over by that time. Iba-1  microglial cells with phagocytic cytology are numerous at the graft site 1 week after grafting (Fig. 3A). At 1 month, microglial reaction remains strong, but many of these Iba-1  cells exhibit cytologies consistent with activated microglia (Fig. 3B).( q4 [+ }! ^; V6 |$ U1 U2 R: Q
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Combined treatment with FK506 and rapamycin has an impressive effect on graft survival, based on the presence of numerous dense, nuclear HNu  profiles in all our experimental subjects (Figs. 1C, 1D, 2B, 3C, 3D; supplemental online Fig. 1B). CD4 and CD8  cell infiltration is markedly diminished; many such cells appear to have died based on the early presence of CD4 or CD8  debris. Microglial response is moderate; the overwhelming majority of Iba-1  cells have the appearance of activated, but not phagocytic, microglia (Fig. 3; compare insets between panels C and A). A very low-level of CD4 or CD8  response is present at 1 month after grafting (Fig. 1D). Microglial reaction is still strong, and there is an apparent increase in the presence of macrophages at 1 month (Fig. 3D) and well into end-stage disease (not shown). The addition of MMF to the regimen of FK506 plus rapamycin had no apparent effects in graft survival and infiltration by CD4/CD8  cells (Fig. 1E) or by microglia/macrophages (Fig. 3E) compared with the double immunosuppressive regimen.8 V, o% ^& r) I( S

The use of CD4 antibodies also significantly promoted the viability of HNu  cells. Invasion of the graft by blood-borne lymphocytes (Fig. 1F) or microglial cells (Fig. 3F) was significantly attenuated. As with combined immunosuppressive treatments, the cytology of most Iba-1  cells resembled that of activated, but not phagocytic, microglia (Fig. 3F, inset). In concert, the combination of FK506 and rapamycin with or without MMF or the use of CD4 antibodies suppressed CD4- and CD8-cell infiltration and also significantly attenuated and delayed microglia-mediated phagocytosis.

Combined Immunosuppressive Agents and CD4 Antibodies Improve Disease Outcomes in SOD1-G93A Mice Grafted with Human NSCs8 B7 @3 G9 g' Z- Q. n

When treated with combined immunosuppressive drugs or anti-CD4 antibodies, SOD1-G93A mice grafted with live NSCs showed delayed disease onset, improved motor scores, and longer life spans compared with FK506 monotherapy (Fig. 4). Mice treated with FK506 alone had an average disease onset of 13.2 ¡À 1.8 weeks. Treatments with the GK1.5 antibody, FK506 rapamycin, and less so with FK506 rapamycin MMF all delayed time to disease onset (15.5 ¡À 1.3, 15.3 ¡À 1.0, and 14.5 ¡À 0.9 weeks, respectively) compared with the FK506 group (Fig. 4A). Post hoc testing reveals that individual comparisons between the anti-CD4 or FK506 rapamycin groups and FK506 group are significant, but the difference between FK506 rapamycin MMF and FK506 groups is not. Treatment with anti-CD4 antibody, FK506 rapamycin, and FK506 rapamycin MMF significantly extended the life span of NSC-grafted animals compared with FK506 treatment alone (20.3 ¡À 1.5, 19.7 ¡À 1.9, and 20.4 ¡À 1.7 weeks as compared with 17.6 ¡À 2.0 weeks) (Fig. 4B). Motor score testing also showed a delayed loss of muscle strength in the anti-CD4, FK506 rapamycin, and FK506 rapamycin MMF groups compared with FK506 treatment group (Fig. 4C). Log-rank testing of Kaplan-Meier survival curves also revealed significant differences among anti-CD4, FK506 rapamycin, FK506 rapamycin MMF, and FK506 groups. The use of Log-rank testing within pairs of treatment groups revealed significant differences between FK506 monotherapy and any of the other treatment groups (Fig. 4D). Based on the above data, the combined FK506 rapamycin regimen appears to optimize prevention of graft rejection, at least for the purpose of generating clinically meaningful differences in the time framework of motor neuron disease in SOD1-G93A mice.
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Figure 4. Combined immunosuppressive drugs or CD4 antibodies delay disease onset, improve motor scores, and extend life span of SOD1-G93A mice grafted with human neural stem cells and treated with combined immunosuppressants as compared with FK506 monotherapy. (A, B): These two graphs indicate that combined immunosuppressive treatments or CD4 antibodies delay disease onset (A) and extend the life span (B) of SOD1 mice. Variance in both measures is significant (analysis of variance : p = .0029 and p = .068, respectively). Fischer least significant difference (LSD) post hoc testing shows that the significance originates in differences between the combined treatment groups or the CD4 antibody group with the FK506 monotherapy group. Note that the FK506 rapamycin MMF group is not significantly different from the FK506 group with respect to disease onset. (C, D): Variance in the progression of muscle weakness (C) and in survival (D) among treatment groups. Muscle strength was scored with open-field testing as explained in Materials and Methods. Repeated-measures ANOVA followed by Fisher LSD post hoc testing of individual differences in (C) reveals significant differences comparing FK506 rapamycin or FK506 rapamycin MMF or anti-CD4 groups to FK506 group (p = .007, .008, and .023, respectively). Log-rank testing of Kaplan-Meier survival curves in (D) shows an overall significant variance (p = .011, 2 = 11.11). Using Log-rank testing for comparisons between pairs of groups reveals significant differences between anti-CD4 or FK506 rapamycin or FK506 rapamycin MMF and FK506 monotherapy (p = .023, .016, and .001, respectively). Group data are displayed as mean ¡À SD. (**, p  .01 based on post hoc testing). Abbreviations: FK, FK506; MMF, mycophenolate mofetil; Rapa, rapamycin.

To control for the potential effects of immunosuppressants on clinical parameters of motor neuron disease, we compared disease onset, motor score, and life span data between animals grafted with live-versus-dead human NSCs, all of which were treated with FK506 rapamycin. In animals grafted with live cells, disease onset was delayed by 2.1 weeks (Fig. 5A), and life span was extended for 1.7 weeks (Fig. 5B) compared with animals that had received dead cells (disease onset: 15.3 ¡À 1 vs. 13.1 ¡À 1.9 weeks; life span: 19.7 ¡À 1.9 vs. 18.0 ¡À 1.8 weeks). Motor score testing shows a later onset and slower progression of weakness in animals treated with live NSCs (p = .02) (Fig. 5C), and Kaplan-Meier survival analysis shows a significant difference between live- and dead-cell groups (p = .001) (Fig. 5D). These differences indicate that immunosuppressants influence key clinical parameters of motor neuron disease in SOD1-G93A mice via their protection of NSC survival and not via a direct protective effect on host motor neurons.% i. z9 \' z5 @$ s! k* b% l3 H
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Figure 5. Differences in clinical parameters of motor neuron disease between animals grafted with live or dead human neural stem cells (NSCs), all of which were optimally immunosuppressed with FK506 plus rapamycin. (A, B): Live NSC grafts delay disease onset (A) and extend the life span (B) of SOD1 mice compared with dead-cell grafts. On both measures, differences between the two groups are significant (Student¡¯s t test: p = .0025 and p = .012, respectively). (C, D): Differences in the progression of motor weakness (C) and in survival (D) between live- and dead-cell grafted groups. Repeated-measures analysis of variance followed by Fisher least significance difference post hoc testing of variance in (C) shows significant differences between live- and dead-cell grafted groups (p = .02). Log-rank testing of Kaplan-Meier survival curves (D) also shows significant differences between the two groups (p = .001, 2 = 11.11). Group data are displayed as mean ¡À SD. (*, p  .05; **, p  .01). Abbreviations: FK, FK506; Rapa, rapamycin.
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Grafted Human NSCs Survive to End-Stage Disease, Differentiate Predominantly into Neurons, and Establish Synaptic Contacts with Host Neurons

In end-stage animals (more than 2 months after grafting) treated with combinations of immunosuppressive agents or CD4 antibodies, the vast majority of human NSCs had differentiated into TUJ1  neurons (Fig. 6A). Confocal microscopy confirmed the colocalization of HNu (red) with TUJ1 (green) in the same cells (Fig. 6A'). Although numerous glial fibrillary acidic protein (GFAP)  astrocytic processes were present in the graft site (Fig. 6B), the colocalization of GFAP immunoreactivity with HNu  nuclei within the same cells was a rare phenomenon in the majority of grafts located within the spinal cord parenchyma (Fig. 6B). This profile suggests that most GFAP  processes at the graft site are of host origin. However, in portions of grafts located very close to meninges (pia), the frequency of HNu  cells colocalizing GFAP increased, including the occasional clustering of double-labeled cells (Fig. 6B'); this pattern shows a degree of plasticity in NSC differentiation determined by local factors in the spinal cord microenvironment. HNu  nuclear profiles did not colocalize with O4 or Rip immunoreactivity within the same cells, a pattern suggesting that human NSCs did not differentiate in the oligodendrocyte or Schwann cell lineage in our experimental settings (data not shown). A small number of human NSCs remained undifferentiated as nestin  cells (Fig. 6C, 6C').3 t) \9 x3 c: n3 l, b; K
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Figure 6. Differentiation of human neural stem cells (NSCs) into neurons and astrocytes in vivo. (A-A'): These two sections were dually stained for HNu (red) and TUJ1 (green) and illustrate the very high frequency of neurons derived from human NSCs using epifluorescence (A) and confocal microscopy (A'). (A) is a low-power image illustrating the marked enrichment of TUJ1 immunoreactivity in the graft compared with the surrounding host tissue. Confocal images in (A') illustrate the typical filamentous cytoplasmic TUJ1 immunoreactivity; image in main frame was optically resectioned in the x- and y-planes to confirm the intimate apposition of the two immunoreactivities within these densely clustered neuronal cell bodies. (B-B'): These sections were stained for HNu (red) and GFAP (green) and showcase the sparse astrocytic differentiation of human NSCs by epifluorescence (B) and confocal microscopy (B'). Note the presence of rare GFAP  cell bodies with enclosed HNu  nuclei in the graft (arrow in ) despite the presence of numerous GFAP  processes. Confocal microscopy (B') shows a small cluster of human NSC-derived GFAP  cells located close to the pial surface. Confocal sections have been processed as in (A'). (C, C'): These sections were stained for HNu (red) and nestin (green) and illustrate that some human NSCs retain stem cell properties, at least as indicated by their expression of high levels of nestin immunoreactivity by epifluorescence (C) and confocal microscopy (C'). Confocal images have been processed as in (A'). Arrows in (C) point to selected nestin  cells. Scale bars = 50 µm (A), 20 µm (B, C), and 10 µm (A', B', C'). Abbreviations: GFAP, glial fibrillary acidic protein; HNu, human nuclear antigen.
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When synaptic terminal markers specifying graft origin were combined with generic neuronal markers, large numbers of human synaptophysin  boutons (representing terminals of graft-derived neurons) were found apposed to host neurons, especially surviving -motor neurons (Fig. 7A). Confocal analysis of specimens from 1 month after grafting and end-stage animals shows anatomical patterns typical of synaptic contacts (Fig. 7A'). Conversely, HNu and TUJ1  cells (i.e., graft-derived neurons) were closely apposed to VGLUT1/2  terminals of mouse origin (Fig. 7B); the synaptic significance of such appositions was difficult to determine by confocal microscopy because of the high cell density of grafts (Fig. 7B'). Together, these patterns of reciprocal innervation between host and graft-derived neurons indicate a substantial degree of structural integration of NSC grafts within the host circuitry. This phenomenon is likely to account for the beneficial effects of NSCs on clinical outcomes laid out in the previous section.2 @9 I+ }' k: \# |

Figure 7. Reciprocal innervation between graft-derived and host neurons based on triple immunocytochemistry for human Syn, two VGLUT epitopes (1 and 2), and TUJ1. Human synaptophysin immunoreactivity is used as a selective marker for graft-derived synapses. VGLUT1/2 is an excitatory synaptic marker specifying host origin because of the absence of glutamatergic phenotypes in differentiated neural stem cells (NSCs). TUJ1 is a generic neuronal marker. Color representations of various immunoreactivities are specified on top of panels. (A, A'): These epifluorescent (A) and confocal (A') images taken through the ventral horn of a SOD1-G93A mouse 1 month after grafting show a TUJ1  (green) host -motor neuron (outlined by the three arrows in ). Confocal images have been optically resectioned in the x- and y-planes as explained in Figure 6A'. Scale bars = 20 µm (A, B), 10 µm (A'), and 5 µm (B'). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; HNu, human nuclear antigen; Syn, synaptophysin.

DISCUSSION

The goal of this study was to establish effective immunosuppressive regimens in order to prevent the rejection of human NSCs grafted in SOD1-G93A mice. Our findings indicate that combinations of immunosuppressive drugs have significantly better outcomes compared with FK506 monotherapy in preventing graft rejection and, apparently via their promotion of graft survival, improving key parameters of motor neuron disease in SOD1-G93A mice. Similar results were obtained with CD4 antibodies. The effective suppression of NSC graft rejection allowed sufficient time for the differentiation of grafted cells into neurons and the establishment of networks linking host and graft neurons. Based on our data, combined immunosuppressive regimens or CD4 antibodies appeared to protect NSC grafts by suppressing CD4- and CD8-cell recruitment into the graft area and attenuating the microglial phagocytic response from surrounding spinal cord tissues. NK cells were rarely seen at the graft sites, and this is consistent with previous findings pointing to the low significance of these cells in xenograft rejection in the brain . Within the clinical framework of our experimental design, the FK506 rapamycin combination was optimal due to its efficacy and relative simplicity.
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Xenograft rejection is a serious problem when studying cell grafts of human or other highly discordant mammal origin in rodent models. For example, human spinal cord-derived progenitors are rejected 4 weeks after grafting into the adult rat spinal cord despite the use of cyclosporine A .6 Y/ d4 v* \4 L2 e' z; R, Z
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Host rejection of xenografts is a complex response that is not fully understood. Although antibody-mediated mechanisms and participation of the complement have been implicated . Based on our findings, CD4 antibodies are a viable alternative to immunosuppressive drugs to prevent rejection of human xenografts. However, the efficacy of antibodies is comparable with that of combined immunosuppressants, and the cost of producing large antibody quantities by inducing ascites in nude mice is a significant disadvantage for their routine use in large-scale experiments.
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The use of combinations of immunosuppressants exploits some differences in their mechanisms of action without exposing animals to the side effects of extremely high doses of single compounds . However, the optimization of immunosuppressive treatments to prevent rejection of discordant neural xenografts in experimental animals has not been systematically studied. A potential concern is whether these immunosuppressive compounds can cross the blood-brain barrier (BBB). FK506 and rapamycin can readily cross the BBB, but there is no published work, nor does the manufacturer have any data, on the permeability of the BBB to MMF. However, CNS bioavailability is unlikely to influence the efficacy of these drugs whose primary mode of action is suppression of T-cell proliferation in sites outside the CNS.5 J6 P0 @! \4 {% ]" L

Besides their prevention of xenograft rejection, immunosuppressive compounds may also exert neuroprotective effects under certain conditions . As far as we know, rapamycin and MMF have not yet been tested. In our hands, cyclosporine A was ineffective in altering the course of motor neuron disease in SOD1-G93A mice. In addition, when FK506 and rapamycin were given in combination to mice grafted with dead human NSCs, they had no obvious effects in clinical outcomes. Therefore, in the context of the present grafting experiment, the therapeutic contributions of the immunosuppressive compounds themselves to Tg motor neuron disease were negligible.

CONCLUSION) Q! a* w# m3 W; q1 C

Treatments with immunosuppressant compounds or CD4 antibodies can be optimized in relatively simple regimens to allow for long-term survival and differentiation of human NSCs in the spinal cord of SOD1-G93A mice with motor neuron disease. The combination of FK506 with rapamycin is optimal with respect to efficacy and simplicity of administration. By protecting the neuronal differentiation and networking of human cells in the host spinal cord, immunosuppressive regimens allow the detection of significant clinical effects of human NSCs which merit further characterization in future studies.
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DISCLOSURES

T.H. and K.J. own stock in, have acted as consultants for, have performed contract work for, have a financial interest in, and have served either as an officer or member of the Board of NeuralStem, Inc.1 Y0 X% G1 }( y: w# T% w2 h* J0 l
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ACKNOWLEDGMENTS" H0 N: L6 I4 |; `; _3 v

We thank Dr. William Baldwin, Department of Pathology, Johns Hopkins Medical Institutions, for advice with the immunological aspects of the paper and gift of GK1.5 hybridoma cells. This work was supported by grants from the Muscular Dystrophy Association (MDA3493), the U.S. Public Health Service (NS45140-03), and the Robert Packard Center for ALS Research at Johns Hopkins. J.Y. and L.X. contributed equally to this work.+ ]- T  O- G5 q
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