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Abstract

Allografts continue to be used in clinical neurotransplantation studies; hence, it is crucial to understand the mechanisms that govern allograft tolerance. We investigated the impact of transplantation site within the brain on graft survival. Mouse [Friend leukemia virus, strain B (FVB)] glial precursors, transfected with luciferase, were injected (3 × 10⁵) into the forceps minor (FM) or striatum (STR). Immunodeficient rag2^−/- and immuno-competent BALB/c mice were used as recipients. Magnetic resonance imaging (MRI) confirmed that cells were precisely deposited at the selected coordinates. The graft viability was assessed noninvasively with biolumi-nescent imaging (BLI) for a period of 16 days. Regardless of implantation site, all grafts (n = 10) deposited in immunodeficient animals revealed excellent survival. In contrast, immunocompetent animals only accepted grafts at the STR site (n = 10), whereas all the FM grafts were rejected (n = 10). To investigate the factors that led to rejection of FM grafts, with acceptance of STR grafts, another group of animals (n = 19) was sacrificed during the prerejection period, on day 5. Near-infrared fluorescence imaging with IRDye 800CW–polyethylene glycol probe displayed similar blood–brain barrier disruption at both graft locations. The morphological distribution of FM grafts was cylindrical, parallel to the needle track, whereas cells transplanted into the STR accumulated along the border between the STR and the corpus callosum. There was significantly less infiltration by both innate and adaptive immune cells in the STR grafts, especially along the calloso-striatal border. With allograft survival being dependent on the transplantation site, the anatomical coordinates of the graft target should always be taken into account as it may determine the success or failure of therapy.

Keywords

Transplantation Brain Rejection Stem cells Glial progenitors

Introduction

Cell therapy is a promising approach to overcome the limitations of current treatment methods of neurological disorders (25). Autologous cell sources for neurotrans-plantation including bone marrow or umbilical cord blood have been shown to result in therapeutic effects (16,36), but overall they have a low therapeutic efficacy and, as such, are unacceptable for broad clinical use. A greater potential for clinical neurotransplantation has been assigned to the use of primary neural progenitors (13,18,19,33). The derivation of such progenitors from autologous sources is not feasible, and hence its use relies on allografting. Despite the immunoprivileged environment of the brain, allotransplantation is associated with the risk of graft rejection (9). The differentiation of pluri-potent stem cells toward somatic cells offers the potential for the generation of patient-specific cells of any type and can be expected to eliminate the immune response as an impediment to successful cell transplantation (40). Unfortunately, it has been shown that an autografted progeny of induced pluripotent stem cells can elicit a host-immune response, subsequently leading to graft loss (45). Thus, further investigations designed to prevent allograft rejection are necessary before widespread clinical studies can be implemented.

There appears to be no general consent about allograft survival within the central nervous system (CNS). Although some studies reported an excellent survival (4), others clearly observed rejection of allotransplanted cells (5,10,22,24,34,41). Immunogenicity of neural progenitors has been demonstrated in vitro by targeting with allogeneic T and natural killer (NK) cells (35). Previous clinical histopathological studies did not reveal a robust ongoing immune response against allografts; however, tissues were obtained several years posttransplantation (14,20,28). It has recently been shown for a patient who died early after transplantation that allografted cells are subject to robust immune cell infiltration with direct contact of transplanted cells and leukocytes in the Virchow– Robin spaces (7). Host alloimmunization against cerebral allografts is present in almost half of patients with neu-rodegenerative disease, despite immunosuppression (21). Thus, host–graft interactions within the CNS may be more complex than previously thought, and initial immune response can have long-lasting consequences, even with gradual clearance of immune cells from the graft site (6).

Fetal-derived human glial-restricted progenitors (GRPs) have been shown to induce a robust myelination in hypomyelinated immunodeficient animals, ultimately restoring their normal life span (43). However, their clinical translation is in need of a thorough preclinical evaluation of allografting in order to minimize the risk of complications. We previously reported an excellent survival of grafts deposited into the corpus callosum for immunodeficient recombination activating gene 2 knockout (rag2^−/-) mice. In contrast, in immunocompe-tent animals, graft survival was compromised in many animals despite the use of immunosuppressive drug (15). Interestingly, up to 30% of grafts survived in immunocom-petent, nonimmunosuppressed animals as demonstrated by bioluminescent imaging (BLI). Whereas graft survival occurred throughout the first week posttransplantation, the majority of grafts were acutely rejected during the second week. In a few animals, however, the grafts continued to demonstrate an increased BLI signal, similar to the allografts in immunodeficient recipients. The corpus callosum in mice is composed of a very thin strip of white matter located between two relatively large gray matter structures [i.e., the cortex and the striatum (STR)]. Due to the small size of the corpus callosum, it cannot accommodate the entire volume of transplanted cells without overspill into the neighboring gray matter structures. We hypothesized that the variability of cell survival may be related to the different immune responses that may exist between gray and white matter. In the current study, we therefore compared the survival of GRPs allografted into either the forceps minor (FM) as the largest white matter structure, or the STR as gray matter structure.

Materials and Methods

Cell Preparation

Mouse GRPs were derived from Friend leukemia virus, strain B (FVB; Taconic, Hudson, NY, USA), mouse spinal cord at embryonic day 13.5 (E13.5). Cells were immortalized using a lentivirus encoding the simian virus 40 (SV40) large T-antigen (kindly provided by Lingzhao Cheng, Johns Hopkins University) and selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA; cat. No. P8833). Subsequently, cells were transduced with a lentivector (Invitrogen, Carlsbad, CA, USA; cat. No. V498-10) encoding the firefly luciferase (Promega, Madison, WI, USA; cat. No. E6651) under a constitutive cytomegalovirus (CMV) promoter and then clonally selected on the basis of the intensity of BLI. Cells were maintained in serum-free Dulbecco's modified Eagle's medium (DMEM))/F12 (Life Technologies, Grand Island, NY, USA; cat. No. 10565042) supplemented with N2 (Life Technologies; cat. No. 17502-048), B27 (Life Technologies; cat. No. 17504-044), bovine serum albumin (Sigma-Aldrich; cat. No. A7030), and basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ, USA; cat. No. 100-18B), as described elsewhere (23). Cells were labeled overnight with superparamagnetic iron oxide [SPIO, 25 μg Fe/ml, Molday Ion Rhodamine B, BioPhysics Assay Laboratory (BioPAL), Worcester, MA, USA] for magnetic resonance imaging (MRI). Before transplantation, cells were trypsinized (Gibco, Gaithers-burg, MD, USA; cat. No. 25300-54), washed, and suspended in culture medium (as above) at a concentration of 100,000 cells/μl.

Animals

Immunodeficient, graft-accepting rag2^−/- male mice (8 weeks old, n = 10; Taconic) and immunocompetent male Balb/c mice (8 weeks old, n = 44; Jackson Labs, Bar Harbor, ME, USA) were each divided into two groups based on graft site (FM and STR). For graft survival evaluation, mice (Balb/c FM, n = 10, and STR, n = 10; rag2^−/- FM, n = 5; STR, n = 5) were evaluated by BLI over a period of 16 days. To detect the difference in immuno-logical response to the graft, animals (Balb/c FM, n = 5; STR, n = 5) were sacrificed in the prerejection period (5th day). For MRI visualization of graft placement, five Balb/c (FM = 3, STR = 2) mice were sacrificed immediately after cell injection. For near-infrared fluorescence (NIRF) imaging, animals (Balb/c STR, n = 5; FM, n = 4) were imaged in the prerejection period–transplantation period. Animals were housed under an artificial light– dark (12 h/12 h) cycle and had access to food and water ad libitum. All experimental procedures were in accordance with the guidance provided in the Rodent Survival Surgery manual and were approved by our Institutional Animal Care and Use Committee.

Cell Transplantation

Anesthesia was induced with 5% isoflurane gas and maintained throughout the surgical procedure with a mixed flow of 2% isoflurane and 98% oxygen delivered at a rate of 2 L/min. Mice were stabilized in a Cunningham adaptor mounted on a stereotactic frame (both from Stoelting, Wood Dale, IL, USA), and a 7.0-mm incision was made along the midline of the skull. The bregma was identified and burr holes were placed according to the coordinates for the targeted structures that were selected based on a mouse brain stereotactic atlas. The coordinates (in mm) were as follows: for FM: AP = 1.8, ML = 1.0, DV = 2.0; and for STR: AP = 0.0, ML = 2.2, and DV = 2.2. Cells were loaded into a 10-μl Hamilton syringe (Reno, NV, USA) with an attached 31-gauge needle (Hamilton) and lowered into the brain according to the coordinates. Three microliters of cell suspension was injected at a rate of 1 μl/min using a nanoinjector (Stoelting). The syringe was removed and the wound was closed with sutures (Silk 5.0; Ethicon, San Angelo, TX, USA).

Imaging of Graft Location and Cell Survival

For MRI visualization of the topography of the graft site, transplanted cells were labeled with SPIO (see above), and rag2^−/- mice (n = 5) were sacrificed directly after surgery to avoid artifacts related to graft loss or proliferation (3). After transcardial perfusion with 10 mM phosphate-buffered saline (PBS; Invitrogen; cat. No. 10010049) and then 4% paraformaldehyde (PFA; Sigma; cat. No. P6148), the entire head was dissected, postfixed overnight in 4% PFA, and transferred to PBS. The tissue was immersed in Fomblin (Solvay, Brussels, Belgium), and ex vivo images were obtained at 11.7T (Bruker Biospin, Ettlingen, Germany) using a volume coil and a T2-weighted spin echo sequence [echo time (TE) = 12 ms, repetition time (TR) = 2000 ms, average (AV) = 4, field of view = 2.0 × 2.0 cm, matrix = 256 × 256, rapid acquisition with refocused echoes (RARE) factor = 2].

BLI was initiated the day after cell transplantation and continued weekly until the end of the study (16 days). Anesthesia was induced with 5% isoflurane and maintained with 2% isoflurane/98% oxygen. Luciferin (Goldbio.com, cat. No. Luck-1G) was administered intraperitoneally at 150 mg/kg. Animals (Balb/c, n = 20; rag2^−/-, n = 10) were placed inside an IVIS Spectrum optical imager (Caliper, Mountain View, CA, USA) and imaged 10 and 15 min after luciferin injection. The acquisition time was set at 1 min, and the data were expressed as photon flux (p/s). To account for baseline differences across animals, each animal's recordings were standardized to the signal measured at day 1. Cell survival curves were generated using standardized data. A drop in BLI signal to the background level (signal generated by other parts of the body) was interpreted as rejection of transplanted cells.

Imaging of Blood–Brain Barrier Disruption

The blood–brain barrier (BBB) integrity was evaluated using NIRF during the prerejection period—5 days after cell transplantation. Balb/c mice (FM graft, n = 4; STR graft, n = 5) were injected intravenously with IRDye 800CW conjugated to polyethylene glycol (LI-COR^®, Lincoln, NE, USA). The dose was selected according to the manufacturer's instructions (1 nmol per mouse). NIRF imaging (Pearl Imager, LI-COR^®) was performed immediately after injection of the probe to confirm that the agent was properly injected, and then after 24 h to evaluate BBB integrity. At that time, animals were sacrificed and the brains were dissected for ex vivo NIRF imaging. For both in vivo and ex vivo imaging, the length of exposure was 1 s with an 800-nm acquisition channel. Quantification of photon flux (p/s) was performed using dedicated Pearl Imager software. The results are presented as mean ± standard deviation (SD) for each group. The extent of BBB disruption was correlated with graft viability as measured by BLI.

Immunohistochemical Analysis

After transcardial perfusion with 4% PFA, brains were removed and postfixed overnight in 4% PFA, cryopre-served in 30% sucrose (Sigma; cat. No. S5016) solution, and then snap-frozen. Thirty-micrometer-thick coronal tissue sections were cryosectioned and immunostained with primary antibodies against cluster of differentiation 45 (CD45), CD8 (1:100, both Serotec, Raleigh, NC, USA), CD4 (1:100 Santa Cruz, Dallas, TX, USA), and CD11b (1:100, Biolegend, San Diego, CA, USA) for evaluation of the immune response, and against luciferase (1:1000, AbCam, Cambridge, MA, USA) to assess graft survival. Secondary antibodies included goat anti-rabbit conjugated to Alexa Fluor-488 (1:500, Invitrogen), and goat anti-mouse IgG conjugated to Alexa Fluor-594 (1:300, Invitrogen). Cell nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; Sigma; cat. No. D9542). Cells were manually counted on every 10 sections using stereology. An Olympus BX 400 microscope (Olympus, Center Valley, PA, USA) was used to acquire fluorescent images.

Statistical Analysis

Regression analysis is reported as type III tests of fixed effects, with the lowest mean square (LMS) difference test used for comparison between means (PROC MIXED, SAS 9.2, Cary, NC, USA). A coefficient of determination was calculated, and the data were subjected to logistic transformation to maximize the model fit. For correlational analysis, a Pearson's r test was employed (PROC CORR, SAS 9.2). Values are shown as the means ± SD.

Results

Topography of Graft Deposits

The precision of the allograft injection as related to the targeted brain region was evaluated by ex vivo MRI. We confirmed that the STR and FM coordinates corresponded, on axial sections, to the upper STR (Fig. 1A) and the FM (Fig. 1B), respectively. Interestingly, on coronal sections, there was a major difference in graft shape between the two targets, as evaluated by histology. FM transplants were cylindrical, with cells distributed along the needle track (Fig. 1C). STR transplants, in contrast, assumed a unique, wedge-shaped, semilunar pocket, with cells spreading laterally and located between the STR and the corpus callosum (calloso-striatal border) (Fig. 1D). Such a distribution is most likely due to the area of lower resistance between the STR and the corpus callosum, with the cell suspension easily entering that space.

Figure 1.

Topography of graft location. Axial magnetic resonance imaging (MRI) sections depicting the location of the striatum (STR) (A) and the forceps minor (FM) (B) graft (black arrows). Immunohistochemistry of coronal section through the FM graft (green) reveals a cylindrical shape parallel to the needle track (C), whereas the STR graft (green) is characterized by a backflow of cell suspension from the site of the needle and a lateral spread between the STR and the corpus callosum (D). Note the limited graft leakage to the lateral ventricle (LV). Green, anti-luciferase; blue, 4′,6-diamidino-2-phenylindole (DAPI) stain. Scale bars: 200 μm.

BLI of Graft Survival

In immunodeficient rag2^−/- mice, the grafts survived for at least 16 days for both the FM and the STR grafts. There was no significant difference between the two implantation sites (p = NS). In immunocompetent animals, all STR grafts survived throughout the time course of the experiment. The time course of the BLI signal did not differ from the grafts in immunodeficient animals (p = NS). In contrast, all FM grafts in the immunocom-petent mice were rejected (maximized model fit, type III test, p < 0.001) (Fig. 2).

Figure 2.

Serial bioluminescence imaging (BLI) of graft survival. In immunodeficient animals, both STR (closed circles) and FM (open circles) grafts survived. In immunocompetent animals, only the STR grafts survived (closed squares), whereas the FM grafts (open squares) were rejected.

NIRF of BBB Integrity

The BBB integrity was evaluated in the prerejection period (5th day posttransplantation) to determine whether a potential disruption might contribute to graft rejection. In vivo NIRF 24 h after contrast injection demonstrated disruption of the BBB in all animals but did not show any difference between the FM and STR grafts (p = NS, data not shown). Because it is possible that surgery-related factors, such as skin wound healing, might lead to an accumulation of the NIRF probe, animals were sacrificed immediately following in vivo imaging, and brains were removed and imaged ex vivo. Ex vivo NIRF was in good agreement with the in vivo data. The measured photon flux did not show a statistically significant difference between the graft sites (p = NS), although there was a trend toward a larger disruption of the BBB in the FM grafts. The focal increase of fluorescence was found to correspond to the graft location. Thus, ex vivo NIRF revealed BBB disruption in all brains, with a considerable variability of results between animals (Fig. 3). Most importantly, the range of BBB disruption did not correlate with cell survival/rejection, as measured by BLI signal (Pearson's r = 0.39, p = 0.3).

Figure 3.

Ex vivo near-infrared fluorescence (NIRF) imaging of blood–brain barrier (BBB) integrity. IRDye 800CW conjugated to polyethylene glycol was injected at 4 days posttransplantation. In all STR (A) and FM (B) grafts, leakage of dye occurred, indicating a disruption of the BBB in all animals.

Immunohistochemical Analysis of Immune Response

According to the BLI data at 16 days posttransplan-tation, all FM grafts in immunocompetent animals were fully rejected, whereas all STR grafts survived. Despite good survival, immunostaining against the pan- leukocyte marker (CD45) revealed a substantial infiltration of immune cells, although the variability of the inflammatory cell infiltration between individual animals in the STR group was high (Fig. 4). There was a trend toward higher infiltration of more compact, round grafts (Fig. 4A, B) compared to grafts that were thinner and more laterally distributed (Fig. 4C, D). Presumably, these grafts distributed between the corpus callosum and the STR with less overall traumatic injury resulting from the injection procedure.

Figure 4.

Variability of the immune response against STR grafts at 16 days posttransplantation. Examples are presented from four different animals, sorted from the strongest (A) to the weakest (D) immune response. Red, anti-luciferase; green, anti-cluster of differentiation 45 (CD45). CC, corpus callosum; LV, lateral ventricle; STR, striatum. Scale bars: 100 μm.

Because at the postrejection period (16 days post-transplantation) the effectors of the immune system are largely cleared from the tissue, we performed additional experiments in immunocompetent mice (n = 10) at 5 days posttransplantation. At this time point, grafted cells at both the FM and the STR sites were still viable, as verified by BLI (data not shown). We observed that grafts were infiltrated with cells belonging to both the innate and the adaptive immune system. There was a higher cell count for markers of innate (CD11b) than adaptive (CD4, CD8) immune cells (CD4 < CD8 < CD11b, type III test, p < 0.001). Comparison between the FM and the STR site displayed a higher infiltration by CD8 (p = 0.02) and CD11b (p = 0.04) cells for FM grafts (Fig. 5). CD4⁺ cells were rare and equally distributed regardless of graft location (p = 0.85). In addition to infiltration of graft itself, it is noteworthy that grafts injected in the FM exhibited a “cuffed pattern” of microglia (CD11b) (Fig. 6A); these were absent for the STR (Fig. 6B). Furthermore, STR grafts that contained most of the CD8⁺ cells were in the proximity of the needle track (Fig. 6C, arrowheads), whereas almost no immune cells were visible in the space between the STR and the corpus callosum (Fig. 6C, arrows). Occasionally, there were visible features of central graft necrosis in some transplants in both the FM and the STR grafts (Fig. 6D, asterisks).

Figure 5.

Quantitative assessment of graft infiltration for various immune cell phenotypes. Gray bars, STR grafts; black bars, FM grafts.

Figure 6.

Postmortem analysis of STR and FM grafts at day 5 posttransplantation. Anti-CD11b staining (red) demonstrates micro-glial cuffing around the FM graft (A, costained with anti-luciferase green), whereas no activation of an innate immune response was observed around the calloso-striatal pocket of the STR graft (B). STR graft infiltration by CD8 cells (red) occurred in the vicinity of the needle track (arrowheads) but was absent at the lateral part of the graft in the space between the corpus callosum and the STR (C). Central graft necrosis was visible in some grafts as a loss of signal (D, asterisks). Nuclei were counterstained with DAPI stain (blue). Scale bars: 100 μm (A, B), 200 μm (C, D).

Discussion

To the best of our knowledge, this is the first demonstration that allograft survival within the CNS of an immunocompetent, nonimmunosuppressed host may be highly dependent on the implantation site. As shown in our previous work (3,15), the BLI signal correlates very well with the survival of transplanted cells, as assessed by histology. Interestingly, the shape of the graft was found to be determined by the anatomical location of the cell deposit. The FM graft was cylindrical, with cells distributed along the needle track, which is in good agreement with previous reports (26,30). Surprisingly, cells from the STR grafts distributed more laterally, with an accumulation along the border between the STR and the corpus callosum, forming a wedge-shaped, semilunar “pocket.” Thus, the majority of transplanted cells were located between the STR and the corpus callosum and not within the STR itself. This pattern of cell distribution occurred in all animals. Despite our careful injection procedures, we were unable to deposit all cells in a homogeneous manner within the upper STR.

The cylinder-shaped cellular deposit in the FM is most likely related to the damage to the brain parenchyma caused by the microinjection procedure, providing an impetus for immune cell activation. This was in stark contrast to STR grafts, which were mostly located between the STR and the corpus callosum, limiting brain microlaceration, and subsequently limiting the exposure of the graft to activation of host cells. This may explain the robust microglial cuffing of FM grafts, whereas such phenomena were essentially absent in the STR grafts. Within the STR grafts that spread laterally between the corpus callosum and the STR, host immune cells were located mostly in close proximity to the needle track. The migration of transplanted cells was not observed during the study, and the results might have been different if highly migratory cells were used. Our observation that immunorejection depends on the graft implantation site has important ramifications. It may be the primary cause for the variability in allograft survival reported by different research groups (4,5,10,22,24,34,41). The selection of a specific anatomical structure as an implantation site, including natural pockets, to accommodate an injection volume without damage to the brain parenchyma and/ or vasculature is directly relevant to successful clinical translation of cell-based therapy.

We found that allografts are infiltrated by cells belonging to both the innate and the adaptive immune system. Although it appears counterintuitive, it has been shown previously that in the early phase after cell transplantation, the immune response against allo- and xenografts is similar (17). This is an indicator that surgical injury may be the leading factor that initiates the rejection process. Lymphocytes contribute to macrophage recruitment within inflamed tissue (32), which may explain the lack of macrophage infiltration in immunodeficient animals. Whereas lymphocytes precede the macrophage/microg-lia at the inflammation site, after a few days, the latter cell population constitutes the most prominent fraction of immune-infiltrating cells (31). This was also observed for the few days posttransplantation in our study. The use of rag 2^−/- mice, which are devoid of lymphocytes, previously enabled us to determine that the innate immunity does not negatively affect xenograft survival (15). Reactive microg-lia may play a mediating role in the cytotoxic response by presenting antigens to lymphocytes (2). Thus, minimally traumatic allograft transplantation, preventing activation of microglia and limiting foreign antigen presentation, may contribute to long-term allograft survival.

We previously observed that if grafts were not rejected within the first 2 weeks, long-term survival was likely to occur (3). Nevertheless, despite the excellent survival of the STR grafts, there was a marked variability of inflammatory cell infiltration between the animals for each group. The presence of immune cells within the graft may endanger proper graft function with long-lasting consequences (44). BLI is an excellent method by which to follow graft survival, but it cannot provide information about the graft infiltration by immune cells. We did not observe a living graft in any case where BLI showed a loss of signal, but in all cases with BLI signal detected we did find a living transplant. The dense packing of cells within a graft precluded a precise estimation of cell number that could be correlated directly with the BLI signal on an animal-to-animal basis. We used NIRF to test whether the BBB disruption might be an indicator of an active immune response; however, it did not correlate with the process of immune rejection. This indicates the involvement of a cellular rather than a humoral response against the graft.

The postmortem assessment of grafts in the prerejection period revealed small areas of graft necrosis located in the central parts of the grafts. The amount of necrosis was similar for the FM and STR grafts and appeared to be a transient phenomenon as it could only be observed at day 5 and not at day 15 posttransplantation. Because necrosis occurred even in noninfiltrated graft areas, we hypothesize that this is related to nutrient/oxygen deprivation and not related to any immune response. In this context, the amount of transplanted cells has certain limitations to meet the demand for essential nutrients. Specific preconditioning of grafts, such as culture in a low-oxygen environment before transplantation, has been shown to improve graft adaptation to hostile host environments (39).

The accumulation of transplanted cells in the pocket between the two natural bordering regions of the brain, with minimal disruption of tissue integrity, appears to be the most plausible explanation for the dramatic difference in the allograft rejection rate. Another aspect that should be taken into consideration is that the STR grafts were transplanted closer to the ventricles than the FM grafts. Studies on malignant brain tumors have revealed that the prognosis for tumors of similar grade is much worse if the tumor is located in close proximity to the lateral ventricles rather than more peripherally (8). The vicinity of the lateral ventricles appears to represent a favorable microen-vironment characterized by weaker immune survey.

Although the deposition of cellular transplants within specific sites of the brain offers a way to manipulate a possible immune attack toward the allograft, several other approaches are currently being investigated to tackle the limitation of immunorejection. Most widely used is the administration of conventional immunosup-pressive drugs. However, cyclosporine A was shown not to be consistently effective (15). Double-drug regimens are more potent, even for xenotransplant protection, but at the cost of severe side effects (42). An alternative method is the costimulation blockade recently introduced for the immunoprotection of transplanted organs (38). A high dose of cyclophosphamide administered early after transplantation is able to kill lymphocytes that proliferate in response to graft injection, prolonging graft survival (29). Protection of intracerebral grafts by local immune suppression has also been shown to be effective (1). Microchimerism has also been reported to increase graft tolerance under very specific conditions, but, on the contrary, may also sensitize the host to the graft (12,27). The induction of tolerance through thymic transplantation is another alternative (11,37). The findings of our study are relevant to any of the above strategies aimed at alleviating allograft immunorejection.

Conclusions

STR allografts were found to survive dramatically better than FM grafts. The specific site of implantation may thus direct the overall fate of the graft. The topographical and anatomical characteristics of the target site for allografting should be carefully considered when designing cell therapies for neurological disorders at both the preclinical and the clinical level.

Footnotes

Acknowledgments

This study was supported by grants MSCRFII0193, MSCRFII0052, NIH 1RO1 NS076573, and NIH 2RO1 NS045062. The authors thank Mary McAllister for editorial assistance. M.J. was supported by a Kolumb Fellowship from Foundation for Polish Science. The authors declare no conflict of interest.

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Survival of Neural Progenitors Allografted into the CNS of Immunocompetent Recipients is Highly Dependent on Transplantation Site

Abstract

Keywords

Introduction

Materials and Methods

Cell Preparation

Animals

Cell Transplantation

Imaging of Graft Location and Cell Survival

Imaging of Blood–Brain Barrier Disruption

Immunohistochemical Analysis

Statistical Analysis

Results

Topography of Graft Deposits

BLI of Graft Survival

NIRF of BBB Integrity

Immunohistochemical Analysis of Immune Response

Discussion

Conclusions

Footnotes

Acknowledgments

References