Marrow Stromal Cells Transplanted to the Adult Brain Are Rejected by an Inflamma - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:00 |只看该作者 |倒序浏览 |打印

作者：Thomas M. Coynea,b,c, Akiva J. Marcusa,b, Dale Woodburya, Ira B. Blacka作者单位：aThe Ira B. Black Center for Stem Cell Research and Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA;bM.D./Ph.D. Program, Graduate School of Biomedical Sciences, University of Medicine and
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      【摘要】" `+ c* g7 n* i4 f9 U
   ABSTRACT: The remarkable plasticity of marrow stromal cells (MSCs) after transplantation to models of neurological disease and injury has been described. In this report, we investigated the plasticity and long-term survival of MSCs transplanted into the normal brain. MSCs were isolated from green fluorescent protein (GFP) transgenic rats and double-labeled with 5-bromo-2-deoxyuridine (BrdU) and bis benzamide (BBZ) prior to transplantation into the adult hippocampus or striatum. Surgery elicited an immediate inflammatory response. MSC grafts were massively infiltrated by ED1-positive microglia/macrophages and surrounded by a marked astrogliosis. By 14 days, graft volume had retracted and GFP immunoreactivity was absent, indicating complete donor rejection. Consequently, MSCs did not exhibit plasticity formerly identified in other studies. However, BrdU- and BBZ-labeled cells were detected up to 12 weeks. Control transplants of nonviable MSCs demonstrated the transfer of donor labels to host cells. Unexpectedly, BrdU labeling was colocalized to host phagocytes, astrocytes, and neurons in both regions. Our results indicate that MSCs transplanted to the intact adult brain are rejected by an inflammatory response. Moreover, use of the traditional cell labels BrdU and BBZ may provide a misleading index of donor survival and differentiation after transplantation. ! P; ^1 B! F* u6 Z; T3 {) z
      【关键词】 Mesenchymal stem cells Bone marrow stromal cells Cell transplantation Mesenchymal stem cell transplantation Graft rejection9 U! w( F- G3 b: X
   INTRODUCTION+ ^! Y! @; k! J  Q7 w7 I
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Numerous studies over the past decade have demonstrated the potential of cell transplantation for the treatment of neurological disease and injury. However, logistical, immunological, and ethical limitations have complicated the use of fetal and embryonic cell sources .6 B# j; R3 ^7 @+ l/ u( R' H

We and others have differentiated marrow stromal cells (MSCs) into presumptive neurons in vitro . Donor cells entered ventricular germinal zones, expressed neural progenitor traits, and migrated to distant brain regions. Furthermore, MSCs populated widespread areas in the fetal and neonatal brain, expressed site-specific neuronal genes, and demonstrated robust survival. These observations indicated that MSCs exhibit extensive plasticity in the embryonic brain, responding appropriately to local developmental cues. To examine whether similar potentials are displayed in a mature environment, we have now transplanted MSCs to the adult brain.
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In the adult brain, in contrast to the developing brain, factors that regulate neuronal ontogenetic processes are significantly reduced. As such, MSCs transplanted to the adult brain may not exhibit potentials formerly identified in the embryo. However, two distinct neurogenic regions, the hippocampal dentate gyrus and ventricular subependymal zone, exhibit plasticity into adulthood. Compared with parenchymal regions, these adult germinal zones contain morphogenetic signals that regulate proliferation, differentiation, and survival of resident progenitor cells . Therefore, donor MSCs can also serve as probes to evaluate the host response of the mature brain to transplantation.

Once considered immunologically privileged, tissue grafted to the adult brain is subject to considerable immune surveillance, as is now evident .
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However, in vitro and in vivo evidence has suggested that MSCs are not intrinsically immunogenic, do not stimulate alloreactivity, and exert suppressive effects on T-cell proliferation, stimulation, and mixed lymphocyte reactions .5 P# Y' m3 f' U1 O% i
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Despite these advances, unanswered questions remain regarding the fate and consequences of MSC transplantation. Most generally, do MSCs, which demonstrate plasticity in the developing and lesioned brain, display similar potentials in the normal adult brain? Specifically, what is the nature of MSC survival, migration, and phenotypic expression in the mature brain? Does the adult brain possess regional differences in the ability to direct plastic responses? In addition, what is the nature of the host immune response to transplanted MSCs?( q6 C& T2 h: Y$ J+ {

To address these questions and provide a standard assessment of plasticity in the normal adult brain, MSCs were transplanted to the hippocampus or striatum, neurogenic or non-neurogenic regions. Unexpectedly, transplantation elicited an inflammatory response leading to the rapid rejection of grafted tissue. As a result, MSCs did not exhibit plasticity formerly identified in other systems. Moreover, we demonstrate transfer of the traditional cell labels 5-bromo-2-deoxyuridine (BrdU) and bis benzamide (BBZ) to host phagocytes, astrocytes, and neurons. These findings raise questions concerning the survival and maturation of MSCs transplanted to the brain.
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MATERIALS AND METHODS
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Animal Welfare
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Animal studies were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (Piscataway, NJ). Animal subjects were housed in pairs and maintained on a 12-hour light/dark cycle with food and water ad libidum.. g: m+ H2 _. c
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Isolation and Enrichment of MSCs8 ~( m" \1 j& \
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MSCs were isolated from the femurs of adult male Sprague-Dawley enhanced green fluorescent protein (GFP) transgenic rats (kind gift from Dr. Donming Sun, Rutgers University, Piscataway, NJ) expressing GFP under the control of the cytomegalovirus enhancer and the chicken ß-actin promoter . Briefly, adult rats were sacrificed by CO2 asphyxiation. Femurs were dissected from attached musculature and connective tissue, ends were removed, an 18-gauge needle fitted to a 5-ml syringe was inserted into the shaft, and marrow was aspirated with Dulbecco's modified Eagle's medium (DMEM)/20% fetal bovine serum (FBS). The marrow aspirate was triturated with a fire-polished Pasteur pipette, and the cell suspension was resuspended in 12 ml of DMEM/20% FBS with 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). To enrich for MSCs, 2 ml of the marrow cell suspension was added per well to six-well plates (Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences) fitted with 3-µm-pore filter inserts. Filters were transferred to new six-well plates at 24 and 72 hours, and 1 ml of DMEM/20% FBS was added to each filter insert to promote passage of smaller, nonadherent lymphohematopoietic cells through the filter membrane. At 7 days, size-selected MSCs were removed from the filter membrane with trypsin/EDTA and transferred to T75 flasks at 50 cells per cm2." D0 c; |/ b  \

Labeling and Harvesting MSCs for Transplantation- J3 G- n$ q' S
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MSCs were maintained in DMEM/20% FBS without further supplementation and passaged at 80% confluency. Seventy-two hours prior to transplantation, MSCs were supplemented with 2 µM BrdU (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to label dividing cells. In addition, MSCs were labeled with 1 µg/ml BBZ (Sigma-Aldrich) 24 hours prior to surgery. Cells were harvested with trypsin/EDTA, washed twice with DMEM, and resuspended at a concentration of 50,000 cells per µl in DMEM. Cells between passages 10 and 15 were used for all transplantations.

Transplantation
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Adult female Sprague-Dawley rats (Hilltop Lab Animals, Inc., Scottdale, PA, http://www.hilltoplabs.com), weighing 225 g at the start of the experiment, were used for all surgeries. Animals were anesthetized with an i.m. injection of ketamine (50 mg/ml), xylazine (2.6 mg/ml), and acepromazine (0.65 mg/kg) and placed in a small animal stereotaxic apparatus affixed with a microinjector unit (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). Each subject received a 1 µl cell suspension, unilaterally, into either the hippocampus (HP) or striatum (ST) according to the following coordinates: HP, anterior/posterior (AP) = ¨C2.0, medial/lateral (ML) =  1.6, ventral (V) = ¨C3.4; ST, (AP) =  2.0, (ML) =  3.0, (V) = ¨C5.0. The bite bar was set at  5.0, and all ventral coordinates were taken from dura. Cells were infused with a 10-µl syringe fitted with a 30-gauge needle (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com). After infusion, the needle was left in place for 3 minutes before retraction. For both HP and ST transplant groups, subjects were sacrificed at 3 days, 7 days, 2 weeks, or 12 weeks (n = 4 per group). All subjects analyzed at 2 weeks or earlier were immunosuppressed with daily injections of cyclosporine (i.p., 10 mg/kg; Novartis International, Basel, Switzerland, http://www.novartis.com) from 24 hours prior to surgery until sacrifice.
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Nonviable Cell Transplantation
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MSCs were harvested as described above. Prior to transplantation, the cell suspension was subjected to 12 to 15 rapid freeze-thaw cycles on dry ice to promote lysis. Nonviability was confirmed by replating. Transplantation was performed as described above. In parallel, MSCs, rendered nonviable by microwave fixation or media, containing 2 µM BrdU and 1 µg/ml BBZ, were infused. For microwave fixation, the cell suspension was irradiated in an 800-W microwave oven for 30 seconds, which rendered cells nonviable albeit intact. Subjects were sacrificed at 2 and 12 weeks (n = 4 per group).  I4 u( k( }3 U- p( j
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Tissue Processing

Subjects were anesthetized with sodium pentobarbital (70 mg/kg) and transcardially perfused with 0.9% saline, followed by fixation with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were postfixed with 30% sucrose in PFA for 48 hours and then transferred to 30% sucrose in 0.1 M phosphate-buffered saline for cryopreservation. Serial coronal sections were cut on a cryostat at 10 µm, mounted on Superfrost slides (VWR International, Inc., Bridgeport, NJ, http://www.vwr.com), and stored at 4¡ãC.* y/ D" _6 W. Q: [* A, k
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Immunohistochemistry and Histology

Immunohistochemistry was performed as described , 1:1,000; Covance, Princeton, NJ, http://www.covance.com), GFP (ms, 1:100; Chemicon International), GFP (rb, 1:50; Chemicon International), glial acidic fibrillary protein (GFAP) (rb, 1:1,000; DAKO, Glostrup, Denmark, http://www.dako.com), ED1 (ms, 1:100; Serotec Ltd., Oxford, U.K., http://www.serotec.com), CD8 (ms, 1:100; Serotec Ltd.), and fibronectin (rb, 1:750; Sigma-Aldrich). Secondary antibodies included Oregon Green 488 or Alexa Fluor 594 (1:1,000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Sections were visualized with a Zeiss Axiovert 200M fluorescent microscope and a Zeiss LM510 confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Images were processed using Axiovison (Carl Zeiss) and Photoshop (Adobe Systems Incorporated, San Jose, CA, http://www.adobe.com). Colocalization of BrdU with phenotype markers was confirmed through analysis of x40 and x63 z-series confocal reconstructions and corresponding orthogonal planes. Negative controls for immunohistochemical staining were performed by omitting the primary antibody. In addition, contralateral brain regions served as internal controls to confirm primary antibody specificity.

RESULTS3 k# Y' k8 p9 Z  W, y7 E0 V
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Graft Survival and Morphology
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To assess the survival and plasticity of MSCs in the adult brain, we transplanted cells to the hippocampus and striatum, adult germinal and parenchymal regions. For the explicit identification of grafted cells, MSCs were isolated from a GFP transgenic rat. Isolated MSCs uniformly expressed GFP, and stable reporter gene expression was maintained for at least 30 cell passages, the longest time examined (supplemental online Fig. 1). However, to control for possible GFP downregulation in vivo, MSCs were double-labeled with BrdU, a thymidine analog, and BBZ, a nuclear fluorescent marker, prior to transplantation. The double-labeling protocol did not produce negative effects on cell proliferation or viability (supplemental online Fig. 1).

We initially characterized the survival and localization of transplanted MSCs. In all sections examined, the donor cells were identified with GFP-specific antibodies. A single deposit of 50,000 donor cells was transplanted into the striatum or hippocampus, and the brains were examined 3, 7, and 14 days postoperatively.

In the striatum, MSCs were transplanted into the head of the caudate-putamen. Three days postoperatively, GFP  MSCs formed an elongated mass along the needle tract, establishing a graft core (Fig. 1A). Migratory subpopulations were not observed in the surrounding neuropil. A few scattered MSCs were observed within 25 µm of the core, which we attributed to dispersal from donor cell infusion and/or cannula retraction.

Figure 1. Donor marrow stromal cells (MSCs) exhibit poor survival in the normal, adult striatum and hippocampus. (A): Green fluorescent protein (GFP)  MSC grafts (arrow; diaminobenzidine , black) were visible as a large cluster of cells in the striatum at 3 days. (B): Graft volume (arrow; DAB, black) was drastically reduced by 7 days. Inset micrograph (region denoted by arrow) depicts few remaining GFP  MSCs in the graft core (arrows). (C): At 14 days, GFP  MSCs were absent, and the grafts were identified as a scar along the needle tract (arrowheads). (D): GFP  MSCs were visible as a mass apposed to the dorsal dentate granule layer in the hippocampus (arrow; DAB, black). (E): By 7 days, graft volume (arrow) was decreased, and few GFP  MSCs were present (inset, arrows). (F): At 14 days, the hippocampal graft site presented as a scar (arrowheads) with no detectable GFP  donor cells. In both sites, regional blood vessels appear dilated (asterisks), diagnostic of an inflammatory reaction. Nuclei (blue) were counterstained with Hematoxylin QS. Scale bars = 250 µm. Magnification = x40 (insets). Abbreviations: HP, hippocampus; ST, striatum.6 B" b1 Y( c6 e4 o9 e3 H( o& ]
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In the hippocampus, MSCs were transplanted centrally into the hilus of the dorsal dentate gyrus. At 3 days, GFP  MSCs were visible as a cluster apposed to the dorsal leaf of the dentate gyrus (Fig. 1D). Similar to the striatum, migratory populations were not observed.1 W: d) R3 i3 f7 e

Few GFP  cells were detected in either graft site 7 days postoperatively (Fig. 1B, 1E). Examination of serial hematoxylin-counterstained sections indicated a dramatic reduction in graft density. By 14 days, GFP  MSCs were no longer detected in either region. The transplant sites were visible as scars corresponding to the needle tract (Fig. 1C, 1F). These data suggested donor MSCs were rapidly rejected. We next examined the nature of graft destruction.3 N; ^0 D; K' Y5 z. n% @6 ^/ l

Transplantation of MSCs Elicits an Inflammatory Response! h; w- p5 D# L7 m& ~
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To define the nature of the host response, serial sections from both hippocampal and striatal graft sites were immunostained for ED1, a marker specific for activated, rat microglia and monocytes/macrophages. Immunostaining demonstrated a marked inflammatory response at 3 days postoperatively. Both striatal and hippocampal MSC grafts were massively infiltrated with ED1  cells (Fig. 2A, 2B). ED1-reactive cells described a radial pattern around the graft core extending up to 1.0 mm from the needle tract. Moreover, ED1  cells surrounded dilated blood vessels in the graft region, suggesting infiltration of circulating myeloid cells (Fig. 2C).% b: V/ e- P# @% q* H6 C# t5 v/ L

Figure 2. Transplantation of marrow stromal cells (MSCs) elicits an inflammatory response. (A, B): Both striatal (A) and hippocampal (B) MSC grafts (green) were massively infiltrated by ED1-positive (red) microglia/macrophages 3 days postoperatively. (A', B'): Panels depict the intimate relationship with GFP  MSCs (green) and ED1  cells (red) in the graft core. (C): A vessel lumen (asterisk) surrounded with ED1-postive cells (red), dorsal to the 3-day hippocampal graft core, suggesting the influx of circulating myeloid cells. (D): At 3 days, sections stained with hematoxylin and eosin revealed cells with granulocytic (arrows), lymphocytic (arrowheads), and monocytic (double arrowheads) nuclear profiles in perivascular regions (lumen marked by asterisk), indicative of inflammation. (E): Rare T lymphocytes (CD8, red, arrows) were present in the graft regions at all times. Nuclei (blue) in (A, B) were counterstained with 4'6-diamidino-2-phenylindole. Scale bars = 100 µm (A, B), 10 µm (A', B'), 25 µm (C), and 50 µm (D, E). Abbreviations: dapi, 4'6-diamidino-2-phenylindole; GFP, green fluorescent protein; HP, hippocampus; ST, striatum.2 U/ }$ o4 B2 ~# y9 E! m
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Corresponding sections from both sites were stained with hematoxylin and eosin to examine the histology of the graft regions. The histology of both transplant sites was consistent with inflammation. Numerous blood vessels in the surrounding graft regions were dilated, with cells exhibiting monocytic, lymphocytic, and granulocytic nuclear morphology in the adventitial regions (Fig. 2D). Scattered lymphocytes and neutrophils were present in the neuropil, yet few were visible within the grafts. Limited CD8  T cells were detected at all times examined, consistent with the cyclosporine immunosuppression we employed (Fig. 2E).1 }3 J8 }7 D' ], I1 |- M
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The inflammatory response persisted for 7 days postoperatively. Unstained sections examined with fluorescent microscopy revealed cavitations within both striatal and hippocampal grafts. They were visible as dense cores of lipofuscin with a few surviving GFP  MSCs (Fig. 3A, 3B). Serial immunostained sections identified the hippocampal and striatal transplants as a mass of ED1-reactive cells (Fig. 3C, 3F). Furthermore, the grafts were enveloped in a fibronectin-positive matrix, indicating scar formation (Fig. 3D, 3G). A distinct astrogliosis surrounded the transplant sites (Fig. 3E, 3H). The increased density of GFAP  astrocytes delineated a defined graft-host border extending 2.0 mm distant to the graft cores.; E, p7 W5 ?; Q1 B$ ]3 |, ?: i( X

Figure 3. Marrow stromal cell (MSC) graft rejection is nearly complete 7 days postoperatively. (A, B): Unstained sections reveal the striatal (A) and hippocampal (B) graft sites and were lipofuscin (orange)-dense with cavitations, suggesting cell destruction. Few GFP  MSCs (green) were detected within the striatal (A, C¨CE) or hippocampal (B, F¨CH) grafts at 7 days. (C, F): ED1-reactive cells (red) densely populated the graft regions. (D, G): A fibronectin-positive matrix (red) had enveloped the MSC grafts, indicating scar formation. (E, H): A marked astrogliosis, identified by GFAP reactivity (red), was present along the graft-host border. The histology suggested graft rejection with concomitant scar formation. Nuclei were counterstained with 4'6-diamidino-2-phenylindole (blue). Scale bars = 50 µm. Abbreviations: GFAP, glial acidic fibrillary protein; GFP, green fluorescent protein; HP, hippocampus; ST, striatum.
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By 14 days, inflammation was reduced with ED1 reactivity confined to the needle tract. The graft sites were identified as GFAP- and fibronectin-reactive scars, indicating complete graft rejection. These data suggest that transplantation of MSCs into the normal adult striatum or hippocampus elicited an inflammatory response leading to donor cell destruction.

Widespread Distribution of BrdU- and BBZ-Labeled Cells% b$ S+ e' T0 m, G1 u
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Despite the histological evidence of graft destruction, both BrdU and BBZ labels were detected in the striatum and hippocampus up to 12 weeks, the longest time examined. Although GFP immunoreactivity was absent, labeled cells were widely distributed in both graft sites at 14 days. In the striatum, BrdU- and BBZ-labeled cells were visible as an elongated mass along the needle tract, with subpopulations located 1.5 mm distant to the core (Fig. 4A). In the hippocampus, BrdU  and BBZ  cells were clustered along the dorsal leaf of the dentate gyrus, a pattern commonly observed after intradentate transplantation of neural stem cells (Fig. 4B) . In addition, labeled cells were distributed throughout the dentate hilus and granule layers, with subpopulations located more than 1.0 mm from the needle tract in the hippocampal pyramidal fields and the underlying ependyma, choroid plexus, and thalamic nuclei. This pattern persisted up to 12 weeks, the longest time examined (Fig. 4C, 4D). These data suggested robust survival of MSCs in both regions, in marked contrast to the histological evidence.

Figure 4. Distribution of 5-bromo-2-deoxyuridine (BrdU)-labeled donor cells in the striatum and hippocampus at 2 and 12 weeks postoperatively. (A, B): Confocal tile scans of the striatal (A, C) and hippocampal (B, D) graft site at 2 (A, B) and 12 (C, D) weeks. The graft cores in the striatum and dentate hilus were densely populated with BrdU-labeled cells (green) at both times with apparent migratory subpopulations located at least 1 mm (asterisks) from the needle tract. The BrdU immunohistochemistry suggested that the donor cells exhibit robust survival in the striatum and hippocampus. The insets (A¨CD) depict the corresponding pattern of bis benzamide-labeled cells at each time point. ß-III-tubulin immunohistochemistry (red) was used as a background stain. Scale bars = 500 µm. Magnification = x10 (insets). Abbreviations: cpu, caudate putamen (striatum); dg, dentate gyrus; HP, hippocampus; ST, striatum.0 @/ C8 i: [# k- k5 d

Phenotypic Expression of BrdU-Labeled Cells

To determine the phenotype of BrdU-labeled cells, we evaluated the expression of specific neuroectodermal genes at 7 days, 14 days, and 12 weeks postoperatively. The phenotype of BBZ-labeled cells could not be determined, because the label was not retained after immunohistochemical processing. Despite differences in graft location, early phenotypic expression was similar in both regions.
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As described previously, the hippocampal and striatal grafts were densely populated with ED1-reactive microglia/macrophages with few surviving GFP  MSCs 7 days postoperatively. Corresponding sections stained for BrdU revealed that the majority of ED1  cells were double-labeled with BrdU (Fig. 5A, 5E). Few BrdU/ED1  cells were present outside of the core region. Along the graft-host border, and extending up to 1.0 mm distally, numerous BrdU  cells were colocalized with GFAP, an intermediate filament protein specific for astrocytes (Fig. 5B, 5F). Colocalization with neuronal markers was not detected at early times.% r& G6 v" a% [- f( V
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Figure 5. Phenotypic expression of 5-bromo-2-deoxyuridine (BrdU)-labeled donor cells in the striatum and hippocampus. (A): At 7 days, nearly all BrdU-labeled cells (green) in the graft core are ED1-positive (red). Inset depicts representative BrdU/ED1 double-labeled cells. (B): Astrogliosis (red, glial acidic fibrillary protein ) surrounded the graft/inflammatory cell infiltrate in the striatum 7 days postoperatively. Numerous BrdU/GFAP  cells (arrowheads) were visible along the graft-host border. Inset depicts representative BrdU/GFAP double-labeled cells. (C, D): Rare BrdU-labeled cells (green, arrows) were colocalized with the mature neuronal marker NeuN (red) in the striatum at 12 weeks. (E): Nearly all BrdU  cells (green) in the dentate graft core were colocalized with ED1 (red) at 7 days. (F): Astrogliosis (red, GFAP) defined the graft-host border with numerous BrdU/GFAP  cells (arrowheads) along the periphery. (G): BrdU-labeled cells (green) were clustered along the dorsal granule layer (red, NeuN) with subpopulations distributed throughout the hilar region at 12 weeks postoperatively. Boxed regions correspond to (H, L). (H): Confocal three-dimensional (3D) reconstruction of a BrdU/NeuN  cell located in the ventral granule layer. (I¨CK): Confocal z-series of the cell in (H) indicates colocalization of both labels. (L): Confocal 3D reconstruction demonstrates multiple BrdU (green) and NeuN (red) double-labeled cells (arrows) in the dorsal granule layer. (M¨CO): Confocal z-series of cells in (L) demonstrates double-labeled cells. Scale bars = 50 µm (A, B, E, F), 250 µm (G), and 10 µm (C, D, H¨CJ). Abbreviations: HP, hippocampus; ST, striatum.
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The density of BrdU/ED1  cells within the graft core/needle tract was significantly reduced by 14 days. Scattered ED1  cells were present within the needle tract in the striatum, and along the dorsal, dentate granule layer, a region damaged by passage of the transplant cannula. The majority of BrdU-labeled cells within the vicinity of the graft core were colocalized with GFAP. Numerous BrdU/GFAP  cells with characteristic astrocytic morphology were present within the gliotic scar along the needle tract in both regions. In addition, colocalization of BrdU with NeuN, a mature neuronal marker, was observed in rare cells within the dentate granule layer.
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BrdU/ED1  cells were not detected in either region 12 weeks postoperatively. The distribution of BrdU/GFAP  cells was similar to that at 14 days. However, the number of BrdU  cells double-labeled with NeuN had increased. Subpopulations of BrdU/NeuN  cells were visible within the dentate apex, dorsal, and ventral leaves (Fig. 5G¨C5O). Surprisingly, limited BrdU/NeuN  cells were observed within the non-neurogenic striatum (Fig. 5C, 5D).# |5 v5 c$ ], h3 V- D4 A

The BBZ and BrdU data suggested that MSCs exhibit remarkable maturation in the adult brain. However, the histological evidence and GFP immunohistochemistry indicated graft rejection. To determine whether the discrepancy between the BrdU, BBZ, and GFP histological data was the result of label transfer from rejected donor cells, nonviable BBZ- and BrdU-labeled MSCs were transplanted as controls.

BrdU and BBZ Labels Transfer to Host Cells
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For control transplantations, MSCs were expanded under standard presurgical culture conditions and harvested as normal. Prior to transplantation, MSCs were lysed with 12 to 15 rapid freeze-thaw cycles. Fifty thousand nonviable MSCs were transplanted into the striatum or hippocampus, and the graft sites were examined 2 and 12 weeks postoperatively.. j8 e  m6 f  B" A/ D7 ]6 b

Surprisingly, the transplantation of lysed MSCs resulted in the transfer of both BrdU and BBZ to host cells. Labeled cells were detected in both striatal and hippocampal graft sites up to 12 weeks. Moreover, the localization was virtually identical to that observed after the transplantation of live MSCs (Fig. 6A¨C6D).
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Figure 6. Distribution of 5-bromo-2-deoxyuridine (BrdU) and bis benzamide labels after transplantation of nonviable marrow stromal cell (MSCs). (A, B): Confocal tile scans depict the distribution of BrdU (green) after the transplantation of freeze-thaw-lysed MSCs within the striatum (A) and hippocampus (B) at 12 weeks. The boxed areas denote regions depicted in (C, D). (C, D): The distribution of bis benzamide (blue) in the striatum (C) and hippocampus (D) at 12 weeks. The localization of BrdU and bis benzamide after the infusion of lysed MSCs mirrored the pattern observed after the transplantation of live MSCs. ß-III tubulin immunohistochemistry (red) was used as a background stain in (A, B). Scale bars = 500 µm (A, B) and 100 µm (C, D). Abbreviations: dg, dentate gyrus; HP, hippocampus; ST, striatum.

Phenotypic expression was also similar to that of live MSC transplants. Two weeks postoperatively, the majority of BrdU-labeled cells within the striatal and hippocampal graft cores were colocalized with ED1 (Fig. 7A¨C7C). As with live MSC transplants, BrdU/ED1  cells were not detected at 12 weeks. Numerous BrdU/GFAP-labeled cells were observed in both graft regions at 2 and 12 weeks; cells predominated along the core/needle tract with populations located diffusely throughout the surrounding parenchyma (Fig. 7D¨C7F). Remarkably, BrdU/NeuN  cells were detected in both graft regions. Multiple BrdU/NeuN  cells were observed within the dentate granule layers at 2 and 12 weeks postoperatively (Fig. 7G¨C7J). In the striatum, rare BrdU/NeuN  cells were distributed randomly within the neuropil at 12 weeks (Fig. 7K¨C7N). Colocalization with NeuN was not observed in the striatum at 14 days. The latent increase in BrdU/NeuN  cells at 12 weeks suggests the initial transfer of BrdU to dividing precursor populations.

Figure 7. Phenotypic expression of 5-bromo-2-deoxyuridine (BrdU)-labeled cells after the transplantation of nonviable marrow stromal cell (MSCs) to the striatum and hippocampus. The pattern of phenotypic expression after the transplantation of nonviable MSCs was very similar to that of live MSCs. (A, B): Nearly all BrdU-labeled cells (green) in the striatal (A) and hippocampal (B) graft core were colocalized with ED1 (red). (C): Representative BrdU/ED1  cells in the hippocampus with classic microglial morphology. (D, E): Increased density of BrdU (green) and glial acidic fibrillary protein (GFAP) (red) double-labeled cells (arrowheads) in the striatal needle tract scar (D) and damaged dorsal granule layer (E) at 12 weeks. (F): BrdU/GFAP  cell in the hippocampus at 12 weeks demonstrating classic astrocytic morphology. (G): Confocal three-dimensional (3D) reconstruction of two BrdU (green) and NeuN (red) double-labeled cells (arrows) in the dentate gyrus 12 weeks after transplantation of lysed MSCs. (H¨CJ): Confocal z-series of cells in (G) demonstrating colocalization. (K): Confocal 3D reconstruction of a BrdU/NeuN  cell (arrow) in the striatum 12 weeks after the transplantation of lysed MSCs. (L¨CN): Confocal z-series of the cell depicted in (K) (arrows) demonstrating colocalization. Scale bars = 50 µm (A, B, D, E) and 10 µm (C, F¨CN). Abbreviations: HP, hippocampus; ST, striatum.- Z& U  T7 d! O& n
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To confirm the label transfer findings, control transplants were repeated with microwave-irradiated MSCs. Similar to freeze-thaw lysis, microwave fixation rendered the donor cells nonviable, yet the cells remain intact. The same pattern of label distribution and phenotypic expression was observed, which indicates that fragmentation of donor cells prior to transplantation is not required for BrdU or BBZ transfer. In contrast, the direct infusion of both markers alone did not result in cell labeling in vivo (supplemental online Fig. 2). These data suggest that the apparent long-term survival and maturation of transplanted MSCs is the result of label transfer rather than actual survival and differentiation.
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DISCUSSION

To determine the long-term survival, migration, and phenotypic expression of MSCs in the normal adult brain, we transplanted donor cells to the hippocampus or striatum, neurogenic or non-neurogenic regions, respectively. Previous reports have described the remarkable plasticity of MSCs after administration to the developing, normal, and lesioned adult brain . These attributes should foster long-term survival and maturation after transplantation to the adult brain. On the contrary, we now report that transplantation of MSCs into the intact adult brain elicited an inflammatory response leading to the rapid rejection of donor tissue.
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Immunologic rejection is a major obstacle to the survival and stable integration of stem cells transplanted to the adult brain . However, the temporal and histological profile of graft rejection in the present study was not consistent with an adaptive immune response. Minimal CD8  cytotoxic T cells were present in the graft regions at all times examined, and graft rejection was near complete by 7 days. The histological evidence suggested that MSCs were rejected by an inflammatory response.; j% y7 e. v3 @, G" ?" t

In the adult brain, the innate immune system responds vigorously to injury, initiating inflammation .+ R! Y$ v( i% k

In the present study, we observed the similar response of host microglia after transplantation. Intracerebral transplantation produces significant mechanical trauma to the brain, as passage of the cannula through the parenchyma damages local neuronal and vascular structures producing inflammation . The evidence supports our conclusion that MSCs were rejected by an inflammatory response.2 }3 I& x7 C  x5 u3 J6 l

Although innate immune cells initiate postoperative inflammation, the response is generally not sufficient to produce graft destruction . Perhaps, expression of these factors by MSCs in situ augmented postoperative inflammation, leading to graft destruction.

Although the histological and immunohistochemical evidence clearly indicated graft rejection, the persistence of BrdU- and BBZ-labeled cells initially suggested MSC survival and maturation. One explanation for this discrepancy is the downregulation of GFP expression in vivo. Transgene silencing has been a documented problem for cells transfected with retroviral or lentiviral vectors . Moreover, the histological evidence for graft rejection is compelling.1 H( E+ e- r) {- }' K2 b. x
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To investigate whether this discrepancy was the result of label transfer, MSCs labeled with both markers were rendered nonviable by rapid freeze-thaw lysis or microwave irradiation and transplanted into the hippocampus and striatum. Surprisingly, BrdU- and BBZ-labeled cells were widely distributed throughout both transplant regions up to 12 weeks postoperatively. The localization and phenotypic expression of labeled cells was nearly identical to that observed after transplantation of live MSCs.2 f' `- [0 L0 w) d" ]

At 7 days, the majority of BrdU was colocalized within ED1-positive microglia/macrophages in both regions. By 14 days, BrdU was colocalized to GFAP  astrocytes clustered around the needle tract scars and at distances greater than 1.0 mm away. Double-labeled cells with classic astrocytic morphologies densely populated both graft regions up to 12 weeks. The pattern of BrdU-labeled glial cells is consistent with the proliferative response exhibited by microglia and astrocytes after mechanical or traumatic brain injury .

Remarkably, BrdU/NeuN  neurons were detected in both regions 12 weeks postoperatively. In the hippocampus, double-labeled cells were concentrated within the dentate gyrus, a region where neurogenesis persists in the adult brain. In addition, rare BrdU/NeuN  cells were detected in the striatum. Although the striatum is a non-neurogenic region, recent evidence suggests that recruitment and subsequent proliferation of neural progenitors may occur in the cortex and striatum after brain injury . The trauma and resultant inflammation from the transplant procedure may have provided the stimulus for a similar response.8 Y* j3 @1 Q" q" p/ j$ b3 ~

Our results provide evidence for the transfer of BrdU and BBZ to host cells in vivo. More importantly, we demonstrate the transfer of BrdU to host astrocytes and neurons in the intact, adult hippocampus and striatum. During the preparation of this manuscript, Burns et al. elegantly described the transfer of thymidine analogs to neurons and glia in the developing, neurogenic, and lesioned brain . Based on these data, we conclude that the apparent plasticity suggested by BrdU- and BBZ-labeled cells was the result of label transfer to endogenous precursors rather than actual survival and differentiation of donor cells. The present findings contradict previous reports detailing MSC plasticity in the brain.

Pioneering studies by Kopen et al. demonstrated the extensive migration and adoption of astrocytic fates of MSCs infused into the lateral ventricles of neonatal mice .' f  l8 j4 [+ A! X
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The lack of survival and maturation of transplanted MSCs described in the current study may be attributed to the nature of the normal, adult brain. Evidence from transplantation studies indicates that the intact, adult brain has a limited capacity to direct the differentiation of transplanted stem cells, whereas the developing brain and injured brain express morphogenic cues that appear to foster donor plasticity . The fate of transplanted donor cells may be dependent upon critical variables, including host developmental age and background pathophysiology.
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On the other hand, many of the previous studies describing MSC plasticity and therapeutic potential have evaluated donor survival and phenotypic expression using BrdU, BBZ, or other fluorescent nuclear markers as the exclusive cell label . The current findings raise the possibility that label transfer may have compromised previous interpretations of MSC survival and plasticity in the brain.
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Recent evidence from transplant studies using GFP-expressing MSCs or human MSCs, identified with human-specific antibodies, has demonstrated limited survival commensurate with our present findings .

The collective inconsistencies regarding MSC fate in vivo and the evidence for label transfer warrant a re-evaluation of MSC plasticity and therapeutic utility in the brain. Moreover, the unequivocal identification of donor cells after transplantation is paramount to an analysis of stem cell survival and differentiation. We therefore caution against the continued use of BrdU and BBZ as donor cell labels.
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS- N: k3 q1 d+ j) ^$ Z) J/ n9 L
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This work was supported by National Institutes of Health Grant PO1 HD23315 and Albert Zofchak Fund Grant 181-01R. T.M.C. is supported by a National Institute of Environmental Health Sciences Training Grant in Environmental Toxicology (ES07148). We thank Kathleen Reynolds and Noriko Kane-Goldsmith for technical assistance and Dr. Emanuel DiCicco-Bloom for helpful discussions. We dedicate this manuscript to the memory of our fellow scientist, mentor, and friend, Dr. Ira Black.8 z* j1 O% G8 Q" t, Q
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