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TISSUE-SPECIFIC STEM CELLS |
aThe Ira B. Black Center for Stem Cell Research and the 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 Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA;
cThe Joint Graduate Program in Toxicology, Rutgers University, Piscataway, New Jersey, USA
Key Words. Mesenchymal stem cells • Bone marrow stromal cells • Cell transplantation • Mesenchymal stem cell transplantation • Graft rejection
Correspondence: Thomas M. Coyne, Ph.D., UMDNJ-RWJMS, SPH Bldg., Rm. 366, 683 Hoes Lane, Piscataway, New Jersey 08854-5635, USA. Telephone: 732-235-5387; Fax: 732-235-4990; e-mail: coynetm{at}umdnj.edu
Received on March 23, 2006;
accepted for publication on July 17, 2006.
First published online in STEM CELLS EXPRESS July 27, 2006.
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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We and others have differentiated marrow stromal cells (MSCs) into presumptive neurons in vitro [15–17]. MSCs assumed classic neuronal morphologies and expressed neuron-specific genes and proteins. To determine whether plasticity is also exhibited in vivo, we recently transplanted MSCs into the developing brain [18]. 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.
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 [19, 20]. In turn, these signals may foster similar responses in stem cells grafted to these regions. The coexistence of stable parenchymal regions with dynamic germinal niches provides a unique opportunity to assess the survival and plasticity of engrafted MSCs. However, the adult, unlike the embryo, has a developed humoral and cellular immune system, which may influence the fate of donor cells [21]. 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 [22]. The transplantation procedure invariably leads to inflammation and the initiation of effector mechanisms that coordinate innate and adaptive immune responses. The dynamic interactions between immune cells and the injured, neural parenchyma enable the brain to respond vigorously to the grafted tissue. As a consequence, the majority of cell types transplanted to the adult brain exhibit poor survival [21, 23].
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 [24]. Thus, these cells may be well suited for neural transplantation strategies in the adult brain. In fact, pioneering studies by Azizi et al. have described MSC plasticity after transplantation to the adult rat striatum [25]. Donor cells migrated extensively and expressed astrocytic and neuronal phenotypic characteristics. MSCs have also been administered to models of neurodegenerative disease and injury, providing therapeutic benefit and exhibiting neuroglial traits in vivo [26–30].
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?
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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Isolation and Enrichment of MSCs
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 [31]. MSCs were enriched by a method modified from Hung et al. [17]. 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.
Labeling and Harvesting MSCs for Transplantation
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
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) = –2.0, medial/lateral (ML) = +1.6, ventral (V) = –3.4; ST, (AP) = +2.0, (ML) = +3.0, (V) = –5.0. 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.
Nonviable Cell Transplantation
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).
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°C.
Immunohistochemistry and Histology
Immunohistochemistry was performed as described [18]. For immunofluorescence, sections were counterstained with 1 µg/ml DAPI (4'6-diamidino-2-phenylindole) (Sigma-Aldrich). For light microscopy, GFP immunohistochemistry was performed using an avidin-biotin system (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) with nickel-enhanced diaminobenzidine (Vector Laboratories) as the chromagen. Sections were counterstained with Hematoxylin QS (Vector Laboratories). Hematoxylin and eosin staining was performed as described [32]. Primary antibodies used were BrdU (mouse [ms], 1:50; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), BrdU (rat, 1:75; Accurate Chemical & Scientific Corporation, Westbury, NY, http://www.accuratechemical.com), NeuN (ms, 1:200; Chemicon International, Temecula, CA, http://www.chemicon.com), ß-III-tubulin (rabbit [rb], 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.
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RESULTS |
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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.
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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.
Transplantation of MSCs Elicits an Inflammatory Response
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).
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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.
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Widespread Distribution of BrdU- and BBZ-Labeled Cells
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) [33, 34]. 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.
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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.
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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–5O). Surprisingly, limited BrdU/NeuN+ cells were observed within the non-neurogenic striatum (Fig. 5C, 5D).
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
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.
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–6D).
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DISCUSSION |
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Immunologic rejection is a major obstacle to the survival and stable integration of stem cells transplanted to the adult brain [21]. The rejection of allogeneic donor tissue is traditionally mediated by the adaptive or acquired immune system [23, 36]. Adaptive responses commence 10–14 days post-transplantation after antigen presentation, selection, and clonal expansion of the responding T-cell population. Rejection proceeds as cytotoxic T cells infiltrate the graft site, recognize donor antigen, and facilitate cell destruction [23, 36]. 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.
In the adult brain, the innate immune system responds vigorously to injury, initiating inflammation [37, 38]. The inflammatory response is primarily coordinated by microglia/macrophages, tissue-resident innate immune cells [39]. Recent time-lapse microscopy has demonstrated the dynamic response of microglia to mechanical brain injury. Microglia rapidly accumulated to form a barrier around the lesion site, separating healthy and injured tissue [40, 41].
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 [42, 43]. Consistent with this contention, we observed the rapid accumulation of ED1+ microglia/macrophages in both striatal and hippocampal grafts 3 days postoperatively. At 7 days, a fibronectin-reactive matrix and astrogliosis formed a barrier around the few remaining donor cells, sequestering them from the host neuropil. The astrogliosis observed at 7 days is consistent with microglia/macrophage-mediated inflammation [44, 45]. The evidence supports our conclusion that MSCs were rejected by an inflammatory response.
Although innate immune cells initiate postoperative inflammation, the response is generally not sufficient to produce graft destruction [36]. Remy and colleagues recently described an interesting disparity in host responsiveness after the transplantation of xenogeneic endothelial cells or fetal neurons into the adult rat striatum [46]. Analogous to our current results, grafted endothelial cells were rapidly rejected by an inflammatory response mediated by microglia/macrophages. In contrast, the fetal neural grafts were rejected after 3 weeks by an adaptive response predominated by T cells. These findings suggest that the nature of the host immune response may be directed by intrinsic characteristics of the donor cells. MSCs and endothelial cells produce GM-CSF (granulocyte/macrophage-colony stimulating factor) and other cytokines that promote the proliferation and maturation of granulocytes and macrophages [47, 48]. Furthermore, MSCs express SDF-1 (stromal-derived factor-1), which is chemotactic for cells of the myeloid lineage [49]. 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 [50, 51]. However, it is unlikely that reporter gene downregulation is responsible for the absence of GFP fluorescence reported herein. The MSCs used in the present study were isolated from GFP- transgenic rats. Transplantation studies using donor cells derived from GFP-transgenic mice or rats have demonstrated stable, long-term expression of reporter genes in vivo [52–54]. Moreover, the histological evidence for graft rejection is compelling.
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.
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 [55].
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 [56, 57]. The trauma and resultant inflammation from the transplant procedure may have provided the stimulus for a similar response.
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 [58]. In addition, a recent study demonstrated the transfer of a nuclear fluorescent marker to unlabeled MSCs in vitro [59]. 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 [35]. We have described the developmental potential of MSCs after infusion into the embryonic rat ventricles in utero [18]. In addition, MSC plasticity has also been reported after i.v. or intracerebral infusion into animal models of neurological disease and injury [26, 28, 30, 60, 61].
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 [2]. The fate of transplanted donor cells may be dependent upon critical variables, including host developmental age and background pathophysiology.
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 [18, 25–30, 35]. In fact, thymidine analogs and fluorescent nuclear markers have been used extensively in the last two decades to assess the plasticity of multiple donor cell types transplanted to the brain. The use of these reagents for the preimplantation labeling of donor cells is still recommended in the literature [29, 62, 63]. The current findings raise the possibility that label transfer may have compromised previous interpretations of MSC survival and plasticity in the brain.
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 [64, 65]. In addition, another current report suggests that the therapeutic benefit attributed to MSC transplantation may occur independently of donor cell survival [66].
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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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REFERENCES |
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