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BDNF Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model
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doi:10.1016/j.ymthe.2003.10.012
BDNF Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model Kazuhiko Kurozumi,1,2 Kiminori Nakamura,1 Takashi Tamiya,2 Yutaka Kawano,1,3 Masayoshi Kobune,1,3 Sachie Hirai,1 Hiroaki Uchida,1 Katsunori Sasaki,1 Yoshinori Ito,1 Kazunori Kato,1 Osamu Honmou,4 Kiyohiro Houkin,4 Isao Date,2 and Hirofumi Hamada1,* 1 Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan 3 Fourth Department of Internal Medicine and 4 Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan 2
*To whom correspondence and reprint request should be addressed at the Department of Molecular Medicine, Sapporo Medical University, South-1, West-17, Chuo, Sapporo 060-8556, Japan. Fax: +81-11-611-2136. E-mail: [email protected].
Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSCBDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke. Key Words: cerebral infarction, mesenchymal stem cell, gene therapy, adenoviral vector, BDNF, MRI
INTRODUCTION Neural stem cells from the mammalian CNS have recently been isolated, propagated in culture, and implanted into CNS lesions in an effort to replace dead and dying neurons [1]. Such cells have also been used to deliver glial cell line-derived neurotrophic factor in a mouse model of Parkinson disease [2], and attempts have been made to transplant neural stem cells from fetal tissue into the adult brain to treat a variety of neurodegenerative disorders [3,4]. While ethical and political issues have limited the use of fetal stem cells,
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these issues do not pertain to the use of adult stem cells. Therefore, the focus has recently shifted to the use of adult bone marrow stromal cells containing mesenchymal stem cells (MSC) for clinical transplantation, in particular autotransplantation. MSC, which exist primarily in the bone marrow, can differentiate into osteoblasts, chondrocytes, adipocytes [5,6], and hepatocytes [7]. It has also been suggested that they might be able to differentiate into other phenotypes as well, including neuronal cells [8]. Chen et al. [9] reported that the transplantation of MSC into local ischemic regions of the rat brain re-
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Strategies are currently being developed for introducing growth factor genes into MSC. To date, transfection efficiency into these cells using plasmid, retroviral, and adenoviral vectors (Adv) has been shown to be 1, 18 – 65, and 20%, respectively [24 – 26]. Fiber-modified Adv (Adv-F/RGD) [27,28], which has an RGD-containing
FIG. 1. BDNF production by cultured MSC. MSC transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at m.o.i. of 100, 300, 1000, and 3000 pu/cell secreted BDNF at a rate of 0.230 F 0.110, 0.434 F 0.122, 0.931 F 0.101, and 1.860 F 0.41 ng/105 cells/48 h, respectively. Nontransfected MSC also produced BDNF (0.0407 F 0.0059 ng/105 cells/48 h).
duced their functional deficits after experimentally induced stroke. Both local, intra-arterial injection and systemic intravenous administration of MSC [10,11] promoted functional improvement in these animals. Human MSC were shown to reduce functional deficits after cerebral ischemia in rats [12], and Zhao et al. [13] further demonstrated that human MSC could differentiate into neuronal cell types. Although MSC express neuronal proteins when grafted into the ischemic brain microenvironment [9], they were not often found to differentiate into cells with a neuronal phenotype [14]. Thus, it seems likely that MSC differentiation into phenotypic neurons was not required by these cells to provide a functional benefit. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor, vascular endothelial growth factors, and hepatocyte growth factor were all reported to be produced by MSC [14]. Thus, the release of such factors from transplanted MSC within the host brain may have contributed to the recovery of function following stroke in recipient animals [15,16]. BDNF promotes the survival and differentiation of neuronal tissue by acting on receptor kinases [17 – 19]. Epileptic, hypoglycemic, ischemic, and traumatic insults to the brain induce marked changes in BDNF gene expression in cortical and hippocampal neurons, which were suggested to represent neuroprotective responses [20,21]. Intraventricular BDNF administered before focal cerebral ischemia [22] and intraparenchymal infusion of BDNF after the ischemia [23] each significantly reduced infarct volume in the cortex, and intravenous BDNF reduced infarct size after temporary focal cerebral ischemia [17].
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FIG. 2. Assessment of brain ischemia-induced neurological deficits. (A) Deficits in limb placement were evaluated using the following scale: 0, severe neurological deficits; 16, no neurological deficits. One day after MCAO but prior to intracerebral MSC injection, there was no statistical difference in limb placement score among the four ischemic groups. Eight days after MCAO, rats that received MSC-BDNF achieved significantly higher limb placement scores compared to control DMEM rats ( P = 0.0001) and rats that received fibroblasts ( P = 0.003). Fifteen days after MCAO, the score of rats that received MSC-BDNF was similarly elevated compared to the DMEM group ( P = 0.024). (B) Prior to MCAO, mean treadmill speeds were comparable between groups. Eight days after MCAO, rats in the MSC-BDNF group achieved significantly higher speeds compared to animals in the control DMEM- ( P = 0.001) and fibroblast-treated ( P = 0.017) groups. These differences were maintained on day 15, with speeds in the MSC-BDNF group being significantly higher than in the control DMEM ( P = 0.002) and fibroblast groups ( P = 0.023).
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peptide in the HI loop of the fiber knob domain of the adenovirus type 5 (Ad5), reportedly demonstrated enhanced effectiveness for the treatment of melanoma [29], arthritis [30], and head and neck cancers [31]. It was recently shown that transfection efficiency of Adv F/RGD into MSC was 12-fold greater than that seen with a vector containing the wild-type fiber (Adv-F/wt) [32]. Recently, we established a human hTERT-transduced MSC line [33], the cells of which have a differentiation potential similar to that of primary MSC. These cells can also be more easily expanded ex vivo than primary MSC. In this study, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether such cells could influence functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model.
RESULTS Detection of Immunoreactive Human BDNF and Quantitative Analysis In Vitro MSC transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at m.o.i. of 100, 300, 1000, and 3000 particle units (pu)/ cell secreted BDNF at a rate of 0.230 F 0.110, 0.434 F 0.122, 0.931 F 0.101, and 1.860 F 0.410 ng/105 cells/48 h, respectively. Nontransfected MSC also produced BDNF protein (0.0407 F 0.0059 ng/105 cells/48 h). The level of BDNF production from MSC-BDNF transfected at an m.o.i. of 1000 pu/cell was 23-fold greater than that seen in uninfected MSC (Fig. 1). Therapeutic Effects of MSC-BDNF (Experiment 1) Limb placement test (LPT). Prior to MCAO, neurological scores were similar among all animals. One day after MCAO but prior to intracerebral MSC injection, there
FIG. 3. (A) T2-weighted (T2W) images taken 2, 7, and 14 days after MCAO in rats injected with DMEM, fibroblasts, MSC, or MSC-BDNF. Seven days after MCAO, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to rats that received DMEM ( P = 0.002), fibroblasts ( P = 0.015), or MSC ( P = 0.028). After 14 days, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to those that received DMEM ( P = 0.011). (B) Representative T2W images obtained 2 and 7 days after MCAO in rats injected with DMEM, MSC, or MSC-BDNF. A reduction in the volume of ischemic damage was detected in the MSC-BDNF group compared to the other groups on day 7.
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was no statistical difference in limb placement score among the four ischemic groups. Eight days after MCAO, rats that received MSC-BDNF achieved significantly higher limb placement scores (8.43 F 1.52) compared to control Dulbecco’s modified essential medium (DMEM) rats (3.71 F 0.49; P = 0.0001) and rats that received fibroblasts (5.00 F 1.10; P = 0.003). Fifteen days after MCAO, the score of rats that received MSC-BDNF was 9.14 F 2.61, which was significantly higher than that seen in the DMEM group (5.00 F 1.73; P = 0.024). On the other hand, neither on day 8 nor on day 15 did rats that received MSC achieve higher scores than control rats that received DMEM or fibroblasts (Fig. 2A). Treadmill test. Prior to MCAO, mean treadmill speeds were comparable between groups. Eight days after MCAO, rats in the MSC-BDNF group achieved significantly higher speeds (23.4 F 2.6 m/s) compared to animals in the control DMEM- (9.57 F 5.6 m/s; P = 0.001) and fibroblast-treated (11.8 F 6.2 m/s; P = 0.017) groups. These differences were maintained on day 15, with speeds in the MSC-BDNF, control DMEM, and control fibroblast groups being 36.6 F 9.5, 12.1 F 9.4 ( P = 0.002), and 15.8 F 11.3 m/s ( P = 0.023), respectively. Rats treated with MSC did not demonstrate enhanced recovery on day 8 or 15 (Fig. 2B). Infarcted volume reduction after MSC-BDNF treatment, as determined by MRI. We summed the areas of hyperintensity on T2-weighted images over the central six MR images and expressed lesion volume as a percentage of contralateral hemispheric volume (%HLV). In all groups, the %HLV decreased from day 2 to day 14. Two days after MCAO, there were no significant differences in %HLV between the MSC-BDNF (35.0 F 4.8%), DMEM (38.7 F 4.9%), fibroblast (37.9 F 3.8%), and MSC (37.8 F 2.8%) groups, though the %HLV in the MSC-BDNF group was reduced somewhat relative to these other groups. On the other hand, 7 days after MCAO, there was a significant reduction in %HLV in rats in the MSC-BDNF group (25.4 F 2.8%) compared to rats in the control DMEM (32.8 F 4.9%; P = 0.002), control fibroblast-treated (31.6 F 2.2%; P = 0.015), and control MSC-treated (30.8 F 4.3%; P = 0.028) groups. After 14 days, there was a significant reduction in %HLV in rats in the MSC-BDNF group (23.7 F 3.2%) compared to DMEM controls (29.6 F 3.6%; P = 0.011). Rats treated with MSC did not show any significant recovery in %HLV on either day 7 (30.8 F 4.3%) or day 14 (26.2 F 2.9%) compared to the control DMEM and fibroblast groups (Figs. 3A and 3B). In Vivo Levels of BDNF Production (Experiment 2) Using a sandwich ELISA, we examined the levels of BDNF in local brain tissue 7 days after MCAO. BDNF levels were significantly increased in the ischemic hemisphere of MSC-BDNF-transplanted rats (45.2 F
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FIG. 4. In vivo levels of BDNF production. BDNF levels were significantly elevated in the ischemic hemisphere of MSC-BDNF-transplanted rats 7 days after MCAO compared to rats that received DMEM ( P = 0.0002) or MSC ( P = 0.0006). Levels of BDNF were also significantly increased in the ischemic hemisphere of MSC compared to DMEM-treated rats ( P = 0.0124).
14.8 pg/mg protein) compared to rats injected with DMEM (12.5 F 1.9 pg/mg protein; P = 0.0002) or MSC (19.3 F 5.5 pg/mg protein; P = 0.0006). BDNF levels were also significantly increased in the ischemic hemisphere of MSC-treated rats compared to DMEMtreated rats ( P = 0.0124) (Fig. 4). Nuclear DNA Fragmentation in MSC-BDNF-Treated Animals (Experiments 3A and 3B) There were significantly fewer terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL)-positive cells (green) in animals injected with MSC-BDNF in the ischemic boundary zone 7 days after MCAO compared to those injected with DMEM (275 F 73 vs 55.0 F 41.0, respectively; P = 0.013). On the other hand, these cells were not significantly less common in animals injected with MSC than in animals injected with DMEM (173.0 F 64.9 vs 55.0 F 41.0; P = 0.20) (Figs. 5A, 5B, and 5C). We detected many Discosoma red fluorescent protein (DsR)-positive MSC within less than 2 mm of the injection site. TUNEL-positive cells of transplanted MSC were decreased in animals treated with MSC-BDNF in the injection site compared with the MSC group (Fig. 5D). Moreover, the TUNEL-positive cells near MSC were decreased in animals treated with MSC-BDNF in the injection site compared with the MSC group. MSC Phenotype (Experiment 4) We examined MSC in the ischemic area morphologically 7 days after MCAO to determine whether they assumed a neuronal phenotype. Some transplanted MSC were immunopositive for the neuronal marker NeuN and the astrocyte marker GFAP. Some displayed fibrous projections while others were round in shape. Transplanted
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FIG. 5. Cells with DNA fragmentation were present in the ischemic penumbra and injection site after MCAO. (A) Fewer TUNEL-positive cells were detected in rats administered MSC-BDNF than in those administered DMEM (FITC, green, TUNEL-positive; PI, red, nuclear; original magnification 200) (B) Original magnification 630. (C) TUNEL-positive cells were significantly reduced in number in animals treated with MSC-BDNF in the ischemic boundary zone compared to animals injected with DMEM ( P = 0.013). (D) Fewer positive cells were detected in rats administered MSC-BDNF compared to those that received MSC. Many DsR-positive MSC were detected within <2 mm of the injection site. FITC (green, TUNEL positive), DsR (red, MSC). MOLECULAR THERAPY Vol. 9, No. 2, February 2004 Copyright D The American Society of Gene Therapy
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FIG. 6. Photomicrographs showing the morphologic features of exogenous MSC and endogenous brain cells in the rat brain. Using double immunofluorescence staining, EGFP cells were localized near the injection site. Laser scanning confocal microscopy showed EGFP cells (green), neuronal nuclear antigen (NeuN; A), and glial fibrillary acidic protein (GFAP; B) in a recipient rat brain. Scale bar, 20 Am.
MSC-BDNF showed features similar to those of MSC (data not shown) (Fig. 6).
DISCUSSION In this study, rats that were intracerebrally injected with MSC-BDNF showed significantly more functional recovery than did rats in the control groups; rats transplanted with nontransfected MSC did not show such improvement. This improvement correlated with the presence of fewer apoptotic cells in the boundary zone. In contrast to our findings, Chen et al. [9] reported a more significant recovery in animals transplanted with MSC compared to control animals; these differences may be attributable to differences between the stroke models used in these studies. Thus, in our model, ligation of the common carotid artery probably resulted in a greater hemodynamic disturbance and more damage than did Chen’s methodology. With regard to MSC spreading, in our study, many MSC were detected within less than 2 mm of the injection site on day 7. Chen et al. [9] reported that their transplanted rat MSC were detected at least 2 mm from the graft site along white matter tracts, including the corpus callosum, 14 days after transplantation. Comparing the discrepancy between the data of Chen et al. [9] and ours, it should be noted that Chen et al. [9] performed MSC treatment with rodent MSC, while we used human MSC. The time point of observation (14 days after MCAO by Chen et al. [9] vs 7 days after
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MCAO by us) might be important. In addition, Chen et al. [9] did not employ immunosuppression, while we administered cyclosporin A. In our study, we transfected MSC with the BDNF gene using Adv-F/RGD and confirmed the production of BDNF protein by these cells in vitro using an ELISA. MSC-BDNF transfected at an m.o.i. of 1000 pu/cell secreted BDNF at a rate of 2.3 ng/5 105 cells/24 h. This secretory rate was quite high and was a reflection of the success of transfection using Ad-F/RGD [29,32]. MSC express low levels of coxsackie adenovirus receptor but high levels of integˆ v [32]; this latter finding probably accounts for the rin a high effectiveness of transfection using this vector, since Adv-F/RGD is known to infect cells by interacting with their cell surface integrins [30]. The production of BDNF protein by transfected MSC was significantly increased in vivo 7 days after MCAO, suggesting that the MSC-BDNF continued to produce BDNF protein for 7 days. It is assumed, but cannot be proven as yet, that this continuous production of BDNF was responsible for limiting the number of cells that underwent DNA fragmentation and for the significant improvement in recovery in these animals. Yamashita et al. [23] and Schabitz et al. [22] reported that the intracranial infusion of BDNF using an osmotic minipump significantly reduced infarct volume following stroke. Our study showed that transplanted MSC carrying the BDNF gene could be used to maintain high levels of BDNF during the critical postischemic period.
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A small number of our grafted MSC expressed the neuronal marker NeuN and the astrocyte marker GFAP (Fig. 6). Chen et al. [9] and Zhao et al. [34] similarly demonstrated the expression of differentiation markers in their transplanted MSC. Two recent reports [35,36] suggested the possibility that Y-positive cells of bone marrow origin fuse with embryonic stem cells rather than transdifferentiating into cells characteristic of various tissues. Further studies must be done to evaluate definitively whether MSC differentiate into parenchymal tissue. Clearly, there are still many obstacles to the widespread use of the technique described in this paper, not the least of which is the intracerebral injection itself, which becomes even more difficult if the target sites are large, widespread, and numerous. While it was reported that MSC injected into the carotid artery appeared to distribute over a wide area of the ischemic core and penumbra [10], intra-arterial injection carries with it the increased risk of embolic events; as such, it would be important to ensure that the MCA and its branches are patent prior to cell injection. It was reported that the intravenous injection of MSC promoted functional improvement after cerebral ischemia [11,12]. Intravenous injection is certainly less invasive and can be more readily carried out in the clinic. However, Chen et al. reported that <1% of intravenously injected cells [37] reached and survived in the brain lesion of rats. Thus, many more cells would be needed to treat patients via intravenous injection compared to the intracranial route. In conclusion, the intracerebral injection of MSC transfected with the BDNF gene using a fiber-mutant adenovirus vector resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data indicate a possible usefulness of this gene-modified cell therapy as an approach for the treatment of stroke.
MATERIALS AND METHODS Cell preparations. Human bone marrow (BM) from three healthy adult volunteers was obtained by aspiration from the posterior iliac crest after informed consent was obtained; this study was approved by the Institutional Review Board at our university. BM mononuclear cells were plated in 150-cm2 plastic tissue culture flasks and incubated overnight. After the free cells were washed away, the adherent cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL, Rockville, MD) in a humidified atmosphere of 5% CO2 at 37jC. After reaching confluence, the cells were harvested and used for gene transfection with a retroviral vector, BABE-hygro-hTERT [38]. MSC within 40 population doublings were used in this study. The morphological features of the MSC were the same as those previously described by Kobune et al. [33]. Adult normal human dermal fibroblasts (NHDF-Ad) were obtained from Takara Bio, Inc., in Japan and were cultured in DMEM containing 10% FBS as described above. Adenoviral vectors. Adenoviral vectors (AxCAEGFP-F/RGD) carrying a humanized variant of Aequoria victoria green fluorescent protein (enhanced GFP:EGFP) together with the gene for the RGD-mutated fiber under the control of a CA promoter (CMV-IE enhancer, with the chicken
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h-actin promoter) were constructed as described previously [29,39]. The EGFP gene fragment was isolated from the pEGFP vector (BD Biosciences Clontech, Palo Alto, CA) and inserted into the pCAcc vector (pCAEGFP) [40]. The cosmid vector pWEAxCAEGFP-F/RGD so generated, together with ClaI- and EcoT22I-digested DNA-TPC from Ad5dlx-F/RGD, was cotransfected into human embryonic kidney 293 cells. The adenoviral EGFP expression vector, AxCAEGFP-F/RGD, obtained from isolated plaques, was expanded in these cells and purified by cesium chloride ultracentrifugation [41]. Adenoviral vectors (AxCADsR-F/RGD) carrying humanized DsR together with the gene for RGD-mutated fiber under the control of a CA promoter were constructed as described above. Human BDNF cDNA was cloned using the RT-PCR method using the total RNA extracted from primary MSC as the template. The identity of BDNF cDNA obtained in this manner was confirmed by sequencing and comparing it to the GenBank sequence XM_006027. The human BDNF primer sequence was Forward, 5V-CGGAATTCCACCATGACCATC C T T T T C C T T A C T A T G G T T A - 3 V, a n d R e v e r s e , 5 V- C C A G A T C TATCTTCCCCTTTTAATGGTCAATGTA-3V. The BDNF cDNA was inserted between the EcoRI site and the BglII site in the pCAcc vector and the resulting plasmid was designated pCAhBDNF. The plasmid pCAhBDNF was digested with ClaI, and the BDNF expression unit was cloned into the adenoviral expression cosmid, resulting in pWEACAhBDNF-F/RGD. The recombinant adenovirus, ACAhBDNF-F/RGD, was generated by transfection with PAC I-digested pWEACAhBDNF-F/RGD. Before being used, the above viral vectors were evaluated for their viral concentration and titer, and viral stocks were examined for potential contamination with replication-competent viruses. To determine viral concentration (pu/ml), the viral solution was incubated in 0.1% sodium dodecyl sulfate and A 260 was measured [42]. The viral titers of AxCAhBDNF-F/RGD, AxCAEGFP-F/RGD, and AxCADsR-F/RGD were 4.35 1011, 5.38 1011, and 1.03 1012 pu/ml, respectively. Adenovirus infection. Adenovirus-mediated gene transfection was performed as previously described [32]. Briefly, the cells were seeded at a density of 2 106 cells per 15-cm plate. MSC were exposed to the infectious viral particles in 7.5 ml DMEM at 37jC medium for 60 min; cells were infected with AxCAhBDNF-F/RGD, AxCAEGFP-F/RGD, or AxCADsR-F/RGD at an m.o.i. of 1 103, 4 103, or 4 103 pu/cell, respectively. The medium was then removed, and the cells were washed once with DMEM and then recultured with normal medium for 24 h, after which intracerebral transplantation was performed. Transient MCAO animal model and intracerebral transplantation. The use of animals in this study was approved by the animal care and use committee of Sapporo Medical University and all procedures were carried out in accordance with institutional guidelines. Rats were anesthetized with 3.5% halothane and unconsciousness was maintained with 1.0 to 2.0% halothane in 70% N2O and 30% O2 using a face mask. Body temperature was maintained at 37jC after surgery by placing the animals under heat lamps. Focal cerebral ischemia was induced in male Wistar rats (250 – 300 g each) by endovascular middle cerebral artery occlusion [43]. A No. 5-O monofilament nylon suture with a silicone-coated tip was inserted through an arteriotomy in the right common carotid artery and gently advanced into the internal carotid artery to a point approximately 18 mm distal to the bifurcation of the carotid artery. After 90 min of transient occlusion, the cerebral blood flow was restored by withdrawal of the nylon thread. Intracerebral transplantation of donor MSC was carried out according to the methods described by Goto et al. [44]. After the induction of ischemic brain injury was confirmed using the behavioral tests described below, the animals were randomized for transplantation. They were anesthetized with an intraperitoneal (ip) injection of ketamine (2.7 – 3 mg/100 g) and xylazine (0.36 – 0.4 mg/100 g) and positioned in a Narishige stereotaxic frame (Model SR-6N; Narishige Co., Japan). Using a 26gauge Hamilton syringe, 5 105 MSC in 5 Al of serum-free DMEM were
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then injected into the right dorsolateral striatum 4 mm beneath the skull surface and 3 mm lateral to bregma over a 2.5-min period [45]. This position approximated the ischemic boundary zone. Rats were administered cyclosporin A (10 mg/kg daily) ip to prevent them from rejecting their human MSC implants. Experimental groups. Experiment 1 was designed to test the therapeutic effectiveness of MSC-BDNF over the course of 14 days following MCAO. The experimental groups included group 1 (control)—rats injected with DMEM (n = 7), group 2—rats transplanted with fibroblasts (NHDF-Ad) (n = 6), group 3—rats transplanted with MSC (n = 7), and group 4—rats transplanted with MSC-BDNF (n = 7) into the ischemic boundary zone 24 h after MCAO. LPTs were performed on days 1, 8, and 15 after MCAO, and treadmill tests were performed on days 8 and 15 after MCAO; MRIs were performed on days 2, 7, and 14. In Experiment 2, growth factor was measured in the following groups of animals: group 1 (control), in which rats were healthy (n = 3); group 2 (control), in which rats were injected with DMEM (n = 3); group 3, in which rats were injected with MSC (n = 3); and group 4, in which rats were injected with MSC-BDNF (n = 3) into the ischemic boundary zone 24 h after MCAO. Rats were sacrificed 7 days after MCAO and the concentration of BDNF in local brain tissue was determined. Experiment 3A was performed to assess the degree of DNA fragmentation of brain cells on day 7 postischemia. Experimental groups were the same as those described above for Experiment 2. Rats were sacrificed 7 days after MCAO and brain tissue was assessed by TUNEL staining. In Experiment 3B, DNA fragmentation on day 7 was investigated in control animals (group 1; n = 3) as well as in animals transplanted with MSC-DsR (group 2; n = 3) or MSC-BDNF-DsR (group 3; n = 3). Experiment 4 was carried out to examine cellular morphology on day 7. Experimental groups included control rats injected with DMEM (group 1; n = 3), rats transplanted with MSC-EGFP (group 2; n = 3), and rats transplanted with MSC-BDNF-EGFP (group 3; n = 3). Rats were sacrificed 7 days after MCAO and brain tissue was assessed morphologically. Behavioral test. The LPTs included eight subtests, described by Johansson and co-workers [46], and were performed 24 h after ischemia. Briefly, the rats’ four limbs were evaluated using the top and edges of a counter top. For each subtest, animals received a score of 0 if they were unable to place their limbs, 1 if they displayed partial and/or delayed (more than 2 s) placement of their limbs, and 2 if they exhibited immediate and correct limb placement. For the treadmill test, rats were placed on an accelerating treadmill (Model MK-680; Muromach Kikai Co., Japan) [47], and their task was to run and maintain their median position on the belt as its speed was steadily increased by 10 m/s for 10 s to a maximum of 70 m/s. A trial was officially ended if a rat could not run; the maximum speed at which each animal could run was measured. Rats were tested on days 8 and 15 after MCAO. MRI studies and measurement of infarct volume. MRIs were performed on all animals on days 2, 7, and 14 after MCAO; animals were anesthetized prior to the procedure as described above. The MRI machine consisted of a 7-T, 18-cm-bore superconducting magnet interfaced to a UNITYINOVA console (Oxford Instruments, UK, and Varian, Inc., Palo Alto, CA). The animals were kept in the same position during imaging. Multislice T2-weighted spin-echo MR images (TR 3000 ms, TE 40 ms, field of view 40 30 mm, slice thickness 2 mm, gapless) were obtained. The disposition of the ischemic area was evaluated by calculating hemispheric lesion volumes (%HLV) from T2-weighted images using imaging software (Scion Image, version Beta 4.0.2; Scion Corp.). For each slice, ischemic tissue was marked and infarct volume calculated taking slice thickness (2 mm/slice) into account. To avoid overestimation of the infarct volume, the corrected infarct volume, or CIV, was calculated as follows, as described by Neumann-Haefelin et al. [48], CIV = (LT (RT RI)) d, where LT was the area of the left hemisphere in square millimeters, RT was the area of the right hemisphere in square millimeters, RI
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was the infarcted area in square millimeters, and d was the thickness of the slices (2 mm). Relative infarct volumes (%HLV) were expressed as a percentage of right hemispheric volume. BDNF ELISA. Forty-eight hours after MSC were transfected in vitro at various m.o.i. (pu/cell), culture supernatants were collected for analysis. Furthermore, 7 days after MCAO, rats were anesthetized with ketamine (4.4 – 8 mg/100 g) and xylazine (1.3 mg/100 g) ip, their brains were removed, and coronal sections (200 mg) from 1.0 to 1.0 mm to bregma in the ischemic hemisphere were dissected on ice and were stored at 80jC until use. Subsequently, each tissue sample was suspended in an equal weight of homogenate buffer (1 ml; 137 mM NaCl, 20 mM Tris, 1% NP-40, 1 mM PMSF, 10 Ag/ml aprotinin, 1 Ag/ml leupeptin, 0.5 mM sodium vanadate) and homogenized with a Dounce homogenizer. The homogenate was centrifuged (10,000 g) for 10 min at 4jC, and the supernatant (5 Ag/Al) was collected for analysis. Commercial BDNF ELISA kits (Promega, Madison, WI) were used to quantify the concentration of BDNF in each of the samples, which were analyzed in triplicate. TUNEL staining and immunohistochemical assessment. Seven days after MCAO, rats were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Their brains were harvested and immersed in 4% PFA in PBS for 2 days, after which 30-Am frozen sections (coronal coordinates bregma from 1.0 to 1.0 mm) were cut with a cryostat at 20jC. Cellular DNA fragmentation in the ischemic boundary zone was detected using the TUNEL method by means of an in situ Apoptosis Detection Kit (TaKaRa Biomedicals, Shiga, Japan) [49]. Specifically, after proteinase digestion, the sections were incubated in a mixture containing terminal deoxynucleotidyltransferase and fluorescein isothiocyanate-labeled dUTP (Green). Sections were counterstained with PI (propidium iodide), which stains red; MSC transfected with AxCADsR-F/RGD stained red in these sections. The total number of positive, red-stained cells was counted in three 1 1mm2 regions of the inner boundary zone [50]. One-hundred-micrometer sections, prepared using a Vibratome, were incubated with primary antibodies diluted in PBS containing 3% BSA and 0.1% Triton X-100 overnight at 4jC. The primary antibodies used in this study were anti-neuronal nuclear antigen (NeuN; mAb377; Chemicon, Temecula, CA) and anti-glial fibrillary acidic protein (GFAP; G3893; Sigma) antibodies. After being rinsed in PBS, the sections were incubated with a fluorescent second antibody (Alexa Fluor 594 goat anti-mouse IgG(H+L), A-11032; Molecular Probes) for 1 h at room temperature. Data analysis. Data were presented as the means F standard deviation. Data from the limb placement and treadmill tests were analyzed using a one-way ANOVA followed by a Games Howell post hoc test. HLV data were analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. The Student t test was used to compare the ELISA data between groups. The number of TUNEL-positive cells in each group was compared using a one-way ANOVA followed by a Scheffe´ post hoc test. Significance was assumed if the P value was <0.05.
ACKNOWLEDGMENTS We thank Hiroshi Isogai and Noriko Kawano for their help with the animal experiments; Hanae Inoue, Satoshi Iiboshi, and Kuniaki Harada for their technical assistance; Akira Takeuchi for helpful discussions; and Tomoko Sonoda for her analysis assistance. This work was supported by grants-in-aid to H.H. from the Japan Ministry of Education and Science (Nos. 14370433 and 14571312). RECEIVED FOR PUBLICATION SEPTEMBER 29, 2003; ACCEPTED OCTOBER 29, 2003.
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BDNF Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model Kazuhiko Kurozumi,1,2 Kiminori Nakamura,1 Takashi Tamiya,2 Yutaka Kawano,1,3 Masayoshi Kobune,1,3 Sachie Hirai,1 Hiroaki Uchida,1 Katsunori Sasaki,1 Yoshinori Ito,1 Kazunori Kato,1 Osamu Honmou,4 Kiyohiro Houkin,4 Isao Date,2 and Hirofumi Hamada1,* 1 Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan 3 Fourth Department of Internal Medicine and 4 Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan 2
*To whom correspondence and reprint request should be addressed at the Department of Molecular Medicine, Sapporo Medical University, South-1, West-17, Chuo, Sapporo 060-8556, Japan. Fax: +81-11-611-2136. E-mail: [email protected].
Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSCBDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke. Key Words: cerebral infarction, mesenchymal stem cell, gene therapy, adenoviral vector, BDNF, MRI
INTRODUCTION Neural stem cells from the mammalian CNS have recently been isolated, propagated in culture, and implanted into CNS lesions in an effort to replace dead and dying neurons [1]. Such cells have also been used to deliver glial cell line-derived neurotrophic factor in a mouse model of Parkinson disease [2], and attempts have been made to transplant neural stem cells from fetal tissue into the adult brain to treat a variety of neurodegenerative disorders [3,4]. While ethical and political issues have limited the use of fetal stem cells,
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these issues do not pertain to the use of adult stem cells. Therefore, the focus has recently shifted to the use of adult bone marrow stromal cells containing mesenchymal stem cells (MSC) for clinical transplantation, in particular autotransplantation. MSC, which exist primarily in the bone marrow, can differentiate into osteoblasts, chondrocytes, adipocytes [5,6], and hepatocytes [7]. It has also been suggested that they might be able to differentiate into other phenotypes as well, including neuronal cells [8]. Chen et al. [9] reported that the transplantation of MSC into local ischemic regions of the rat brain re-
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Strategies are currently being developed for introducing growth factor genes into MSC. To date, transfection efficiency into these cells using plasmid, retroviral, and adenoviral vectors (Adv) has been shown to be 1, 18 – 65, and 20%, respectively [24 – 26]. Fiber-modified Adv (Adv-F/RGD) [27,28], which has an RGD-containing
FIG. 1. BDNF production by cultured MSC. MSC transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at m.o.i. of 100, 300, 1000, and 3000 pu/cell secreted BDNF at a rate of 0.230 F 0.110, 0.434 F 0.122, 0.931 F 0.101, and 1.860 F 0.41 ng/105 cells/48 h, respectively. Nontransfected MSC also produced BDNF (0.0407 F 0.0059 ng/105 cells/48 h).
duced their functional deficits after experimentally induced stroke. Both local, intra-arterial injection and systemic intravenous administration of MSC [10,11] promoted functional improvement in these animals. Human MSC were shown to reduce functional deficits after cerebral ischemia in rats [12], and Zhao et al. [13] further demonstrated that human MSC could differentiate into neuronal cell types. Although MSC express neuronal proteins when grafted into the ischemic brain microenvironment [9], they were not often found to differentiate into cells with a neuronal phenotype [14]. Thus, it seems likely that MSC differentiation into phenotypic neurons was not required by these cells to provide a functional benefit. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor, vascular endothelial growth factors, and hepatocyte growth factor were all reported to be produced by MSC [14]. Thus, the release of such factors from transplanted MSC within the host brain may have contributed to the recovery of function following stroke in recipient animals [15,16]. BDNF promotes the survival and differentiation of neuronal tissue by acting on receptor kinases [17 – 19]. Epileptic, hypoglycemic, ischemic, and traumatic insults to the brain induce marked changes in BDNF gene expression in cortical and hippocampal neurons, which were suggested to represent neuroprotective responses [20,21]. Intraventricular BDNF administered before focal cerebral ischemia [22] and intraparenchymal infusion of BDNF after the ischemia [23] each significantly reduced infarct volume in the cortex, and intravenous BDNF reduced infarct size after temporary focal cerebral ischemia [17].
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FIG. 2. Assessment of brain ischemia-induced neurological deficits. (A) Deficits in limb placement were evaluated using the following scale: 0, severe neurological deficits; 16, no neurological deficits. One day after MCAO but prior to intracerebral MSC injection, there was no statistical difference in limb placement score among the four ischemic groups. Eight days after MCAO, rats that received MSC-BDNF achieved significantly higher limb placement scores compared to control DMEM rats ( P = 0.0001) and rats that received fibroblasts ( P = 0.003). Fifteen days after MCAO, the score of rats that received MSC-BDNF was similarly elevated compared to the DMEM group ( P = 0.024). (B) Prior to MCAO, mean treadmill speeds were comparable between groups. Eight days after MCAO, rats in the MSC-BDNF group achieved significantly higher speeds compared to animals in the control DMEM- ( P = 0.001) and fibroblast-treated ( P = 0.017) groups. These differences were maintained on day 15, with speeds in the MSC-BDNF group being significantly higher than in the control DMEM ( P = 0.002) and fibroblast groups ( P = 0.023).
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peptide in the HI loop of the fiber knob domain of the adenovirus type 5 (Ad5), reportedly demonstrated enhanced effectiveness for the treatment of melanoma [29], arthritis [30], and head and neck cancers [31]. It was recently shown that transfection efficiency of Adv F/RGD into MSC was 12-fold greater than that seen with a vector containing the wild-type fiber (Adv-F/wt) [32]. Recently, we established a human hTERT-transduced MSC line [33], the cells of which have a differentiation potential similar to that of primary MSC. These cells can also be more easily expanded ex vivo than primary MSC. In this study, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether such cells could influence functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model.
RESULTS Detection of Immunoreactive Human BDNF and Quantitative Analysis In Vitro MSC transfected with AxCAhBDNF-F/RGD (MSC-BDNF) at m.o.i. of 100, 300, 1000, and 3000 particle units (pu)/ cell secreted BDNF at a rate of 0.230 F 0.110, 0.434 F 0.122, 0.931 F 0.101, and 1.860 F 0.410 ng/105 cells/48 h, respectively. Nontransfected MSC also produced BDNF protein (0.0407 F 0.0059 ng/105 cells/48 h). The level of BDNF production from MSC-BDNF transfected at an m.o.i. of 1000 pu/cell was 23-fold greater than that seen in uninfected MSC (Fig. 1). Therapeutic Effects of MSC-BDNF (Experiment 1) Limb placement test (LPT). Prior to MCAO, neurological scores were similar among all animals. One day after MCAO but prior to intracerebral MSC injection, there
FIG. 3. (A) T2-weighted (T2W) images taken 2, 7, and 14 days after MCAO in rats injected with DMEM, fibroblasts, MSC, or MSC-BDNF. Seven days after MCAO, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to rats that received DMEM ( P = 0.002), fibroblasts ( P = 0.015), or MSC ( P = 0.028). After 14 days, there was a significant reduction in %HLV in rats treated with MSC-BDNF compared to those that received DMEM ( P = 0.011). (B) Representative T2W images obtained 2 and 7 days after MCAO in rats injected with DMEM, MSC, or MSC-BDNF. A reduction in the volume of ischemic damage was detected in the MSC-BDNF group compared to the other groups on day 7.
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was no statistical difference in limb placement score among the four ischemic groups. Eight days after MCAO, rats that received MSC-BDNF achieved significantly higher limb placement scores (8.43 F 1.52) compared to control Dulbecco’s modified essential medium (DMEM) rats (3.71 F 0.49; P = 0.0001) and rats that received fibroblasts (5.00 F 1.10; P = 0.003). Fifteen days after MCAO, the score of rats that received MSC-BDNF was 9.14 F 2.61, which was significantly higher than that seen in the DMEM group (5.00 F 1.73; P = 0.024). On the other hand, neither on day 8 nor on day 15 did rats that received MSC achieve higher scores than control rats that received DMEM or fibroblasts (Fig. 2A). Treadmill test. Prior to MCAO, mean treadmill speeds were comparable between groups. Eight days after MCAO, rats in the MSC-BDNF group achieved significantly higher speeds (23.4 F 2.6 m/s) compared to animals in the control DMEM- (9.57 F 5.6 m/s; P = 0.001) and fibroblast-treated (11.8 F 6.2 m/s; P = 0.017) groups. These differences were maintained on day 15, with speeds in the MSC-BDNF, control DMEM, and control fibroblast groups being 36.6 F 9.5, 12.1 F 9.4 ( P = 0.002), and 15.8 F 11.3 m/s ( P = 0.023), respectively. Rats treated with MSC did not demonstrate enhanced recovery on day 8 or 15 (Fig. 2B). Infarcted volume reduction after MSC-BDNF treatment, as determined by MRI. We summed the areas of hyperintensity on T2-weighted images over the central six MR images and expressed lesion volume as a percentage of contralateral hemispheric volume (%HLV). In all groups, the %HLV decreased from day 2 to day 14. Two days after MCAO, there were no significant differences in %HLV between the MSC-BDNF (35.0 F 4.8%), DMEM (38.7 F 4.9%), fibroblast (37.9 F 3.8%), and MSC (37.8 F 2.8%) groups, though the %HLV in the MSC-BDNF group was reduced somewhat relative to these other groups. On the other hand, 7 days after MCAO, there was a significant reduction in %HLV in rats in the MSC-BDNF group (25.4 F 2.8%) compared to rats in the control DMEM (32.8 F 4.9%; P = 0.002), control fibroblast-treated (31.6 F 2.2%; P = 0.015), and control MSC-treated (30.8 F 4.3%; P = 0.028) groups. After 14 days, there was a significant reduction in %HLV in rats in the MSC-BDNF group (23.7 F 3.2%) compared to DMEM controls (29.6 F 3.6%; P = 0.011). Rats treated with MSC did not show any significant recovery in %HLV on either day 7 (30.8 F 4.3%) or day 14 (26.2 F 2.9%) compared to the control DMEM and fibroblast groups (Figs. 3A and 3B). In Vivo Levels of BDNF Production (Experiment 2) Using a sandwich ELISA, we examined the levels of BDNF in local brain tissue 7 days after MCAO. BDNF levels were significantly increased in the ischemic hemisphere of MSC-BDNF-transplanted rats (45.2 F
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FIG. 4. In vivo levels of BDNF production. BDNF levels were significantly elevated in the ischemic hemisphere of MSC-BDNF-transplanted rats 7 days after MCAO compared to rats that received DMEM ( P = 0.0002) or MSC ( P = 0.0006). Levels of BDNF were also significantly increased in the ischemic hemisphere of MSC compared to DMEM-treated rats ( P = 0.0124).
14.8 pg/mg protein) compared to rats injected with DMEM (12.5 F 1.9 pg/mg protein; P = 0.0002) or MSC (19.3 F 5.5 pg/mg protein; P = 0.0006). BDNF levels were also significantly increased in the ischemic hemisphere of MSC-treated rats compared to DMEMtreated rats ( P = 0.0124) (Fig. 4). Nuclear DNA Fragmentation in MSC-BDNF-Treated Animals (Experiments 3A and 3B) There were significantly fewer terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL)-positive cells (green) in animals injected with MSC-BDNF in the ischemic boundary zone 7 days after MCAO compared to those injected with DMEM (275 F 73 vs 55.0 F 41.0, respectively; P = 0.013). On the other hand, these cells were not significantly less common in animals injected with MSC than in animals injected with DMEM (173.0 F 64.9 vs 55.0 F 41.0; P = 0.20) (Figs. 5A, 5B, and 5C). We detected many Discosoma red fluorescent protein (DsR)-positive MSC within less than 2 mm of the injection site. TUNEL-positive cells of transplanted MSC were decreased in animals treated with MSC-BDNF in the injection site compared with the MSC group (Fig. 5D). Moreover, the TUNEL-positive cells near MSC were decreased in animals treated with MSC-BDNF in the injection site compared with the MSC group. MSC Phenotype (Experiment 4) We examined MSC in the ischemic area morphologically 7 days after MCAO to determine whether they assumed a neuronal phenotype. Some transplanted MSC were immunopositive for the neuronal marker NeuN and the astrocyte marker GFAP. Some displayed fibrous projections while others were round in shape. Transplanted
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FIG. 5. Cells with DNA fragmentation were present in the ischemic penumbra and injection site after MCAO. (A) Fewer TUNEL-positive cells were detected in rats administered MSC-BDNF than in those administered DMEM (FITC, green, TUNEL-positive; PI, red, nuclear; original magnification 200) (B) Original magnification 630. (C) TUNEL-positive cells were significantly reduced in number in animals treated with MSC-BDNF in the ischemic boundary zone compared to animals injected with DMEM ( P = 0.013). (D) Fewer positive cells were detected in rats administered MSC-BDNF compared to those that received MSC. Many DsR-positive MSC were detected within <2 mm of the injection site. FITC (green, TUNEL positive), DsR (red, MSC). MOLECULAR THERAPY Vol. 9, No. 2, February 2004 Copyright D The American Society of Gene Therapy
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FIG. 6. Photomicrographs showing the morphologic features of exogenous MSC and endogenous brain cells in the rat brain. Using double immunofluorescence staining, EGFP cells were localized near the injection site. Laser scanning confocal microscopy showed EGFP cells (green), neuronal nuclear antigen (NeuN; A), and glial fibrillary acidic protein (GFAP; B) in a recipient rat brain. Scale bar, 20 Am.
MSC-BDNF showed features similar to those of MSC (data not shown) (Fig. 6).
DISCUSSION In this study, rats that were intracerebrally injected with MSC-BDNF showed significantly more functional recovery than did rats in the control groups; rats transplanted with nontransfected MSC did not show such improvement. This improvement correlated with the presence of fewer apoptotic cells in the boundary zone. In contrast to our findings, Chen et al. [9] reported a more significant recovery in animals transplanted with MSC compared to control animals; these differences may be attributable to differences between the stroke models used in these studies. Thus, in our model, ligation of the common carotid artery probably resulted in a greater hemodynamic disturbance and more damage than did Chen’s methodology. With regard to MSC spreading, in our study, many MSC were detected within less than 2 mm of the injection site on day 7. Chen et al. [9] reported that their transplanted rat MSC were detected at least 2 mm from the graft site along white matter tracts, including the corpus callosum, 14 days after transplantation. Comparing the discrepancy between the data of Chen et al. [9] and ours, it should be noted that Chen et al. [9] performed MSC treatment with rodent MSC, while we used human MSC. The time point of observation (14 days after MCAO by Chen et al. [9] vs 7 days after
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MCAO by us) might be important. In addition, Chen et al. [9] did not employ immunosuppression, while we administered cyclosporin A. In our study, we transfected MSC with the BDNF gene using Adv-F/RGD and confirmed the production of BDNF protein by these cells in vitro using an ELISA. MSC-BDNF transfected at an m.o.i. of 1000 pu/cell secreted BDNF at a rate of 2.3 ng/5 105 cells/24 h. This secretory rate was quite high and was a reflection of the success of transfection using Ad-F/RGD [29,32]. MSC express low levels of coxsackie adenovirus receptor but high levels of integˆ v [32]; this latter finding probably accounts for the rin a high effectiveness of transfection using this vector, since Adv-F/RGD is known to infect cells by interacting with their cell surface integrins [30]. The production of BDNF protein by transfected MSC was significantly increased in vivo 7 days after MCAO, suggesting that the MSC-BDNF continued to produce BDNF protein for 7 days. It is assumed, but cannot be proven as yet, that this continuous production of BDNF was responsible for limiting the number of cells that underwent DNA fragmentation and for the significant improvement in recovery in these animals. Yamashita et al. [23] and Schabitz et al. [22] reported that the intracranial infusion of BDNF using an osmotic minipump significantly reduced infarct volume following stroke. Our study showed that transplanted MSC carrying the BDNF gene could be used to maintain high levels of BDNF during the critical postischemic period.
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A small number of our grafted MSC expressed the neuronal marker NeuN and the astrocyte marker GFAP (Fig. 6). Chen et al. [9] and Zhao et al. [34] similarly demonstrated the expression of differentiation markers in their transplanted MSC. Two recent reports [35,36] suggested the possibility that Y-positive cells of bone marrow origin fuse with embryonic stem cells rather than transdifferentiating into cells characteristic of various tissues. Further studies must be done to evaluate definitively whether MSC differentiate into parenchymal tissue. Clearly, there are still many obstacles to the widespread use of the technique described in this paper, not the least of which is the intracerebral injection itself, which becomes even more difficult if the target sites are large, widespread, and numerous. While it was reported that MSC injected into the carotid artery appeared to distribute over a wide area of the ischemic core and penumbra [10], intra-arterial injection carries with it the increased risk of embolic events; as such, it would be important to ensure that the MCA and its branches are patent prior to cell injection. It was reported that the intravenous injection of MSC promoted functional improvement after cerebral ischemia [11,12]. Intravenous injection is certainly less invasive and can be more readily carried out in the clinic. However, Chen et al. reported that <1% of intravenously injected cells [37] reached and survived in the brain lesion of rats. Thus, many more cells would be needed to treat patients via intravenous injection compared to the intracranial route. In conclusion, the intracerebral injection of MSC transfected with the BDNF gene using a fiber-mutant adenovirus vector resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data indicate a possible usefulness of this gene-modified cell therapy as an approach for the treatment of stroke.
MATERIALS AND METHODS Cell preparations. Human bone marrow (BM) from three healthy adult volunteers was obtained by aspiration from the posterior iliac crest after informed consent was obtained; this study was approved by the Institutional Review Board at our university. BM mononuclear cells were plated in 150-cm2 plastic tissue culture flasks and incubated overnight. After the free cells were washed away, the adherent cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL, Rockville, MD) in a humidified atmosphere of 5% CO2 at 37jC. After reaching confluence, the cells were harvested and used for gene transfection with a retroviral vector, BABE-hygro-hTERT [38]. MSC within 40 population doublings were used in this study. The morphological features of the MSC were the same as those previously described by Kobune et al. [33]. Adult normal human dermal fibroblasts (NHDF-Ad) were obtained from Takara Bio, Inc., in Japan and were cultured in DMEM containing 10% FBS as described above. Adenoviral vectors. Adenoviral vectors (AxCAEGFP-F/RGD) carrying a humanized variant of Aequoria victoria green fluorescent protein (enhanced GFP:EGFP) together with the gene for the RGD-mutated fiber under the control of a CA promoter (CMV-IE enhancer, with the chicken
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h-actin promoter) were constructed as described previously [29,39]. The EGFP gene fragment was isolated from the pEGFP vector (BD Biosciences Clontech, Palo Alto, CA) and inserted into the pCAcc vector (pCAEGFP) [40]. The cosmid vector pWEAxCAEGFP-F/RGD so generated, together with ClaI- and EcoT22I-digested DNA-TPC from Ad5dlx-F/RGD, was cotransfected into human embryonic kidney 293 cells. The adenoviral EGFP expression vector, AxCAEGFP-F/RGD, obtained from isolated plaques, was expanded in these cells and purified by cesium chloride ultracentrifugation [41]. Adenoviral vectors (AxCADsR-F/RGD) carrying humanized DsR together with the gene for RGD-mutated fiber under the control of a CA promoter were constructed as described above. Human BDNF cDNA was cloned using the RT-PCR method using the total RNA extracted from primary MSC as the template. The identity of BDNF cDNA obtained in this manner was confirmed by sequencing and comparing it to the GenBank sequence XM_006027. The human BDNF primer sequence was Forward, 5V-CGGAATTCCACCATGACCATC C T T T T C C T T A C T A T G G T T A - 3 V, a n d R e v e r s e , 5 V- C C A G A T C TATCTTCCCCTTTTAATGGTCAATGTA-3V. The BDNF cDNA was inserted between the EcoRI site and the BglII site in the pCAcc vector and the resulting plasmid was designated pCAhBDNF. The plasmid pCAhBDNF was digested with ClaI, and the BDNF expression unit was cloned into the adenoviral expression cosmid, resulting in pWEACAhBDNF-F/RGD. The recombinant adenovirus, ACAhBDNF-F/RGD, was generated by transfection with PAC I-digested pWEACAhBDNF-F/RGD. Before being used, the above viral vectors were evaluated for their viral concentration and titer, and viral stocks were examined for potential contamination with replication-competent viruses. To determine viral concentration (pu/ml), the viral solution was incubated in 0.1% sodium dodecyl sulfate and A 260 was measured [42]. The viral titers of AxCAhBDNF-F/RGD, AxCAEGFP-F/RGD, and AxCADsR-F/RGD were 4.35 1011, 5.38 1011, and 1.03 1012 pu/ml, respectively. Adenovirus infection. Adenovirus-mediated gene transfection was performed as previously described [32]. Briefly, the cells were seeded at a density of 2 106 cells per 15-cm plate. MSC were exposed to the infectious viral particles in 7.5 ml DMEM at 37jC medium for 60 min; cells were infected with AxCAhBDNF-F/RGD, AxCAEGFP-F/RGD, or AxCADsR-F/RGD at an m.o.i. of 1 103, 4 103, or 4 103 pu/cell, respectively. The medium was then removed, and the cells were washed once with DMEM and then recultured with normal medium for 24 h, after which intracerebral transplantation was performed. Transient MCAO animal model and intracerebral transplantation. The use of animals in this study was approved by the animal care and use committee of Sapporo Medical University and all procedures were carried out in accordance with institutional guidelines. Rats were anesthetized with 3.5% halothane and unconsciousness was maintained with 1.0 to 2.0% halothane in 70% N2O and 30% O2 using a face mask. Body temperature was maintained at 37jC after surgery by placing the animals under heat lamps. Focal cerebral ischemia was induced in male Wistar rats (250 – 300 g each) by endovascular middle cerebral artery occlusion [43]. A No. 5-O monofilament nylon suture with a silicone-coated tip was inserted through an arteriotomy in the right common carotid artery and gently advanced into the internal carotid artery to a point approximately 18 mm distal to the bifurcation of the carotid artery. After 90 min of transient occlusion, the cerebral blood flow was restored by withdrawal of the nylon thread. Intracerebral transplantation of donor MSC was carried out according to the methods described by Goto et al. [44]. After the induction of ischemic brain injury was confirmed using the behavioral tests described below, the animals were randomized for transplantation. They were anesthetized with an intraperitoneal (ip) injection of ketamine (2.7 – 3 mg/100 g) and xylazine (0.36 – 0.4 mg/100 g) and positioned in a Narishige stereotaxic frame (Model SR-6N; Narishige Co., Japan). Using a 26gauge Hamilton syringe, 5 105 MSC in 5 Al of serum-free DMEM were
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then injected into the right dorsolateral striatum 4 mm beneath the skull surface and 3 mm lateral to bregma over a 2.5-min period [45]. This position approximated the ischemic boundary zone. Rats were administered cyclosporin A (10 mg/kg daily) ip to prevent them from rejecting their human MSC implants. Experimental groups. Experiment 1 was designed to test the therapeutic effectiveness of MSC-BDNF over the course of 14 days following MCAO. The experimental groups included group 1 (control)—rats injected with DMEM (n = 7), group 2—rats transplanted with fibroblasts (NHDF-Ad) (n = 6), group 3—rats transplanted with MSC (n = 7), and group 4—rats transplanted with MSC-BDNF (n = 7) into the ischemic boundary zone 24 h after MCAO. LPTs were performed on days 1, 8, and 15 after MCAO, and treadmill tests were performed on days 8 and 15 after MCAO; MRIs were performed on days 2, 7, and 14. In Experiment 2, growth factor was measured in the following groups of animals: group 1 (control), in which rats were healthy (n = 3); group 2 (control), in which rats were injected with DMEM (n = 3); group 3, in which rats were injected with MSC (n = 3); and group 4, in which rats were injected with MSC-BDNF (n = 3) into the ischemic boundary zone 24 h after MCAO. Rats were sacrificed 7 days after MCAO and the concentration of BDNF in local brain tissue was determined. Experiment 3A was performed to assess the degree of DNA fragmentation of brain cells on day 7 postischemia. Experimental groups were the same as those described above for Experiment 2. Rats were sacrificed 7 days after MCAO and brain tissue was assessed by TUNEL staining. In Experiment 3B, DNA fragmentation on day 7 was investigated in control animals (group 1; n = 3) as well as in animals transplanted with MSC-DsR (group 2; n = 3) or MSC-BDNF-DsR (group 3; n = 3). Experiment 4 was carried out to examine cellular morphology on day 7. Experimental groups included control rats injected with DMEM (group 1; n = 3), rats transplanted with MSC-EGFP (group 2; n = 3), and rats transplanted with MSC-BDNF-EGFP (group 3; n = 3). Rats were sacrificed 7 days after MCAO and brain tissue was assessed morphologically. Behavioral test. The LPTs included eight subtests, described by Johansson and co-workers [46], and were performed 24 h after ischemia. Briefly, the rats’ four limbs were evaluated using the top and edges of a counter top. For each subtest, animals received a score of 0 if they were unable to place their limbs, 1 if they displayed partial and/or delayed (more than 2 s) placement of their limbs, and 2 if they exhibited immediate and correct limb placement. For the treadmill test, rats were placed on an accelerating treadmill (Model MK-680; Muromach Kikai Co., Japan) [47], and their task was to run and maintain their median position on the belt as its speed was steadily increased by 10 m/s for 10 s to a maximum of 70 m/s. A trial was officially ended if a rat could not run; the maximum speed at which each animal could run was measured. Rats were tested on days 8 and 15 after MCAO. MRI studies and measurement of infarct volume. MRIs were performed on all animals on days 2, 7, and 14 after MCAO; animals were anesthetized prior to the procedure as described above. The MRI machine consisted of a 7-T, 18-cm-bore superconducting magnet interfaced to a UNITYINOVA console (Oxford Instruments, UK, and Varian, Inc., Palo Alto, CA). The animals were kept in the same position during imaging. Multislice T2-weighted spin-echo MR images (TR 3000 ms, TE 40 ms, field of view 40 30 mm, slice thickness 2 mm, gapless) were obtained. The disposition of the ischemic area was evaluated by calculating hemispheric lesion volumes (%HLV) from T2-weighted images using imaging software (Scion Image, version Beta 4.0.2; Scion Corp.). For each slice, ischemic tissue was marked and infarct volume calculated taking slice thickness (2 mm/slice) into account. To avoid overestimation of the infarct volume, the corrected infarct volume, or CIV, was calculated as follows, as described by Neumann-Haefelin et al. [48], CIV = (LT (RT RI)) d, where LT was the area of the left hemisphere in square millimeters, RT was the area of the right hemisphere in square millimeters, RI
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was the infarcted area in square millimeters, and d was the thickness of the slices (2 mm). Relative infarct volumes (%HLV) were expressed as a percentage of right hemispheric volume. BDNF ELISA. Forty-eight hours after MSC were transfected in vitro at various m.o.i. (pu/cell), culture supernatants were collected for analysis. Furthermore, 7 days after MCAO, rats were anesthetized with ketamine (4.4 – 8 mg/100 g) and xylazine (1.3 mg/100 g) ip, their brains were removed, and coronal sections (200 mg) from 1.0 to 1.0 mm to bregma in the ischemic hemisphere were dissected on ice and were stored at 80jC until use. Subsequently, each tissue sample was suspended in an equal weight of homogenate buffer (1 ml; 137 mM NaCl, 20 mM Tris, 1% NP-40, 1 mM PMSF, 10 Ag/ml aprotinin, 1 Ag/ml leupeptin, 0.5 mM sodium vanadate) and homogenized with a Dounce homogenizer. The homogenate was centrifuged (10,000 g) for 10 min at 4jC, and the supernatant (5 Ag/Al) was collected for analysis. Commercial BDNF ELISA kits (Promega, Madison, WI) were used to quantify the concentration of BDNF in each of the samples, which were analyzed in triplicate. TUNEL staining and immunohistochemical assessment. Seven days after MCAO, rats were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Their brains were harvested and immersed in 4% PFA in PBS for 2 days, after which 30-Am frozen sections (coronal coordinates bregma from 1.0 to 1.0 mm) were cut with a cryostat at 20jC. Cellular DNA fragmentation in the ischemic boundary zone was detected using the TUNEL method by means of an in situ Apoptosis Detection Kit (TaKaRa Biomedicals, Shiga, Japan) [49]. Specifically, after proteinase digestion, the sections were incubated in a mixture containing terminal deoxynucleotidyltransferase and fluorescein isothiocyanate-labeled dUTP (Green). Sections were counterstained with PI (propidium iodide), which stains red; MSC transfected with AxCADsR-F/RGD stained red in these sections. The total number of positive, red-stained cells was counted in three 1 1mm2 regions of the inner boundary zone [50]. One-hundred-micrometer sections, prepared using a Vibratome, were incubated with primary antibodies diluted in PBS containing 3% BSA and 0.1% Triton X-100 overnight at 4jC. The primary antibodies used in this study were anti-neuronal nuclear antigen (NeuN; mAb377; Chemicon, Temecula, CA) and anti-glial fibrillary acidic protein (GFAP; G3893; Sigma) antibodies. After being rinsed in PBS, the sections were incubated with a fluorescent second antibody (Alexa Fluor 594 goat anti-mouse IgG(H+L), A-11032; Molecular Probes) for 1 h at room temperature. Data analysis. Data were presented as the means F standard deviation. Data from the limb placement and treadmill tests were analyzed using a one-way ANOVA followed by a Games Howell post hoc test. HLV data were analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. The Student t test was used to compare the ELISA data between groups. The number of TUNEL-positive cells in each group was compared using a one-way ANOVA followed by a Scheffe´ post hoc test. Significance was assumed if the P value was <0.05.
ACKNOWLEDGMENTS We thank Hiroshi Isogai and Noriko Kawano for their help with the animal experiments; Hanae Inoue, Satoshi Iiboshi, and Kuniaki Harada for their technical assistance; Akira Takeuchi for helpful discussions; and Tomoko Sonoda for her analysis assistance. This work was supported by grants-in-aid to H.H. from the Japan Ministry of Education and Science (Nos. 14370433 and 14571312). RECEIVED FOR PUBLICATION SEPTEMBER 29, 2003; ACCEPTED OCTOBER 29, 2003.
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