Hideo Shichinohe

Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice

There is increasing evidence that bone marrow stromal cells (BMSC) have the potential to migrate into the injured neural tissue and to differentiate into the CNS cells, indicating the possibility of autograft transplantation therapy. The present study was aimed to clarify whether the mouse BMSC can migrate into the lesion and differentiate into the CNS cells when transplanted into the mice subjected to focal cerebral infarct or spinal cord injury. The BMSC were harvested from mice and characterized by flow cytometry. Then, the BMSC were labeled by bis‐benzimide, a nuclear fluorescence dye, over 24 h, and were stereotactically transplanted into the brain or spinal cord of the mice. The cultured BMSC expressed low levels of CD45 and high levels of CD90 and Sca‐1 on flow cytometry. A large number of grafted cells survived in the normal brain 4 weeks after transplantation, many of which were located close to the transplanted sites. They expressed the neuronal marker including NeuN, MAP2, and doublecortin on fluorescent immunohistochemistry. However, when the BMSC were transplanted into the ipsilateral striatum of the mice subjected to middle cerebral artery occlusion, many of the grafted cells migrated into the corpus callosum and injured cortex, and also expressed the neuronal markers 4 weeks after transplantation. In particular, NeuN was very useful to validate the differentiation of the grafted cells, because the marker was expressed in the nuclei and was overlapped with bis‐benzimide. Similar results were obtained in the mice subjected to spinal cord injury. However, many of the transplanted BMSC expressed GFAP, an astrocytic protein, in injured spinal cord. The present results indicate that the mouse BMSC can migrate into the CNS lesion and differentiate into the neurons or astrocytes, and that bis‐benzimide is a simple and useful marker to label the donor cells and to evaluate their migration and differentiation in the host neural tissues over a long period.

Bone marrow stromal cells protect and repair damaged neurons through multiple mechanisms

A surprising shortage of information surrounds the mechanism by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. To clarify the issue, the BMSC were cocultured with the neurons using two paradigms: the cell‐mixing coculture technique and three‐dimensional coculture technique. The green fluorescent protein (GFP)‐expressing BMSC were cocultured with the PKH‐26‐labelled neurons, using cell mixing coculture technique. GFP‐positive, PKH‐26‐negative cells morphologically simulated the neurons and significantly increased the expression of MAP‐2, Tuj‐1, nestin, and GFAP. GFP/nestin‐positive, PKH‐26‐negative cells increased from 13.6% ± 6.7% to 32.1% ± 15.5% over 7 days of coculture. They further enhanced Tuj‐1 expression when cocultured with neurons exposed to 100 μM of glutamate for 10 min. About 20–30% of GFP‐positive cells became positive for PKH‐26 through coculture with the neurons, but the doubly positive cells did not increase when cocultured with glutamate‐exposed neurons. Alternatively, the BMSC significantly ameliorated glutamate‐induced neuronal damage when cocultured with the three‐dimensional coculture technique. The protective effect was more prominent when coculture was started prior to glutamate exposure than when coculture was started just after glutamate exposure. ELISA analysis revealed that the BMSC physiologically produce NGF, BDNF, SDF‐1α, HGF, TGFβ‐1, and IGF‐1 and significantly enhanced the production of NGF and BDNF when cocultured with glutamate‐exposed neurons. These findings strongly suggest that the BMSC may protect and repair the damaged neurons through multiple mechanisms, including transdifferentiation, cell fusion, and production of growth factors.

Neuroprotective effects of the free radical scavenger Edaravone (MCI-186) in mice permanent focal brain ischemia

The present study was aimed to evaluate the effect of the free radical scavenger Edaravone on infarct volume due to permanent MCA occlusion in mice and, if so, to elucidate the mechanism of its neuroprotective effects. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion and were treated with 3.0 mg/kg of Edaravone or vehicle 30 min before ischemia. Infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) method after 24 h. Furthermore, in situ detection of superoxide in the ipsilateral neocortex was carried out using the superoxide-sensitive dye dihydroethidium (DHE) staining technique. Pretreatment with 3.0 mg/kg of Edaravone ameliorated the tissue damage in the infarct rim and significantly reduced infarct volume to about 77% of the control (p<0.05). Semi-quantitative measurement of red fluorescence emitted from DHE revealed that the superoxide increased in the ischemic core at 1 h after the onset of ischemia and extended towards the infarct rim at 3 and 6 h, and that pretreatment with 3.0 mg/kg of Edaravone significantly inhibited the increase of superoxide in the infarct rim at 3 and 6 h (p<0.01). Double staining with DHE and monoclonal antibody against NeuN showed that the majority of the nuclei positive for DHE were also positive for NeuN. These findings suggest that Edaravone salvages the boundary zone of infarct by scavenging reactive oxygen species especially in the neurons during permanent focal cerebral ischemia.

Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct

Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurological function when transplanted into animal models of cerebral infarction. However, it is still obscure how the transplanted BMSC restore the lost neurological function. In this study, therefore, we aimed to elucidate the role of stromal cell-derived factor-1 (SDF-1) and its specific receptor, CXCR4, in BMSC transplantation into the brain subjected to cerebral infarction. The BMSC were harvested from the wild type (WT) and CXCR4-knockout (CXCR4-KO) mice and were cultured. The mice were subjected to permanent middle cerebral artery occlusion. The WT or CXCR4-KO BMSC was injected into the ipsilateral striatum 7 days after the insult. Motor function of the animals was serially evaluated, using a rotarod treadmill. Using fluorescence immunohistochemistry, we evaluated the distribution and phenotype of the transplanted cells 4 weeks after transplantation. Recovery of motor function in the WT BMSC-transplanted mice was more pronounced than in the CXCR4-KO-transplanted mice and the vehicle-treated ones. SDF-1 was extensively expressed in peri-infarct area. In the WT BMSC-transplanted mice, the transplanted cells were extensively distributed in the ipsilateral hemisphere, and many of them migrated towards the peri-infarct area and expressed the proteins specific for neurons and astrocytes, although these findings were not observed in the CXCR4-KO-transplanted mice. The results suggest that the SDF-1/CXCR4 system may play a critical role in the survival, proliferation and migration of the transplanted BMSC and contribute to recovery of neurological function.

Do bone marrow stromal cells proliferate after transplantation into mice cerebral infarct? : A double labeling study

The present study was aimed to clarify the proliferation capacity of the bone marrow stromal cells (BMSC) transplanted into the brain. The BMSC were harvested from green fluorescence protein (GFP)-transgenic mice, grown to the confluency and passed three times. They were labeled by co-culture with Ferucarbotran, a superparamagnetic iron oxide (SPIO) agent. The proportions of the SPIO-positive cells were evaluated from P3 to P7, using Turnbull blue staining. The GFP-BMSC labeled by Ferucarbotran were transplanted into the ipsilateral striatum of the mice brain subjected to permanent focal ischemia at 7 days after the insult. The distribution and differentiation of GFP- and SPIO-positive cells in the brain were studied 3 months after transplantation, using immunohistochemistry and Turnbull blue staining. As the results, the proportions of the SPIO-positive cells gradually decreased from 93.6% at P3 to 6.5% at P7. Fluorescence immunohistochemistry revealed that the GFP-positive cells were widely distributed around infarct and partially expressed MAP2 and NeuN 3 months after transplantation. However, only a smaller number of SPIO-positive cells could be detected on Turnbull blue staining. The ratio of the SPIO- to GFP-positive cells was approximately 2.7%. The results strongly suggest that the BMSC repeat proliferation many times, migrate into the lesion, and partially express the neuronal phenotype in the host brain during 3 months after transplantation. The double labeling technique would be valuable to prove the proliferation of the transplanted cells in the host tissue because GFP gene and SPIO nanoparticles have different inheritance characteristics.

Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: A novel material for CNS tissue engineering

Recent basic experiments have strongly suggested that cell transplantation therapy may promote functional recovery in patients with spinal cord injury (SCI). However, a safe and efficient transplantation technique still remains undetermined. This study, therefore, was aimed to clarify whether fibrin matrix could be a useful scaffold in bone marrow stromal cell (BMSC) transplantation for the injured spinal cord. To clarify the issue, three‐dimensional structure of fibrin matrix was assessed and the green fluorescent protein (GFP)‐expressing BMSC were cultured in fibrin matrix. The rats were subjected to spinal cord hemisection at T8 level, and the vehicle, BMSC or BMSC‐fibrin matrix construct was implanted into the cavity. Neurologic function was serially evaluated. Using immunohistochemistry, we evaluated the survival, migration and differentiation of the transplanted cells at 4 weeks after transplantation. In the initial in vitro study, the BMSC could survive in fibrin matrix for 2 weeks. The animals treated with the BMSC‐fibrin matrix construct showed significantly more pronounced recovery of neurologic function than vehicle‐ or BMSC‐treated animals. Fibrin scaffold markedly improved the survival and migration of the transplanted cells. There was no significant difference in the percentage of cells doubly positive for GFP and microtubule‐associated protein 2 between the animals treated with BMSC‐fibrin matrix construct and those treated with BMSC, but a certain subpopulation of GFP‐positive cells morphologically simulated the neurons in the animals treated with BMSC‐fibrin matrix construct. These findings strongly suggest that fibrin matrix may be one of the promising candidates for a potential, minimally invasive scaffold for injured spinal cord, and that such strategy of tissue engineering could be a hopeful option in regeneration therapy for patients with SCI.

The effects of neuronal induction on gene expression profile in bone marrow stromal cells (BMSC)—a preliminary study using microarray analysis

Bone marrow stromal cells (BMSC) have been anticipated as a donor for cell type for transplantation therapy in various neurological disorders. However, their neurogenic capacity still remains undetermined. In this study, we aimed to clarify whether in vitro chemical treatment promotes their neuronal differentiation on the level of gene expression. Mice BMSC were cultured with medium supplemented with DMSO, retinoic acid, and basic fibroblast growth factor, and their morphology and expression of neuronal markers were evaluated. Subsequently, using microarray and RT-PCR techniques, the treatment-induced changes in the gene expression profile were analyzed. After exposure to the medium, the BMSC simulated a neuron-like appearance and increased their immunoreactivity for nestin and Tuj-1. Microarray analysis revealed that the BMSC per se express the multilineage cellular genes, including those associated with the neuron. Chemical treatment significantly decreased the expression of genes related to mesenchymal cells and increased the expression of 5 neuron-associated genes. Microarray and RT-PCR analyses also demonstrated that the BMSC express the genes for several growth factors including NGF-beta and BDNF, indicating their therapeutic role in protecting the injured central nervous system. The present results suggest that at least a certain subpopulation of the BMSC have the potential to alter their gene expression profile in response to the surrounding environment and may possibly protect the host tissue by secreting neuroprotective factors.

Neuroprotective effect of a heat shock protein inducer, geranylgeranylacetone in permanent focal cerebral ischemia

Previous studies have strongly suggested that heat shock protein 70 (HSP70) has protective effects in ischemia/reperfusion in tissues such as brain, heart, and liver. This study was performed to assess the efficacy of the HSP70 inducer geranylgeranylacetone (GGA) in experiments involving permanent middle cerebral artery (MCA) occlusion. Male Balb/c mice were subjected to permanent MCA occlusion by direct occlusion through small craniectomy. Vehicle or GGA (200 or 1000 mg/kg) was injected intraperitoneally 1 h prior to the onset of ischemia. Infarct volumes were evaluated at 24 h of ischemia by using 2,3,5-triphenyltetrazolium chloride (TTC) staining. The effect of GGA on the induction of HSP70 was studied at 3 h after ischemia with fluorescence immunocytochemistry. The percentage of infarct volume in the control mice (n=10) was 23.0+/-4.0% (mean+/-SD) of the contralateral hemisphere, while those in the treated groups were 22.6+/-7.3% (200 mg/kg group; n=5, P>0.05) and 15.7+/-3.8% (1000 mg/kg group; n=5, P<0.05). Pretreatments with 1000 mg/kg of GGA enhanced the ischemia-related induction of HSP in the neurons and astrocytes in the boundary zone of infarct. The results demonstrate that GGA significantly reduces infarct volume due to permanent MCA occlusion when given 1 h prior to the induction of ischemia.

Intracerebral, but not intravenous, transplantation of bone marrow stromal cells enhances functional recovery in rat cerebral infarct: an optical imaging study.

Recent studies have indicated that bone marrow stromal cells (BMSC) may improve neurological function when transplanted into an animal model of CNS disorders, including cerebral infarct. However, there are few studies that evaluate the therapeutic benefits of intracerebral and intravenous BMSC transplantation for cerebral infarct. This study was aimed to clarify the favorable route of cell delivery for cerebral infarct in rats. The rats were subjected to permanent middle cerebral artery occlusion. The BMSC were labeled with near infrared (NIR)‐emitting quantum dots and were transplanted stereotactically (1 × 106 cells) or intravenously (3 × 106 cells) at 7 days after the insult. Using in vivo NIR fluorescence imaging technique, the behaviors of BMSC were serially visualized during 4 weeks after transplantation. Motor function was also assessed. Immunohistochemistry was performed to evaluate the fate of the engrafted BMSC. Intracerebral, but not intravenous, transplantation of BMSC significantly enhanced functional recovery. In vivo NIR fluorescence imaging could clearly visualize their migration toward the cerebral infarct during 4 weeks after transplantation in the intracerebral group, but not in the intravenous, group. The BMSC were widely distributed in the ischemic brain and some of them expressed neural cell markers in the intracerebral group, but not in the intravenous group. These findings strongly suggest that intravenous administration of BMSC has limited effectiveness at clinically relevant timing and intracerebral administration should be chosen for patients with ischemic stroke, although further studies would be warranted to establish the treatment protocol.

Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.

Objective. Recent reports have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into models of central nervous system (CNS) disorders, including traumatic spinal cord injury. In this study, the authors aimed to clarify the underlying mechanism through which BMSCs supported CNS regeneration in the spinal cord. Methods. The authors topically applied mouse BMSCs expressing green fluorescence protein (0.4-4 × 104 cells) on the organotypic spinal cord slice culture prepared from 6-day-old rat pups (n = 17). They were co-cultured for 3 weeks after the slice culture started, and the behavior of the applied BMSCs was serially observed using a fluorescence bioimaging technique. The authors completed a histological analysis at the end of the co-cultures and evaluated the profiles of the cultured BMSCs using microarray and immunocytochemistry techniques. Results. The fluorescence bioimaging showed that the BMSCs survived and made a cluster on the slice during the experiments. They also induced a morphological change in the slice within 48 hours of co-culture. Immunohistochemistry analysis showed that the BMSCs promoted a marked neurite extension toward their cluster and some of the BMSCs expressed Tuj-1, an early neuronal marker. Analysis by microarray and immunocytochemistry revealed that BMSCs highly expressed the matrix metalloproteinases (MMPs), stromal cell—derived factor-1, and its specific receptor CXCR4. Conclusions . These findings suggest that the donor BMSCs can support CNS regeneration due to their acquisition of a suitable environment for differentiation and promotion of neurite extension via MMPs and chemokines.