Taku Sugiyama

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.

Near-infrared fluorescence labeling allows noninvasive tracking of bone marrow stromal cells transplanted into rat infarct brain.

BACKGROUND:Noninvasive imaging techniques would be needed to validate the therapeutic benefits of cell transplantation therapy for central nervous system disorders. OBJECTIVE:To evaluate whether near-infrared (NIR)-emitting fluorescence tracer, quantum dots, would be useful to noninvasively visualize the bone marrow stromal cells (BMSC) transplanted into the infarct brain in living animals. METHODS:Rat BMSCs were labeled with QD800. In vitro and in vivo conditions to visualize NIR fluorescence were precisely optimized. The QD800-labeled BMSCs were stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Using the NIR fluorescence imaging technique, the behaviors of BMSCs were serially visualized during the 8 weeks after transplantation. RESULTS:NIR fluorescence imaging could noninvasively detect the NIR fluorescence emitted from the transplanted BMSCs engrafted in the peri-infarct neocortex through the scalp up to 8 weeks after transplantation. The intensity gradually increased and reached the peak at 4 weeks. The results were supported by the findings on ex vivo NIR fluorescence imaging and histological analysis. CONCLUSION:NIR fluorescence imaging is valuable in monitoring the behaviors of donor cells in the rodent brain. The results would allow new opportunities to develop noninvasive NIR fluorescence imaging as a modality to track the BMSCs transplanted into the brain.

Noninvasive transplantation of bone marrow stromal cells for ischemic stroke: preliminary study with a thermoreversible gelation polymer hydrogel.

OBJECTIVERecent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into animal models of cerebral infarct. However, it is still undetermined how the BMSCs should be transplanted to obtain the most efficient therapeutic benefits safely. The aim of this study was to assess whether a thermoreversible gelation polymer (TGP) hydrogel acts as a noninvasive, valuable scaffold in BMSC transplantation for infarct brain. METHODSThe mice were subjected to permanent middle cerebral artery occlusion. Vehicle, BMSC suspension, or the BMSC-TGP construct was transplanted onto the ipsilateral intact neocortex at 7 days after the insult. Neurological symptoms were assessed throughout the experiments. The fate of the transplanted BMSC was examined 8 weeks after transplantation with immunohistochemistry. RESULTSTGP hydrogel completely disappeared and provoked no inflammation in the host brain. Many transplanted cells were widely engrafted in the ipsilateral cerebrum, including the dorsal neocortex adjacent to the cerebral infarct in the BMSC-TGP construct—treated mice. Their number was significantly larger than in the BMSC-treated mice. The majority were positive for both NeuN and MAP2 and morphologically simulated the neurons. CONCLUSIONThe findings suggest that surgical transplantation of tissue-engineered BMSCs onto the intact neocortex enhances the engraftment of donor cells around the cerebral infarct. These data may be useful in developing a noninvasive but efficient paradigm in neural tissue engineering. TGP hydrogel can be a promising candidate for valuable scaffolds in BMSC transplantation for central nervous system disorders because of its unique biochemical properties.

Therapeutic effects of intra-arterial delivery of bone marrow stromal cells in traumatic brain injury of rats--in vivo cell tracking study by near-infrared fluorescence imaging.

BACKGROUND: A noninvasive and effective route of cell delivery should be established to yield maximal therapeutic effects for central nervous system (CNS) disorders. OBJECTIVE: To elucidate whether intra-arterial delivery of bone marrow stromal cells (BMSCs) significantly promotes functional recovery in traumatic brain injury (TBI) in rats. METHODS: Rat BMSCs were transplanted through the ipsilateral internal carotid artery 7 days after the onset of cortical freezing injury. The BMSCs were labeled with fluorescent dye, and in vivo optical imaging was employed to monitor the behaviors of cells for 4 weeks after transplantation. Motor function was assessed for 4 weeks, and the transplanted BMSCs were examined using immunohistochemistry. RESULTS: In vivo optical imaging and histologic analysis clearly demonstrated that the intra-arterially injected BMSCs were engrafted during the first pass without systemic circulation, and the transplanted BMSCs started to migrate from the cerebral capillary bed to the injured CNS tissue within 3 hours. Intra-arterial BMSC transplantation significantly promoted functional recovery after cortical freezing injury. A subgroup of BMSCs expressed the phenotypes of neurons, astrocytes, and endothelial cells around the injured neocortex 4 weeks after transplantation. CONCLUSION: Intra-arterial transplantation may be a valuable option for prompt, noninvasive delivery of BMSCs to the injured CNS tissue, enhancing functional recovery after TBI. In vivo optical imaging may provide important information on the intracerebral behaviors of donor cells by noninvasive, serial visualization.

Therapeutic impact of human bone marrow stromal cells expanded by animal serum-free medium for cerebral infarct in rats.

BACKGROUND:The donor cell culture in animal serum-free medium is important for the clinical application of cell transplantation therapy. Recently, human-derived platelet lysate (PL) gained interest as a substitute for fetal calf serum (FCS), but there are no studies that evaluate the validity of human bone marrow stromal cells (hBMSCs) expanded with PL-containing medium for central nervous system disorders. OBJECTIVE:To test the hypothesis that hBMSCs expanded with FCS-free, PL-containing medium can promote functional recovery after cerebral infarct. METHODS:hBMSCs were cultured in the FCS- or PL-containing medium. Cell-growth kinetics were analyzed. The vehicle or hBMSCs was stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Motor function was assessed for 8 weeks, and the fate of transplanted hBMSCs was examined using immunohistochemistry. RESULTS:There was no significant difference in hBMSC expansion between the 2 groups. Transplantation of hBMSCs expanded with the FCS- or PL-containing medium equally promoted functional recovery compared with the vehicle group. Histological analysis revealed that there were no significant differences in their migration, survival, and neural differentiation in the infarct brain between the 2 groups. CONCLUSION:hBMSCs expanded with PL-containing medium retained their capacity of migration, survival, and differentiation and significantly promoted functional recovery when stereotactically transplanted into the infarct brain. The PL may be a clinically valuable and safe substitute for FCS in expanding hBMSCs to regenerate the infarct brain.

Bone marrow stromal cells and bone marrow-derived mononuclear cells: Which are suitable as cell source of transplantation for mice infarct brain?

There are few studies that denote whether bone marrow stromal cells (BMSC) and bone marrow‐derived mononuclear cells (MNC) show the same therapeutic effects, when directly transplanted into the infarct brain. This study therefore aimed to compare their biological properties and behaviors in the infarct brain. Mouse BMSC were harvested and cultured. Mouse MNC were obtained through centrifugation techniques. Their cell markers were analyzed with FACS analysis. The MNC (106 cells; n = 10) or BMSC (2 × 105 cells; n = 10) were stereotactically transplanted into the ipsilateral striatum of the mice subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their survival, migration, and differentiation in the infarct brain were precisely analyzed using immunohistochemistry 4 weeks after transplantation. The MNC were positive for CD34, CD45, CD90, but were negative for Sca‐1. The BMSC were positive for CD90 and Sca‐1. The transplanted BMSC, but not MNC, extensively migrated into the peri‐infarct area. Approximately 20% of the transplanted BMSC expressed a neuronal marker, NeuN in the infarct brain, although only 1.4% of the transplanted MNC expressed NeuN. These findings strongly suggest that there are large, biological differences between MNC and BMSC as cell sources of regenerative medicine for ischemic stroke.

Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats

This study was aimed to assess whether bone marrow stromal cells (BMSC) could ameliorate brain damage when transplanted into the brain of stroke‐prone spontaneously hypertensive rats (SHR‐SP). The BMSC or vehicle was stereotactically engrafted into the striatum of male SHR‐SP at 8 weeks of age. Daily loading with 0.5% NaCl‐containing water was started from 9 weeks. MRIs and histological analysis were performed at 11 and 12 weeks, respectively. Wistar‐Kyoto rats were employed as the control. As a result, T2‐weighted images demonstrated neither cerebral infarct nor intracerebral hemorrhage, but identified abnormal dilatation of the lateral ventricles in SHR‐SP. HE staining demonstrated selective neuronal injury in their neocortices. Double fluorescence immunohistochemistry revealed that they had a decreased density of the collagen IV‐positive microvessels and a decreased number of the microvessels with normal integrity between basement membrane and astrocyte end‐feet. BMSC transplantation significantly ameliorated the ventricular dilatation and the breakdown of neurovascular integrity. These findings strongly suggest that long‐lasting hypertension may primarily damage neurovascular integrity and neurons, leading to tissue atrophy and ventricular dilatation prior to the occurrence of cerebral stroke. The BMSC may ameliorate these damaging processes when directly transplanted into the brain, opening the possibility of prophylactic medicine to prevent microvascular and parenchymal‐damaging processes in hypertensive patients at higher risk for cerebral stroke.

Biological Features of Human Bone Marrow Stromal Cells (hBMSC) Cultured with Animal Protein-Free Medium—Safety and Efficacy of Clinical Use for Neurotransplantation

The donor cell culture in animal serum-free medium is quite important for the clinical application of cell transplantation therapy. This study was aimed to test the hypothesis that the human bone marrow stromal cells (hBMSC) expanded with fetal calf serum (FCS)-free, platelet lysate (PL)-containing medium retain their biological features favoring central nervous system regeneration. The hBMSC were cultured with 5% PL or 10% FCS. Their phenotypes were analyzed with flow cytometry, and their production of growth factors was quantified with enzyme-linked immunosorbent assay. Their capacity of neural differentiation was verified by immunocytochemistry. There was no significant difference in morphology and cell surface marker between the hBMSC–FCS and hBMSC–PL. Both of them were positive for CD44, CD90, CD105, and CD166 and were negative for CD34, CD45, and CD271. The production of human brain-derived neurotrophic factor, human hepatocyte growth factor, human β-nerve growth factor, and human platelet-derived growth factor-BB did not differ between the two groups, although the hBMSC–PL produced significantly more amount of TGF-β1 than the hBMSC–FCS. There was no significant difference in their in vitro differentiation into the neurons and astrocytes between the two groups. The hBMSC expanded with PL-containing medium retain their biological capacity of neural differentiation and neuroprotection. The PL may be a clinically valuable and safe substitute for FCS in expanding the hBMSC for cell therapy.

Bone Marrow Stromal Cell Transplantation Attenuates Cognitive Dysfunction due to Chronic Cerebral Ischemia in Rats

Aims: This study was aimed to elucidate if bone marrow stromal cells (BMSC) could ameliorate cognitive dysfunction due to chronic cerebral ischemia when transplanted into the brain. Methods: The BMSC were harvested from green fluorescence protein (GFP)-expressing mice. Wistar rats were subjected to bilateral common carotid artery (CCA) ligation. The BMSC (4 × 105 cells) or vehicle were stereotactically injected into the right striatum 24 h after the insult. Cognitive function was evaluated with the Morris water maze task after 3 and 5 weeks. Histological analysis was performed after 6 weeks. Results: Cognitive function was significantly impaired in the vehicle-transplanted animals, when compared with the non-CCA-ligation animals. BMSC transplantation significantly improved it. The BMSC were widely distributed in the ischemic brain, including the neocortex, white matter and hippocampus, and some of them expressed the phenotypes of neurons, astrocytes and endothelium. They also significantly ameliorated white matter damage. Conclusions: These findings strongly suggest that the BMSC may have the potential to attenuate white matter injury and improve cognitive dysfunction due to chronic cerebral ischemia. The present results would shed light on the potential of a novel strategy, cell therapy against ischemia-related cognitive dysfunction.

Occipital-posterior cerebral artery bypass via the occipital interhemispheric approach

Background: The unavailability of the superficial temporal artery (STA) and the location of lesions pose a more technically demanding challenge when compared with conventional STA-superior cerebellar or posterior cerebral artery (PCA) bypass in vascular reconstruction procedures. To describe a case series of patients with cerebrovascular lesions who were treated using an occipital artery (OA) to PCA bypass via the occipital interhemispheric approach. Methods: We retrospectively reviewed three consecutive cases of patients with cerebrovascular lesions who were treated using OA-PCA bypass. Results: OA-PCA bypass was performed via the occipital interhemispheric approach. This procedure included: (1) OA-PCA bypass (n = 1), and combined OA-posterior inferior cerebellar artery and OA-PCA saphenous vein interposition graft bypass (n = 1) in patients with vertebrobasilar ischemia; (2) OA-PCA radial artery interposition graft bypass in one patient with residual PCA aneurysm. Conclusions: OA-PCA bypass represents a useful alternative to conventional STA-SCA or PCA bypass.