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Transplantation of neural stem cells into epileptic brain
International Congress Series 1252 (2003) 489 – 492
Transplantation of neural stem cells into epileptic brain Yoshihito Matsumoto a,*, Atsushi Sindo a, Nobuyuki Kawai a, Katsuzo Kunishio a, Seigo Nagao a, Tetsuji Miyazaki b, Toshifumi Itano b, Yohei Okada c, Takuya Shimazaki c, Hideyuki Okano c a
Department of Neurological Surgery, Kagawa Medical University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan b Department of Biology, Kagawa Medical University, Japan c Department of Physiology II, Keio University, Japan
Abstract Embryonic stem cells (ES cells) differentiate into multiple cell lineages including neurons and glia. Subsequent to the isolation of neural stem cells, they can be expanded, genetically modified and extensively characterized prior to transplantation. These neural stem cells represent an ideal cell type for transplantation purpose in the central nervous system due to their multipotency. In the present study, we optimized the effective method to induce the differentiation of GABAergic neurons from neurospheres that had been derived from ES cells. Moreover, the effect of the transplantation of these cells was investigated on morphological and functional outcome in grafted mice kindled by electrical stimulation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Kindling; Epilepsy; Transplantation; ES cell; GABA
1. Background Up to 30% of the population of persons with epilepsy continue to have seizures despite treatment with antiepileptic drugs. Approximately 60% of patients with intractable epilepsy suffer from partial seizures, particularly complex-partial seizures of temporal lobe origin, the most common and difficult-to-treat type of epileptic seizures in patients. In these patients, * Corresponding author. Tel.: +81-87-891-2207; fax: +81-87-891-2208. E-mail address: [email protected] (Y. Matsumoto). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00004-9
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Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
brain surgery with resection of epileptogenic tissue is currently the most important alternative treatment. However, surgery has risks and costs that have to be considered. In contrast to resective surgery, the use of intracerebral grafting as a potential treatment in drugresistant epilepsy is still in its infancy. Several groups have attempted to develop a new treatment for epilepsy. One of the trials supports the hypothesis that embryonic neural tissue implanted into previously kindled animals alters subsequent seizure susceptibility, and opens the possibility that the neural grafts may be used for the therapy of medically intractable epilepsies [1,2]. Embryonic stem cells (ES cells) were first isolated from cultures of mouse blastocytes approximately two decades ago. ES cells give rise to aggregates of cells that are termed embryoid bodies. Embryoid bodies can change to be a variety of cell types that are descendants of the three cardinal germ layers of the embryo. Selective expansion of neural progenitor cells by culture embryoid bodies in a special media containing insulin, transferrin, selenium, FGF, and so on allows further elaboration of neurosphere that can be specified to become to neurons by the application of FGF [3]. Because ES cells offer distinct advantages as experimental and therapeutic reagents, ES cells may constitute a unique therapeutic resource in the setting of specific classes of neurological disorders associated with dysfunction of multiple areas of the neuraxis and pathologic involvement of complex regional neural cell subpopulations, such as intractable epilepsies.
2. Methods A total of 10 mice was used in this experiment. Stereotaxic implantations of electrodes and guide canula were carried out. Bipolar electrodes were implanted in the left side of amygdala. A guide canula was implanted in the left side dorsal hippocampus area [2]. Four days after surgery, the mice were stimulated twice a day with a biphasic square wave pulse using two electric stimulators. In the kindled mice, the response to succeeding stimulation trials consisted of progressively more clinical seizures as described by Racine. Following stage 5 kindling, transplantation of neural stem cells derived from ES cells was performed in the ipsilateral dorsal hippocampus from the guide canula. Four weeks after transplantation, severity of seizures was classified according to the score of Racine again. After finishing the kindling experiment, all mice were perfusion-fixed for immunohistological analysis. Fig. 1 shows the classification of severity of seizures according to the score of Racine. Stage 5 is the most severe seizure in this classification.
Fig. 1. Classification of severity of seizures by Racine.
Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
491
Four weeks post-transplantation, the mice underwent behavioral testing using the classification scale of Racine. After behavioral test, all mice were perfusion-fixed for morphological studies. Colocalization of GFP with neuronal (Hu), GABAergic (GAD), presynaptic (synaptophysin), and glial markers (GFAP) was conducted by confocal microscope to enable exact definition of each of these antibodies. Transplants were stained for GFP because GFP gene was inserted in the ES cells so as to distinguish transplants from host cells.
3. Results Four mice were classified as stage 5 before transplantation therapy (Fig. 1). They showed loss of balance accompanied by all limbs’ clonus and rearing with clonus. The mice are classified as stage 3 recovering from stage 5 at 2 weeks after transplantation and
Fig. 2. Panels A, D, and G: immunohitological images of neurosphere with antibodies (Hu, GAD, and GFAP, respectively). Panels B and C: confocal images of double-labeled cells with GFP and Hu that transplanted neurosphere in the hippocampus at 1 month after transplantation. Panels E and F: confocal images of doublelabeled cells with GFP and GAD that transplanted neurosphere. Panels H and I: confocal images of doublelabeled cells with GFP and GFAP.
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Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
as stage 1 at 4 weeks after plantation. All mice showed drastic positive effect of the transplantation. We can see clonus in only forelimb. Duration of post-ictal state is shorter than the previous mice. Panels A, D, and G of Fig. 2 respectively depict immunohistological images of neurosphere with antibodies (Hu, GAD, and GFAP). The neurosphere was stained with Hu and GAD, indicating that these cells differentiated neurons, especially GABAergic neurons. Nevertheless, the neurosphere was rarely stained with GFAP, showing that these cells differentiated little astrocytes. Immunohistological analysis was done using these antibodies. Colocalization of GFP with neuronal, synaptic, and glial markers was conducted with confocal microscope to enable exact definition of each of these antibodies. Transplants were stained for GFP because GFP gene was inserted in our ES cells so we can distinguish transplants from host cells. Panels B and C of Fig. 2 are confocal images of double-labeled cells with GFP and Hu that transplanted in the hippocampus at 1 month after transplantation. Transplanted neurospheres stained with GFP and Hu were located in hippocampus, indicating transplants differentiating to neuron. Within hippocampus, GFP-positive cells stained positively for GAD, indicating transplants differentiating to GABAergic neurons (panels E and F of Fig. 2). GFP-positive cells were negatively stained for GFAP, showing transplanted cells rarely differentiating into astrocyte.
4. Conclusions In grafted mice fully kindled by electrical stimulation, there was substantial hippocampal GABAergic reinnervation and seizure-suppressing effects, tested at 1 month postgrafting. While these grafts produced little GFAP-positive glial-like features. The neural stem cells derived from ES cells are useful for standardization of a donor cell population for cell transplantation therapy of clinically intractable epilepsies.
References [1] W. Loscher, U. Ebert, H. Lehmann, C. Rosenthal, G. Nikkhah, Seizure suppression in kindling epilepsy by grafts of fetal GABAergic neurons in rat substantia nigra, J. Neurosci. Res. 51 (1998) 196 – 209. [2] O. Miyamoto, T. Itano, H. Matsui, O. Hatase, Effect of embryonic hippocampal transplantation in amygdaloid kindled rat, Brain Res. 603 (1993) 143 – 147. [3] S. Gokhan, M.F. Mehler, Basic and clinical neuroscience applications of embryonic stem cells, Anat. Rec. 265 (2001) 142 – 156.
Transplantation of neural stem cells into epileptic brain Yoshihito Matsumoto a,*, Atsushi Sindo a, Nobuyuki Kawai a, Katsuzo Kunishio a, Seigo Nagao a, Tetsuji Miyazaki b, Toshifumi Itano b, Yohei Okada c, Takuya Shimazaki c, Hideyuki Okano c a
Department of Neurological Surgery, Kagawa Medical University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan b Department of Biology, Kagawa Medical University, Japan c Department of Physiology II, Keio University, Japan
Abstract Embryonic stem cells (ES cells) differentiate into multiple cell lineages including neurons and glia. Subsequent to the isolation of neural stem cells, they can be expanded, genetically modified and extensively characterized prior to transplantation. These neural stem cells represent an ideal cell type for transplantation purpose in the central nervous system due to their multipotency. In the present study, we optimized the effective method to induce the differentiation of GABAergic neurons from neurospheres that had been derived from ES cells. Moreover, the effect of the transplantation of these cells was investigated on morphological and functional outcome in grafted mice kindled by electrical stimulation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Kindling; Epilepsy; Transplantation; ES cell; GABA
1. Background Up to 30% of the population of persons with epilepsy continue to have seizures despite treatment with antiepileptic drugs. Approximately 60% of patients with intractable epilepsy suffer from partial seizures, particularly complex-partial seizures of temporal lobe origin, the most common and difficult-to-treat type of epileptic seizures in patients. In these patients, * Corresponding author. Tel.: +81-87-891-2207; fax: +81-87-891-2208. E-mail address: [email protected] (Y. Matsumoto). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00004-9
490
Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
brain surgery with resection of epileptogenic tissue is currently the most important alternative treatment. However, surgery has risks and costs that have to be considered. In contrast to resective surgery, the use of intracerebral grafting as a potential treatment in drugresistant epilepsy is still in its infancy. Several groups have attempted to develop a new treatment for epilepsy. One of the trials supports the hypothesis that embryonic neural tissue implanted into previously kindled animals alters subsequent seizure susceptibility, and opens the possibility that the neural grafts may be used for the therapy of medically intractable epilepsies [1,2]. Embryonic stem cells (ES cells) were first isolated from cultures of mouse blastocytes approximately two decades ago. ES cells give rise to aggregates of cells that are termed embryoid bodies. Embryoid bodies can change to be a variety of cell types that are descendants of the three cardinal germ layers of the embryo. Selective expansion of neural progenitor cells by culture embryoid bodies in a special media containing insulin, transferrin, selenium, FGF, and so on allows further elaboration of neurosphere that can be specified to become to neurons by the application of FGF [3]. Because ES cells offer distinct advantages as experimental and therapeutic reagents, ES cells may constitute a unique therapeutic resource in the setting of specific classes of neurological disorders associated with dysfunction of multiple areas of the neuraxis and pathologic involvement of complex regional neural cell subpopulations, such as intractable epilepsies.
2. Methods A total of 10 mice was used in this experiment. Stereotaxic implantations of electrodes and guide canula were carried out. Bipolar electrodes were implanted in the left side of amygdala. A guide canula was implanted in the left side dorsal hippocampus area [2]. Four days after surgery, the mice were stimulated twice a day with a biphasic square wave pulse using two electric stimulators. In the kindled mice, the response to succeeding stimulation trials consisted of progressively more clinical seizures as described by Racine. Following stage 5 kindling, transplantation of neural stem cells derived from ES cells was performed in the ipsilateral dorsal hippocampus from the guide canula. Four weeks after transplantation, severity of seizures was classified according to the score of Racine again. After finishing the kindling experiment, all mice were perfusion-fixed for immunohistological analysis. Fig. 1 shows the classification of severity of seizures according to the score of Racine. Stage 5 is the most severe seizure in this classification.
Fig. 1. Classification of severity of seizures by Racine.
Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
491
Four weeks post-transplantation, the mice underwent behavioral testing using the classification scale of Racine. After behavioral test, all mice were perfusion-fixed for morphological studies. Colocalization of GFP with neuronal (Hu), GABAergic (GAD), presynaptic (synaptophysin), and glial markers (GFAP) was conducted by confocal microscope to enable exact definition of each of these antibodies. Transplants were stained for GFP because GFP gene was inserted in the ES cells so as to distinguish transplants from host cells.
3. Results Four mice were classified as stage 5 before transplantation therapy (Fig. 1). They showed loss of balance accompanied by all limbs’ clonus and rearing with clonus. The mice are classified as stage 3 recovering from stage 5 at 2 weeks after transplantation and
Fig. 2. Panels A, D, and G: immunohitological images of neurosphere with antibodies (Hu, GAD, and GFAP, respectively). Panels B and C: confocal images of double-labeled cells with GFP and Hu that transplanted neurosphere in the hippocampus at 1 month after transplantation. Panels E and F: confocal images of doublelabeled cells with GFP and GAD that transplanted neurosphere. Panels H and I: confocal images of doublelabeled cells with GFP and GFAP.
492
Y. Matsumoto et al. / International Congress Series 1252 (2003) 489–492
as stage 1 at 4 weeks after plantation. All mice showed drastic positive effect of the transplantation. We can see clonus in only forelimb. Duration of post-ictal state is shorter than the previous mice. Panels A, D, and G of Fig. 2 respectively depict immunohistological images of neurosphere with antibodies (Hu, GAD, and GFAP). The neurosphere was stained with Hu and GAD, indicating that these cells differentiated neurons, especially GABAergic neurons. Nevertheless, the neurosphere was rarely stained with GFAP, showing that these cells differentiated little astrocytes. Immunohistological analysis was done using these antibodies. Colocalization of GFP with neuronal, synaptic, and glial markers was conducted with confocal microscope to enable exact definition of each of these antibodies. Transplants were stained for GFP because GFP gene was inserted in our ES cells so we can distinguish transplants from host cells. Panels B and C of Fig. 2 are confocal images of double-labeled cells with GFP and Hu that transplanted in the hippocampus at 1 month after transplantation. Transplanted neurospheres stained with GFP and Hu were located in hippocampus, indicating transplants differentiating to neuron. Within hippocampus, GFP-positive cells stained positively for GAD, indicating transplants differentiating to GABAergic neurons (panels E and F of Fig. 2). GFP-positive cells were negatively stained for GFAP, showing transplanted cells rarely differentiating into astrocyte.
4. Conclusions In grafted mice fully kindled by electrical stimulation, there was substantial hippocampal GABAergic reinnervation and seizure-suppressing effects, tested at 1 month postgrafting. While these grafts produced little GFAP-positive glial-like features. The neural stem cells derived from ES cells are useful for standardization of a donor cell population for cell transplantation therapy of clinically intractable epilepsies.
References [1] W. Loscher, U. Ebert, H. Lehmann, C. Rosenthal, G. Nikkhah, Seizure suppression in kindling epilepsy by grafts of fetal GABAergic neurons in rat substantia nigra, J. Neurosci. Res. 51 (1998) 196 – 209. [2] O. Miyamoto, T. Itano, H. Matsui, O. Hatase, Effect of embryonic hippocampal transplantation in amygdaloid kindled rat, Brain Res. 603 (1993) 143 – 147. [3] S. Gokhan, M.F. Mehler, Basic and clinical neuroscience applications of embryonic stem cells, Anat. Rec. 265 (2001) 142 – 156.