[Pathophysiology of Sandhoff Disease and Novel Thrapeutic Targets].

Sandhoff disease (SD) is a glycosphingolipid storage disease resulting from a genetic mutation in HEXB and associated deficiency in ß-hexosaminidase activity. This defect causes abnormal accumulation of ganglioside GM2 and related glycolipids in lysosomes, resulting in progressive deterioration of the central nervous system. Hexb-knockout (Hexb-/-) mice, an established animal model, show abnormalities similar to the severe phenotype seen in human infants. We used iPS cells derived from this mouse model (SD-iPSCs) to examine abnormal neuronal lineage differentiation and development in vitro during the asymptomatic phase of SD. Differentiation ability along the time axis appears to be altered in SD-iPSCs in which the differentiation ability of neural stem cells is promoted and differentiation into neurons is completed earlier, while the timing of differentiation into astrocytes is accelerated. This abnormal differentiation was suppressed by introducing the Hexb gene. These results indicate that the abnormal differentiation of SD-iPSCs into the nervous system reflects the pathogenesis of SD. Analysis using Hexb-/- mice revealed that activated microglia causes astrogliosis at the early stage of development that can be ameliorated via immunosuppression. Furthermore, reactive astrocytes in the cortex of Hexb-/- mice express adenosine A2A receptors in the late inflammatory phase. Inhibition of this receptor resulted in a decrease in activated microglial cells and inflammatory cytokines/chemokines. These results suggest that the astrocyte A2A receptor is important as a sensor that regulates microglial activation in the late inflammatory phase. Thus, our results provide new insights into the complex pathogenesis of SD.

[Glial cells and pharmacological targets in Sandhoff disease].

Sandhoff disease (SD) is a genetic disorder caused by a mutation in the ß-hexosaminidase B (HexB) gene in humans. This results in the massive accumulation of GM2 gangliosides in the nervous system, causing progressive neurodegeneration. The symptoms of SD include muscle weakness, seizures, and mental illness;along with loss of muscle coordination, vision, and hearing. In the most severe form, the onset begins during early infancy, and death usually occurs within 3-5 years of age. The established animal model, Hexb-deficient (Hexb-/-) mouse, shows abnormalities that resemble the severe phenotype found in human infants. We have previously reported that activated microglia causes astrogliosis in Hexb-/- mouse at the early stage of development that can be ameliorated via immunosuppression. Moreover, within the cerebral cortices of Hexb-/- mouse, reactive astrocytes were found to express adenosine A2A receptors in later inflammatory phases. Inhibiting this receptor with istradefylline decreases the number of activated microglial cells and inflammatory cytokines/chemokines. Thus, we underline the importance of the astrocytic A2A receptor as a sensor, in regulating microglial activation in the late phase of inflammation.

Abnormal organization during neurodevelopment in a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a genetic disorder caused by a mutation of HEXB, which is the ß-subunit gene of ß-hexosaminidase A and B (HexA and HexB) in humans. HEXB mutation reduces HexA and HexB enzymatic activities, and results in the massive accumulation of ganglioside GM2 in the nervous system. Severe phenotypes of SD show progressive neurodegeneration in human infants, and lysosomal dysfunction that may affect the early development of the nervous system. In a previous study, neural stem cells (NSCs) and induced pluripotent stem cells derived from SD model mice, which are Hexb-deficient (Hexb-/-), demonstrated impaired neuronal differentiation. This study investigated early neurodevelopment in vivo using Hexb-/- mice. The structure of adult cerebral cortices of Hexb-/- mice was normal. However, the expression of Sox2, an NSC-related gene, was reduced in the embryonic cerebral cortices of Hexb-/- mice. Moreover, a reduction of early neuronal migration and differentiation was observed in the embryonic cerebral cortices of Hexb-/- mice. In addition, we showed that the production of layer-specific neurons was delayed in somatosensory cerebral cortices of Hexb-/- mice. These findings suggest that the alterations observed in embryonic Hexb-/- mice may contribute to deficits in neurodevelopment of SD.

Donepezil-induced oligodendrocyte differentiation is mediated through estrogen receptors.

Loss of oligodendrocytes, the myelin-forming cells of the central nervous system, and subsequent failure of myelin development result in serious neurological disorders such as multiple sclerosis. Using primary mouse embryonic neural stem cells (NSCs), we previously demonstrated that donepezil, an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates the differentiation of NSCs into oligodendrocytes and neurons, albeit at the expense of astrogenesis. However, the precise mechanisms underlying donepezil-induced differentiation remain unclear. In this study, we aimed at elucidating the molecular pathways contributing to donepezil-induced differentiation of mouse-induced pluripotent stem cell-derived neural stem cells (miPSC-NSCs). We used cell-based reporter gene arrays to investigate effects of donepezil on differentiation of miPSC-NSCs. Subsequently, we assessed the molecular pathway underlying donepezil action on differentiation of miPSC-NSCs into mature oligodendrocytes. Donepezil increased the transcriptional activity of estrogen response element under differentiating conditions. Moreover, estrogen receptors α (ERα) and ß (ERß) were highly expressed in MBP-positive mature oligodendrocytes. The ER antagonist ICI 182,780 abrogated the number of MBP-positive oligodendrocytes induced by donepezil, but showed no effect on the differentiation of miPSC-NSCs into Tuj1-positive neurons and GFAP-positive astrocytes. Furthermore, the donepezil-induced generation of mature oligodendrocytes from miPSC-NSC was significantly attenuated by antagonists and siRNA targeting ERα and ERß. In conclusion, we demonstrated, for the first time, that donepezil-induced oligodendrogenesis is mediated through both ER subtypes, ERα and ERß. Cover Image for this issue: https://doi.org/10.1111/jnc.14771.

Inhibition of astrocytic adenosine receptor A2A attenuates microglial activation in a mouse model of Sandhoff disease.

Astrocyte-microglia communication influences the onset and progression of central nervous system (CNS) disorders. In this study, we determined how chronic inflammation by activated astrocytes affected and regulated CNS functions in Sandhoff disease (SD), a CNS lysosomal storage disorder. SD triggers intense CNS inflammation such as microglial activation and astrogliosis. It is caused by mutation of the HEXB gene, which reduces ß-hexosaminidase (Hex) enzymatic activity in lysosomes, leading to accumulation of the substrate GM2 ganglioside in neuronal cells. Hexb-/- mice display a phenotype similar to human patients that suffer from chronic inflammation characterized by activation of astrocytes and microglia. In Hexb-/- mice, tremors and loss of muscle coordination begins at ~12 weeks. Interestingly, we found that reactive astrocytes expressed adenosine A2A receptor in the cerebral cortices of Hexb-/- mice at the later inflammatory phase. In cultured astrocytes, expression of A2A receptor could be induced by astrocyte defined medium, and then the activation of the A2A receptor induced ccl2 expression. In Hexb-/- mice, inhibition of the A2A receptor antagonized by istradefylline decreased the number of activated microglial cells and inflammatory cytokines/chemokines at 13 weeks. Thus, the astrocytic A2A receptor is an important sensor that regulates microglial activation in the late phase of inflammation.

Improvement in dysmyelination by the inhibition of microglial activation in a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a genetic disorder caused by a mutation of the ß-subunit gene ß-hexosaminidase B (HexB) in humans, which results in the massive accumulation of the ganglioside GM2 and related glycosphingolipids in the nervous system. SD causes progressive neurodegeneration and changes in white matter in human infants. An animal model of SD has been established, Hexb-deficient (Hexb) mice, which shows abnormalities resembling the severe phenotype found in human infants. Previously, we reported that the activation state of microglia caused astrogliosis in the early stage of Hexb mouse development. To study how the symptoms of SD develop, we explored the difference in gene expression between 4-week-old Hexb and Hexb mouse cerebral cortices by microarray analysis. The data indicated not only the upregulation of immune system-related genes but also the downregulation of myelin-related genes in the 4-week-old Hexb mouse cerebral cortices. To test the correlation between inflammation and dysmyelination, we generated double-knockout mice of Hexb and the Fc receptor γ gene (Fcrγ), which is a regulator of autoimmune responses. Dysmyelination recovered in these double-knockout mice. The number of oligodendrocyte progenitors, which expressed platelet-derived growth factor receptor-α, did not change in the 2-week-old mouse brain. These results indicate that microglial activation plays an important role in the myelination process, without influencing the number of oligodendrocyte progenitors, in the development of Hexb mice.

Abnormal differentiation of Sandhoff disease model mouse-derived multipotent stem cells toward a neural lineage.

In Sandhoff disease (SD), the activity of the lysosomal hydrolytic enzyme, ß-hexosaminidase (Hex), is lost due to a Hexb gene defect, which results in the abnormal accumulation of the substrate, GM2 ganglioside (GM2), in neuronal cells, causing neuronal loss, microglial activation, and astrogliosis. We established induced pluripotent stem cells from the cells of SD mice (SD-iPSCs). In the present study, we investigated the occurrence of abnormal differentiation and development of a neural lineage in the asymptomatic phase of SD in vitro using SD mouse fetus-derived neural stem cells (NSCs) and SD-iPSCs. It was assumed that the number of SD mouse fetal brain-derived NSCs was reduced and differentiation was promoted, resulting in the inhibition of differentiation into neurons and enhancement of differentiation into astrocytes. The number of SD-iPSC-derived NSCs was also reduced, suggesting that the differentiation of NSCs was promoted, resulting in the inhibition of differentiation into neurons and enhancement of that into astrocytes. This abnormal differentiation of SD-iPSCs toward a neural lineage was reduced by the glucosylceramide synthase inhibitor, miglustat. Furthermore, abnormal differentiation toward a neural lineage was reduced in SD-iPSCs with Hexb gene transfection. Therefore, differentiation ability along the time axis appears to be altered in SD mice in which the differentiation ability of NSCs is promoted and differentiation into neurons is completed earlier, while the timing of differentiation into astrocytes is accelerated. These results clarified that the abnormal differentiation of SD-iPSCs toward a neural lineage in vitro was shown to reflect the pathology of SD.

FcRγ-dependent immune activation initiates astrogliosis during the asymptomatic phase of Sandhoff disease model mice.

Sandhoff disease (SD) is caused by the loss of ß-hexosaminidase (Hex) enzymatic activity in lysosomes resulting from Hexb mutations. In SD patients, the Hex substrate GM2 ganglioside accumulates abnormally in neuronal cells, resulting in neuronal loss, microglial activation, and astrogliosis. Hexb-/- mice, which manifest a phenotype similar to SD, serve as animal models for examining the pathophysiology of SD. Hexb-/- mice reach ~8 weeks without obvious neurological defects; however, trembling begins at 12 weeks and is accompanied by startle reactions and increased limb tone. These symptoms gradually become severe by 16-18 weeks. Immune reactions caused by autoantibodies have been recently associated with the pathology of SD. The inhibition of immune activation may represent a novel therapeutic target for SD. Herein, SD mice (Hexb-/-) were crossed to mice lacking an activating immune receptor (FcRγ-/-) to elucidate the potential relationship between immune responses activated through SD autoantibodies and astrogliosis. Microglial activation and astrogliosis were observed in cortices of Hexb-/- mice during the asymptomatic phase, and were inhibited in Hexb-/- FcRγ-/- mice. Moreover, early astrogliosis and impaired motor coordination in Hexb-/- mice could be ameliorated by immunosuppressants, such as FTY720. Our findings demonstrate the importance of early treatment and the therapeutic effectiveness of immunosuppression in SD.

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice.

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases.

Impaired neural differentiation of induced pluripotent stem cells generated from a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in ß-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.

Isometric contraction of microvascular pericytes from mouse brain parenchyma.

Pericytes were isolated and cultured from mouse cerebroparenchymal microvessels. A single pericyte clone was three-dimensionally cultured in a collagen gel by adding tensile stress, resulting in the reconstruction of narrow stringy fibers. When the contractility of these fibers was evaluated isometrically, they contracted in response to acetylcholine (ACh)1 or noradrenaline; this was accompanied by an increase in intracellular calcium concentration ([Ca(2+)]i). The fibers that were pre-contracted by ACh were completely relaxed by papaverine, which is a smooth-muscle relaxant. Moreover, the muscarinic ACh receptor-antagonist atropine depressed the [Ca(2+)]i response that was induced by ACh. This study demonstrates for the first time the quantitative measurement of the contractions produced by cultured microvascular pericytes from mouse brain parenchyma.

Angiogenic potential of CD44+ CD90+ multipotent CNS stem cells in vitro.

Although it is now clear that several subpopulations of neural stem cells (NSCs) exist during early development and adulthood, the angiogenic potential of NSCs remains a subject of debate. Here, we report that CD44(+) CD90(+) cells isolated from primary neurospheres can form vascular-tube structures in vitro. NSCs isolated from the mouse embryonic cortex formed neurospheres when cultured in serum-free medium containing 20ng/ml basic fibroblast growth factor (bFGF). CD44(+) CD90(+) cells were enriched from the neurospheres using an EPICS ALTRA flow cytometer, and antibodies against CD44 and CD90. The purified CD44(+) CD90(+) cells generated neurospheres, and differentiated into neurons and astrocytes. When the cells were inoculated into collagen gels and cultured with 20% fetal bovine serum plus bFGF for 7 days, vascular tube-like structures were formed. These results indicate that CD44(+) CD90(+) cells have the ability to generate neurospheres and to form vascular tubes.

Angiogenesis in vitro: vascular tube formation from the differentiation of neural stem cells.

Neural stem cells (NSCs) were isolated from the mouse cortex on embryonic day 12.5 and cultured by neurosphere formation in serum-free medium in the presence of basic fibroblast growth factor (bFGF). When NSCs were inoculated in collagen gels with 10% fetal bovine serum (FBS) and bFGF and incubated for 10 days, vessel-like tube structures consisting of PECAM-1- or VE-cadherin-immunoreactive cells were formed in the gels. Moreover, the formation of vascular tube-like structures with a massive investment of alpha-smooth muscle actin-immunoreactive or GFAP-immunoreactive cells was occasionally observed, indicating angiogenesis identical to cerebral vascular development in vivo. To examine whether NSCs are capable of producing endothelial cells, differentiation was induced by the addition of 10% FBS after bFGF withdrawal. Most of the cells displayed a cobblestone-like morphology. Immunological analyses and RT-PCR indicated that NSCs expressed endothelial cell-specific marker proteins such as PECAM-1, VE-cadherin, and Flk-1; and these expressions were maintained or up-regulated during differentiation. Similar tube structures were also observed when the differentiated cells were inoculated in collagen gels and incubated for 5 days. These results suggested that NSCs give rise to two types of vascular cells, endothelial cells and mural cells in vitro, which have the ability to form vascular tubes.

Carbachol-induced membrane ruffling and its desensitization in rat basophilic leukemia (RBL-2H3) cells transfected with m3 muscarinic acetylcholine receptors.

The changes in the reorganization of actin filaments during desensitization of secretion were investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptors (RBL-m3 cells). Incubation of RBL-m3 cells with 10-100 microM carbachol in Ca2+ -free medium developed membrane ruffling. When the cells were desensitized under the condition where desensitization of carbachol-induced secretion occurred, desensitized cells failed to develop membrane ruffling with the subsequent addition of carbachol. These results suggest that m3 muscarinic receptor-mediated desensitization of secretion involves negative regulation of actin reorganization leading to membrane ruffling.

Carbachol-induced secretion and homologous desensitization in rat basophilic leukemia (RBL-2H3) cells transfected with human m2 muscarinic acetylcholine receptors.

Carbachol (CCh) caused a dose-dependent release of beta-hexosaminidase and an increase in the production of inositol 1,4,5-trisphosphate (IP3) in RBL-2H3 cells transfected with m2 mAChR cDNA (RBL-m2 cells). The secretion was completely inhibited by LaCl3 and pertussis toxin. The secretion was dependent on extracellular Ca2+ and mediated through the pertussis toxin-sensitive G protein. Exposing RBL-m2 cells to 100 microM CCh for 30 min in Ca2+ -free medium (desensitizing treatment) inhibited the secretion induced by the subsequent addition of 10 microM CCh plus Ca2+, but not by stimulating the high affinity IgE receptor (FcepsilonRI). Desensitizing treatment of RBL-m2 cells reduced the affinity of the lipophilic ligand [3H]quinuclidinyl benzilate to m2 mAChR without a reduction of the total m2 mAChR number. The treatment also decreased the cell surface mAChR number to 14% with a slight reduction in its affinity. Desensitizing treatment of RBL-m2 cells inhibited the CCh-induced transient increase in levels of IP3 and intracellular Ca2+ concentration. The results suggested that the CCh-induced desensitization of m2 mAChR-mediated secretion is due to the receptor sequestration followed by blocking the increase in [Ca2+]i and that this desensitizing mechanism is receptor-subtype-specific.

[Effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, 3-hydroxy-3-methyl glutaryl (HMG) CoA reductase inhibitors, amlodipine and epalrestat on cultured basilar artery smooth muscle cell proliferation].

Proliferation of vascular smooth muscle cells (VSMC) stimulated by oxidative stresses and reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. Antiatherosclerotic effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, HMG CoA reductase inhibitors, calcium channel blocker and epalrestat were studied with an in vitro guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system over 3 days incubated with 0 to 10% of fetal bovine serum. Results demonstrated that simvastatin (0.1 mM), fluvastatin (0.3 mM), amlodipine (0.2 mM) and epalrestat (1 mM) elicited significant (p < 0.05 or 0.01) antiproliferative effects, whereas losartan (1 mM), valsartan (1 mM), enalapril (0.1 mM), captopril (1 mM), trandolapril (0.01 mM), pravastatin (0.7 mM) did not. In conclusion, the present in vitro VSMC culture system may serve as a comprehensive screening method for pleiotropic effects of commonly used therapeutic agents.

[Alterations of antiproliferative effects of serum obtained from patients with acute cerebral infarction treated with a radical scavenger, edaravone, with or without amlodipine using an in vitro cultured basilar artery smooth muscle cells].

The guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system in the Dulbecco's modified Eagle's medium for 3 days serves as a useful in vitro model for assessing antiproliferative effects of various therapeutic agents on vessels. With use of this system we studied whether human serum obtained from patients with acute cerebral infarction (n = 16) would have a proliferative effect on vessels and whether an administration of a free radical scavenger, edaravone, with or without amlodipine would elicit antiproliferative effects. The control serum was obtained from 3 healthy human subjects. Time courses of the cell growth and survival were measured colorimetrically by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrzolium bromide (MTT) test. The stimulatory effect on the proliferation of GBa-SM3 cells of patients' serum obtained immediately after infarction was significantly (p < 0.05) greater than those obtained from the same patients after the treatment of edaravone for 2 weeks. In addition, the serum obtained from the patients treated by edaravone and amlodipine (n = 7) showed a significantly (p < 0.05) greater antiproliferative effect than that obtained from those treated by edaravone (n = 9). In conclusion, edaravone may have a clinically beneficial antiproliferative effect on vascular smooth muscle cells. Co-administration of amlodipine, possessing an antioxidative calcium channel blocker, with edaravone may be a promising combination to patients with acute cerebral infarction. Further controlled clinical trials with a large number of patients should be warranted.

Edaravone, a radical scavenger, may enhance or produce antiproliferative effects of fluvastatin, amlodipine, ozagrel, GF109203X and Y27632 on cultured basilar artery smooth muscle cells.

Proliferation of vascular smooth muscle cells stimulated by reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. To clarify mechanisms by which ROS promote vascular atherogenesis, effects of fluvastatin, amlodipine, ozagrel (thromboxane synthetase inhibitor), GF109203X (a protein kinase C inhibitor) and Y27632 (a ROCK inhibitor) on the proliferation of guinea-pig basilar artery smooth muscle cells (GBa-SM3) in a 5% FBS culture medium were studied over 3 d in the presence or absence of a free radical scavenger, edaravone. Viability of cells at the end of incubation was measured by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. Results demonstrated that fluvastatin and amlodipine by themselves possess antiproliferative effects on the GBa-SM3 cells at 10-100 microM and 0.1-1 microM, respectively. While edaravone possessed no antiproliferative effect by itself at 100 microM, it significantly (p<0.05) augmented the antiproliferative effects of fluvastatin and amlodipine. In addition, ozagrel, GF109203X and Y27632 possessed no appreciable effects on the cell growth by themselves. However, coincubation of edaravone at 100 microM with these agents elicited significant antiproliferative effects for ozagrel, GF109203X and Y27632 at 10-100 microM, 1-10 microM and 0.1-1 microM, respectively. In conclusion, edaravone may have clinically beneficial interactions with fluvastatin, amlodipine and ozagrel regarding the prevention of vascular atherosclerosis. The interactions between edaravone and the inhibitors of protein kinase C and ROCK were suggestive of possible contributions of ROS-triggered intracellular signals associated with these enzymes to vascular atherogenesis, but further studies are required for confirmation.

Bromination by means of sodium monobromoisocyanurate (SMBI).

A variety of aromatic compounds with both activating and deactivating substituents were brominated with sodium monobromoisocyanurate (SMBI) 1, diethyl ether, diethyl ether-methanesulfonic acid, trifluoroacetic acid, or sulfuric acid were employed as solvents. Thus nitrobenzene was conveniently brominated in sulfuric acid, benzene was readily monobrominated in diethyl ether-methanesulfonic acid, and phenol was selectively brominated at the ortho position under mild conditions in refluxing diethyl ether. With substituents that are easily protonated, trifluoroacetic acid may be employed as solvent in the reaction with 1, in contrast NBS was ineffective in trifluoroacetic acid. This renders 1 a superior reagent relative to NBS. In addition to aromatics, alkenes, ketones and esters were also brominated with 1. Diethyl malonate was brominated with 1 and then subjected to a Bingel reaction with NaH to afford the desired methanofullerene in reasonable yield.

[Functional differentiation of neural stem cells into endothelial cells].

Neural stem cells (NSCs) were isolated from mouse embryonic day 12.5 (E12.5) cortex and cultured by neurosphere formation in serum-free medium in the presence of 20 ng/ml basic fibroblast growth factor (bFGF). To examine whether NSCs give rise to endothelial cells, differentiation was induced by the addition of 10% fetal bovine serum (FBS) after bFGF withdrawal. Most of the differentiated cells displayed a cobblestone-like morphology. Immunological analyses and RT-PCR indicated that NSCs expressed endothelial cell-specific marker proteins such as PECAM-1, VE-cadherin, and Flk-1, and these expressions were up-regulated during differentiation. When the differentiated cells were inoculated in collagen gels with 10% FBS and bFGF and incubated for 5 days, vessel-like tube structures consisting of PECAM-1- or VE-cadherin-immunoreactive cells were formed in the gels. These results suggested that NSCs give rise to endothelial cells in vitro, which have the ability to form vascular tubes.