Yutaka Suzuki

Multitracer assessment of dopamine function after transplantation of embryonic stem cell-derived neural stem cells in a primate model of Parkinson's disease

The ability of primate embryonic stem (ES) cells to differentiate into dopamine (DA)‐synthesizing neurons has raised hopes of creating novel cell therapies for Parkinsons disease (PD). As the primary purpose of cell transplantation in PD is restoration of dopaminergic neurotransmission in the striatum, in vivo assessment of DA function after grafting is necessary to achieve better therapeutic effects. A chronic model of PD was produced in two cynomolgus monkeys (M‐1 and M‐2) by systemic administration of neurotoxin. Neural stem cells (NSCs) derived from cynomolgus ES cells were implanted unilaterally in the putamen. To evaluate DA‐specific functions, we used multiple [11C]‐labeled positron emission tomography (PET) tracers, including [β‐11C]L‐3,4‐dihydroxyphenylalanine (L‐[β‐11C]DOPA, DA precursor ligand), [11C]‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane ([11C]β‐CFT, DA transporter ligand) and [11C]raclopride (D2 receptor ligand). At 12 weeks after grafting NSCs, PET demonstrated significantly increased uptake of L‐[β‐11C]DOPA (M‐1:41%, M‐2:61%) and [11C]β‐CFT (M‐1:31%, M‐2:36%) uptake in the grafted putamen. In addition, methamphetamine challenge in M‐2 induced reduced [11C]raclopride binding (16%) in the transplanted putamen, suggesting release of DA. These results show that transplantation of NSCs derived from cynomolgus monkey ES cells can restore DA function in the putamen of a primate model of PD. PET with multitracers is useful for functional studies in developing cell‐based therapies against PD. Synapse 63: 541‐548, 2009.

Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey (Macaca fascicularis) in vitro.

Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation.

Self-Contained Induction of Neurons from Human Embryonic Stem Cells

Background Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. Methodology/Principal Findings We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. Conclusion/Significance By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey (Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro

Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.

A conceptual design of a low resistance vaccum vessel for the steady state Tokamak reactor

Abstract A design study on the vacuum vessel of the Steady State Tokamak Reactor has been performed in order to provide a realistic structural concept for a fusion reactor. The vacuum vessel and shield are integrated to form a double-thin-wall structure filled with stainless steel and water resulting in a low one-turn electric resistance of ∼4 μω without insulating breaks or bellows. The reinforcement plates are weleded between the inner and outer skins of the double-thin-wall structure, and shileding units are installed in every chamber with electrical insulation from these skins and plates. As a result, the requirements for the vacuum vessel can be realized by this simple structure alone. Transient electromagnetic and structural analysis has been performed for the three-dimensiona; shell model in the plasma disruption condition of plasma current 12 MA and current decay time 20 ms. An eddy current, about 95% of plasma current, is induced on the vacuum vessel, and a maximum magnetic pressure ∼ 5.8 MPa is caused by coupling with the toroidal field. The maximum stress intensity for the magnetic prerssure is about 216 MPa. This low resistance vacuum vessel is extremely effective in shielding the change of the magnetic field in the superconducting toroidal and poloidal field coils during a plasma disruption. In summary, the feasibility and features of this new type of vacuum vessel concept have been shown in this study.

Engineering Design Study of Quasi-Axisymmetric Stellarator with Low Aspect Ratio

Abstract The engineering design of the quasi-axisymmetric stellarator CHS-qa is described, having a toroidal period number of 2, major radius of 1.5 m, and plasma aspect ratio of 3.2. Although the entire structure of the machine is highly nonaxisymmetric and deformative, the following major engineering concerns for the modular coils and the vacuum vessel have been resolved: (a) modular coil design (curvature and twist of conductors), (b) supporting structures for modular coils, (c) errors due to electromagnetic forces and misalignment in manufacturing processes (analysis shows that the magnetic surface is robust against such disturbances), (d) construction procedure for vacuum vessel and modular coils, and (e) ports for heating and diagnostics.