Aoi Odawara

Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture

Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and cell-based therapy. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. We here used a multi-electrode array (MEA) system to investigate the effects of the co-culture of rat astrocytes with hiPSC-derived neurons on the long-term culture, spontaneous firing activity, and drug responsiveness effects. The co-culture facilitated the long-term culture of hiPSC-derived neurons for >3 months and long-term spontaneous firing activity was also observed. After >3 months of culture, we observed synchronous burst firing activity due to synapse transmission within neuronal networks. Compared with rat neurons, hiPSC-derived neurons required longer time to mature functionally. Furthermore, addition of the synapse antagonists bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione induced significant changes in the firing rate. In conclusion, we used a MEA system to demonstrate that the co-culture of hiPSC-derived neurons with rat astrocytes is an effective method for studying the function of human neuronal cells, which could be used for drug screening.

A three-dimensional neuronal culture technique that controls the direction of neurite elongation and the position of soma to mimic the layered structure of the brain

Reconstruction techniques can mimic tissue structure using three-dimensional (3D) substrates or scaffolds to facilitate functional tissue engineering for transplantation or robust experimental models. Neuron-based tissue engineering is being developed to treat neuronal diseases and to understand neuronal function. However, few 3D neuronal tissue reconstruction techniques are available because structural control is hindered by the complexity and polarity of neurites. In this study, we developed a 3D reconstruction neuronal tissue technique using collagen fiber orientation and polydimethylsiloxane microchambers. This technique mimicked the layered structure of the brain (cerebral cortex) on a chip. We used this method to produce 3D neuronal networks by controlling (1) the position of somata and (2) the direction of neurite elongation in the 3D space. The somata area comprised a three-cell layer, and the cell density was equivalent to living tissue. Intracellular Ca2+ imaging and extracellular recordings using multielectrode arrays chip detected interlayer synchronous firings in a 3D reconstructed neuronal network. We confirmed that the interlayer propagation was chemical synaptic transmission by pharmacological experiments and that the velocity of propagation was equivalent to biological tissue. Furthermore, we demonstrated the reconstruction of 3D neuronal networks using neurons derived from human induced pluripotent stem cells. This 3D neuronal culture technique could be a useful tool for regenerative medicine and a drug screening model.

Thymoquinone protects cultured hippocampal and human induced pluripotent stem cells-derived neurons against α-synuclein-induced synapse damage.

The seeds of Nigella sativa are used worldwide to treat various diseases and ailments. Thymoquinone (TQ) that is present in the essential oil of these seeds mediates most of the plants diverse therapeutic effects. The present study aimed to determine whether TQ protects against α-synuclein (αSN)-induced synaptic toxicity in rat hippocampal and human induced pluripotent stem cell (hiPSC)-derived neurons. Here, we report that αSN decreased the level of synaptophysin, a protein used as an indicator of synaptic density, in cultured hippocampal and hiPSC-derived neurons. However, simultaneous treatment with αSN and TQ protected neurons against αSN-induced synapse damage, as revealed by immunostaining. Moreover, administration of TQ efficiently induced protection in these cells against αSN-induced inhibition of synaptic vesicle recycling in hippocampal and hiPSC-derived neurons as well as against mutated P123H β-synuclein (βSN) in hippocampal neurons, as revealed by experiments using the fluorescent dye FM1-43. Using a multielectrode array, we further demonstrated that the treatment of hiPSC-derived neurons with αSN induced a reduction in spontaneous firing activity, and cotreatment with αSN and TQ partially reversed this loss. These results suggest that TQ protects cultured rat primary hippocampal and hiPSC-derived neurons against αSN-induced synaptic toxicity and could be a promising therapeutic agent for patients with Parkinsons disease and dementia with Lewy bodies.

Pain responses of cultured human iPSC-derived sensory neurons using high-throughput MEA system

Introduction Dorsal root ganglion (DRG) sensory neurons are pain-related neurons and have a variety of sensory receptors that are activated by chemical, thermal, and mechanical stimuli. Establishment of pharmacological assay in pain research and drug screening is important issue. In addition, human induced pluripotent stem cell (hiPSC)-derived sensory neurons may be effectively used for drug discovery and toxicity testing. The purpose of this study was to evaluate the physiological responses against typical pain-related molecules, temperature change and anticancer drug in cultured sensory neurons using high-throughput multi-electrode array (MEA) system.

Bundle Gel Fibers with a Tunable Microenvironment for in Vitro Neuron Cell Guiding

As scaffolds for neuron cell guiding in vitro, gel fibers with a bundle structure, comprising multiple microfibrils, were fabricated using a microfluidic device system by casting a phase-separating polymer blend solution comprising hydroxypropyl cellulose (HPC) and sodium alginate (Na-Alg). The topology and stiffness of the obtained bundle gel fibers depended on their microstructure derived by the polymer blend ratio of HPC and Na-Alg. High concentrations of Na-Alg led to the formation of small microfibrils in a one-bundle gel fiber and stiff characteristics. These bundle gel fibers permitted for the elongation of the neuron cells along their axon orientation with the long axis of fibers. In addition, human-induced pluripotent-stem-cell-derived dopaminergic neuron progenitor cells were differentiated into neuronal cells on the bundle gels. The bundle gel fibers demonstrated an enormous potential as cell culture scaffold materials with an optimal microenvironment for guiding neuron cells.

Non-Invasive and Real-Time Measurement Techniques of Dopamine, APs and Fpsp Using Carbon Nanotube Multi-Electrode Array Chip

We have developed the planar carbon nanotube (CNT)-MEA chips that can measure both electrophysiological responses such as field postsynaptic potentials (fPSPs) and action potentials (APs) as well as the release of the neurotransmitter dopamine. These CNT-MEA chips were fabricated by electroplating the indium-tin oxide (ITO) microelectrode surfaces. Cyclic voltametric and chronoamperometric measurements using these CNT-MEA chips detected dopamine at nanomolar concentrations and successfully measured synaptic dopamine release from spontaneous firings in mouse striatal brain slices. Furthermore, APs and fPSP were measured from cultured hippocampal neurons and hippocampal slices with high temporal resolution and S/N. Planar ITO MEAs contained either 64 electrodes, 50 × 50 µm and 200 × 200 µm, the latter used for detection of dopamine. Multi-wall carbon nanotubes (MWCNTs) were electroplated onto the surface of the ITO electrode. Electrochemical measurements were performed using an ALS 1140A electrochemical analyzer with an Ag/AgCl reference electrode and Pt counter electrode. For in vitro measurements, dopamine was dissolved in phosphate buffered saline (PBS) at pH 7.4. Striatal and hippocampal slices were prepared from 4-weeks-old male mouse. For real-time measurement of synaptic dopamine release, the striatal region of coronal or saggital brain slices was placed on CNT-MEA chip. The chips were connected to a 30°C heating plate and the chamber was continuously perfused with oxygenated ACSF at a rate of 2 ml/min at 29±30°C. To detect released dopamine, the amperometric response was recorded at +0.3 V. For neuronal cultures, hippocampi from E18 rat was used. Electrophysiological recordings were performed using CNT-MEA chips connected to the MED planar MEA recording system. The dopamine sensitivity of CNT-MEAs was assessed by adding dopamine solutions of different concentrations every 100 s and recording the response current at a fixed potential. A linear relationship between current and dopamine concentration was obtained over the range 1 nM to 10 µM. The lower limit of detection was 1 nM (S/N = 3.8) and responses were specific for dopamine as no change in response was observed during sequential superfusion with PBS. We then tested the capacity of these CNT-MEA chips to measure physiological dopamine release by placing striatal slices. Both coronal and sagittal slices through the striatum exhibited spontaneous dopamine release as recorded by amperometric current±time responses at 0.30 V. The mean (±S.D) peak current was 14.7 ± 3.1 pA above the noise of 273 fA, indicating a S/N of 62. In contrast, no change in current output was observed at …