Yuzo Takayama

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.

Induced Current Pharmacological Split Stimulation System for Neuronal Networks

Magnetic stimulation noninvasively modulates neuronal activity through a magnetically induced current. However, despite the usefulness and popularity of this method, the effects of neuronal activity in the nonstimulated regions on the stimulus responses are unknown. Here, we report that the induced current-evoked responses were affected by neuronal activities in the nonstimulated regions. Our experiment used a Mu-metal-based localized induced current stimulation (LICS) system combined with the microfabricated cell culture chamber system and a microelectrode array (MEA). The cell culture chamber system has radiating microtunnels connecting one central and eight outer chambers, which were fabricated using soft lithography and a replica modeling technique with SU-8 photoresist and polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in the chambers and formed functional synaptic connections through the microtunnels. By applying a biphasic alternating pulsed magnetic field to the Mu-metal located in the central chamber, induced currents were mainly generated near the cultured neurons and modified the neuronal activities, which were recorded through MEA. Furthermore, we confirmed that the evoked responses were modified by localized pharmacological stimulation (LPS) in the outer chambers. These results suggest that our system would be promising tool for analyzing the effect of magnetic stimulation on interacting neuronal activity.

Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest‐like precursors

In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small‐molecule cocktails in a step‐by‐step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time‐lapse imaging of neural precursor‐specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest‐like precursor stage. Consistent with these results, the neural crest‐like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.

Signal transfer within a cultured asymmetric cortical neuron circuit

OBJECTIVE Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. APPROACH The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. MAIN RESULTS When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. SIGNIFICANCE These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

A novel postoperative immobilization model for murine Achilles tendon sutures.

The body’s motion and function are all in part effected by a vital tissue, the tendon. Tendon injury often results in limited functioning after postoperative procedures and even for a long time after rehabilitation. Although numerous studies have reported surgical procedures using animal models which have contributed to both basic and clinical research, modeling of tendon sutures or postoperative immobilizations has not been performed on small experimental animals, such as mice. In this study we have developed an easy Achilles tendon suture and postoperative ankle fixation model in a mouse. Right Achilles tendons were incised and 10-0 nylons were passed through the proximal and distal ends using a modified Kessler method. Subsequently, the right ankle was immobilized in a plantarflexed position with novel splints, which were made from readily available extension tubes. Restriction of the tendon using handmade splints reduced swelling, as opposed to fixating with the usual plaster of Paris. Using this method, the usage of the right Achilles tendons began on postoperative days 13.5 ± 4.6, which indicated healing within two weeks. Therefore our simple short-term murine Achilles tendon suture procedure is useful for studying immediate tendon repair mechanisms in various models, including genetically-modified mice.

Micropatterning C2C12 myotubes for orderly recording of intracellular calcium transients

Reconstruction of skeletal muscle myotubes in vitro using myogenic cell lines have been widely carried out to study functional properties and disease-related biological changes of myotubes, such as intracellular calcium dynamics. However, the analysis of biological signals in isolated single myotubes or interactions among several myotubes is quite difficult problem because of the randomness in size, morphology and orientation of differentiated myotubes cultured on a conventional tissue culture dish. In the present study, we attempted to form uniform-size myotubes and detect intracellular calcium dynamics from the fabricated myotubes. We modified surfaces of culture dishes using a poly(-dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to form line patterns for myotube formation. We could form uniform-size and -orientation C2C12 myotubes and detect intracellular calcium dynamics from it. This simple method would be a useful for studying properties in myotubes with specific sizes and morphologies.

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography

Regeneration of the central nervous system (CNS), where proliferative potency is limited, is one of the most important research themes in neuroscience and neuroengineering. Pluripotent stem cell lines have attracted broad attentions as an important model system for regenerating damaged brain. The important key for realizing regenerative medicine using pluripotent stem cells is to induce undifferentiated cell into objective cells with high efficiency and reproducibility. Although the methods to induce embryonic stem (ES) cells into specific neuronal subtype with pharmacological treatment have been proposed, however, the handling is difficult and thus the efficiency rate of objective cells is low (Barberi et al., 2003). The main reason for low differentiation rate of pluripotent stem cells is difficulty in precise control of interaction between pharmacological treatment and cell signalling. To overcome these problems, development of alternative methods for precise control of cell differentiation processes is required. During cell differentiation, both endogenous factors, such as cell-cell signal transmission, and exogenous factors, such as pharmacological application, play important roles. Micro and soft lithography-based surface modification of culture substrate enabled to control stem-cell-aggregation (EB; Embryoid body) sizes and therefore to increase cell differentiation efficiency through promotion of cell-cell signal interactions (Karp et al., 2007; Wang et al., 2009). Particularly, Park et al. reported that small-size EBs of mouse ES cells (100 ~ 200 m of diameter) tended to differentiate into ectoderm and large-size EB (500 m of diameter) into mesoderm (Park et al., 2007). These reports suggested that manipulation of EB size and shape could affect endogenous factors of stem cell EBs and thus was an important approach for precise control of differentiation processes. Another important technique for regulating cell differentiation is applying physical stimulation. It was reported that electrical or magnetic stimulation could induce cellular and molecular responses and affected the gene expressions during differentiation (Kimura et al., 1998; Piacentini et al., 2008). Particularly, Yamada et al. reported that applying electrical stimulation induced mouse ES cells efficiently into ectoderm cells (Yamada et al., 2007). These reports suggested