Characterization of Synchronized Burst in Developing Human iPS Cell-derived Neuronal Networks with a Microtunnel Device
-
1
The University of Tokyo, School of Engineering, Japan
-
2
The University of Tokyo, Research Center for Advanced Science and Technology,, Japan
Motivation
Human induced pluripotent stem (iPS) cell-derived neuron is a useful source for regenerative medicine and drug discovery. These cells are needed to maintain human specific phenotypes in a culture condition. However, little is known about whether phenotypes of human cells are maintained in culture because of a lack of suitable tools. Here, we aimed to evaluate developmental time-course of human iPS cell-derived neuron with a microelectrode array (MEA) which has microtunnel structures on electrodes.
Materials and Methods
Fifty microtunnels were developed on an MEA substrate in a parallel fashion by soft lithography. One electrode was set at the bottom of each microtunnel for recording action potential from axons. The microtunnel-electrode configuration enables a close contact between axons and the electrode, even if cell soma migrates during long-term culture. The human iPS cell line (201B7) [1] was provided by RIKEN Bio Resource Center through the Project for Realization of Regenerative Medicine and the National Bio-Resource Project of the MEXT, Japan. Human iPS cells were induced to differentiate into neuronal cells in dorsal cerebral cortex, including neurons and glial cells, as previously described [2]. These cells were seeded into the culture devices and conventional MEAs after coating with polyethyleneimine and laminin. Spontaneous activity was recorded every 10 days from 60 days after induction (DAI) with an MEA recording system [3]. Neural activities were detected with a threshold, and synchronized bursts were detected as previously described [4].
Results
Human iPS cell-derived neuronal cells survived on MEA substrates and extended their neurites. Their axons entered microtunnels from approximately 60 DAI. Morphology of neuronal cells were evaluated with an immunofluorescent staining at 330 DAI. As a result, a neuron in a cell aggregate extended multiple neurites, and astrocytes with multiple processes existed on an MEA, suggesting that human iPS cells differentiated into neuronal cells and the neuronal cells matured in the environment by 330 DAI. Then, spontaneous activities were recorded with the novel device and conventional MEAs. Neuronal networks composed of human neurons exhibited synchronized bursting from 70 DAI. With the novel device, neural activity can be recorded from a larger number of electrodes than with the conventional MEA. Moreover, while no activity was recorded with conventional MEAs from 310 DAI because of detachment of cells from substrates, the novel device kept the stable recording condition until 420 DAI. These results indicate that the microtunnel-enhanced recording is effective to evaluate developmental changes in neuronal network composed of human neurons which require a longer time for development than mouse ones. Finally, developmental changes in network activity were evaluated. Burst frequency increased, and duration and inter-burst interval decreased until 350 DAI, indicating that mature neuronal network showed shorter bursts more frequently than younger ones.
Discussion
Spontaneous activity of human neurons was recorded for more than one year. Although developmental changes in burst shapes are consistent with those in mouse neurons [5], time-course of the change in human neurons is much longer than that in mouse neurons. One possible reason for this discrepancy is species difference between human and mouse. Further study will be needed to elucidate what is a key factor for generating the difference. Taken together, results suggest that our model system is useful to study human neuronal network which has different properties from mouse networks.
Conclusion
In this study, we developed a method for long-term monitoring of human neurons and evaluated their development time-course. As a result, spontaneous activity of human neuronal network was successfully recorded for almost one year with our novel method. Moreover, it was revealed that human neuronal network showed different developmental time-course compared to mouse one. Therefore, the combination of human iPS cell-derived neuron and our novel device is suggested to be a powerful tool for studying development and properties of human neuronal network.
Acknowledgement
This work was partially supported by the Japan Society for the Promotion of Science (JSPS) through a Grant-in-Aid for JSPS Fellows (254923) and Grants-in-Aid for Scientific Research (26560202).
References
[1] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda and S. Yamanaka, Cell, 131(5), 861-872, 2007.
[2] Y. Shi, P. Kirwan and F. J. Livesey, Nat. Protoc., 7(10), 1836-1846, 2012.
[3] Y. Jimbo, N. Kasai, K. Torimitsu, T. Tateno and H. P. Robinson, IEEE Trans. Biomed. Eng., 50(2), 241-248, 2003.
[4] J. van Pelt, P. S. Wolters, M. A. Corner, W. L. Rutten and G. J. Ramakers, IEEE Trans. Biomed. Eng., 51(11), 2051-2062, 2004.
[5] M. Chiappalone, M. Bove, A. Vato, M. Tedesco and S. Martinoia, Brain Res., 1093(1), 41-53, 2006.
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
oral
Topic:
MEA Meeting 2016
Citation:
Shimba
K,
Iida
S,
Sakai
K,
Kotani
K and
Jimbo
Y
(2016). Characterization of Synchronized Burst in Developing Human iPS Cell-derived Neuronal Networks with a Microtunnel Device.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00113
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Kenta Shimba, The University of Tokyo, School of Engineering, Tokyo, Japan, shimba@neuron.t.u-tokyo.ac.jp