Human Embryonic Stem Cell Derived Neurospheres - A Novel Three Dimensional Model For Neurotoxicological Studies
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1
University of Applied Sciences, BioMEMS Lab, Germany
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2
GSI Helmholtz Centre for Heavy Ion Research, Biophysics division, Germany
At present, most neurotoxicological studies in the field of microelectrode array (MEA) technology are based on in vivo or in vitro animal models, particularly in mice or rats. These models contributed much to our knowledge about molecular and cellular mechanisms of neurotoxins affecting the central nervous system. Yet, animal models are not necessarily capable to forecasting the effectiveness of treatment in clinical trials. Therefore the development and characterization of suitable neuronal cell models derived from human cells is emerging in the field of toxicity testing. In this study, we present neurospheres (NS) as a new three dimensional (3D) neuronal cell model based on human embryonic stem cells (hESC), coupled onto microelectrode arrays (MEA). This cell-based assay serves as platform to investigate effects of neuroactive substances on network communication. In order to characterize the neurospheres’ reaction, the well-known GABA receptor antagonist bicuculline and the sodium channel blocker carbamazepine are applied. Preliminary results reveal anticipated and physiologically reasonable alterations in network activity. Thus, we have the first evidence that hESC derived NS are a promising 3D cell model for neurotoxicity testing. In further studies, this model will be used to investigate effects of ionizing radiation on network signaling.
Motivation
Animal models have contributed much to our knowledge about molecular and cellular mechanisms of neurotoxins affecting the central nervous system [1]. Yet, their use in forecasting the effectiveness of treatment in clinical trials is controversial, as animal studies do not reliably predict human responses [2]. Past experience showed that several substances are teratogenic in animal models but not in humans and vice versa [3]. For this reason, suitable human neuronal models are urgently needed. In this respect three dimensional (3D) neuronal cell models become increasingly important since 2D systems are somehow artificial and differ strongly from the 3D situation in vivo [4]. Moreover, electrophysiological techniques emerge as an important tool to properly screen for neurotoxicity. In the present study, we present neurospheres (NS) derived from human embryonic stem cells (hESC) as a new 3D neuronal cell model suitable for electrophysiological analysis via microelectrode arrays (MEA). Up to now only two groups reported on successful recording of hESC derived 3D neuroaggregates on MEA chips. Smith et al. [5] cultivated human neuronal stem cells (NSC) on Alvetex scaffolds, while Ylä-Outinen [6] prepared dissected pieces of hESC derived neural aggregates on MEA chips, resulting in spontaneously active neuronal networks. In contrast, we plate whole, scaffold free NS onto MEA chips and characterize their functionality.
Material and Methods
We differentiated hESC to NSC, using an established protocol (Gibco / Life Technologies, Eggenstein, Germany). NSC were enzymatically dissociated to single cells with accutase. Subsequently, single cells were seeded in 6-well low attachment plates at a concentration of 100 cells/µl in a total volume of 5 ml to generate NS by re-aggregation of NSC. Cells remained in suspension for 2-3 weeks. NS were plated on the electrode field of MEA60 chips (Multichannel Systems, Reutlingen, Germany). All experiments were carried out with MEA chips containing 59 titanium nitride recording electrodes arranged in an 8x8 grid with an inter-electrode distance of 200 µm and an electrode diameter of 30 µm. Before plating, MEA chips were coated with 0.1 % Polyethyleneimine (PEI) and 20 µg/ml laminin. Each chip referred to one single neurosphere. Pharmacological response to the GABA receptor antagonist bicuculline and the sodium channel blocker carbamazepine was investigated applying a concentration of 10 µM bicuculline or 40 µM carbamazepine. Measurements were performed at a sampling rate of 10 kHz. Data evaluation was conducted, applying the custom made Matlab®-based software tool “DrCell” [4].
Results
One day after NS were placed on MEA chips (n=17) electrical activity in form of single spikes was observed (Fig. 1). Also burst-like events were infrequently seen. A significant increase in number of bursts was detected after five days, representing the matured signaling activity of the network. Yet, a synchronization of these burst-events was rarely observed. The NS were viable and electrically active on MEA chips over a period of about three weeks. In order to investigate their capacity to respond to neuroactive substances, NS were treated with bicuculline and carbamazepine. Our preliminary results clearly revealed altered network activity after substance application. Treatment with bicuculline resulted in increased network activity as well as burst synchronization (Fig. 2A), whereas treatment with carbamazepine caused quiescence of the electrical signals (Fig. 2B).
Conclusion
We present a new 3D cell model based on human embryonic stem cells in combination with the MEA method aiming to provide a functional assay for neuroactive substances. This approach not only provides high physiological relevance for the human brain, but also permits neurotoxicological studies at successive stages of neural development. However, many details of the NS, like grade of self-organization and cell distribution, are not studied yet and many questions remain for future work in this interesting field. In further studies, NS will be used to examine the impact of ionizing radiation on neuronal network development and communication.
References
[1] AliMohammadi, M., et al. (2015) Med J Islam Repub Iran 29:273
[2] Hackam, D.G., et al. (2006) JAMA 296: 1731-1732
[3] Brent, R.L., (2004) Pediatrics 113: 984-995
[4] Frega, M. (2014) Sci rep. 4, 5489
[5] Smith, I., et al. (2015) J Tissue Eng Regen. Med., ISSN 1932-7005
[6] Ylä-Outinen, L., et al. (2010) Front. Neuroeng. 3: 111
[7] Nick, C., et al. (2013) SPIJ 7 (2): 96-109
Figure Legend
Figure 1: Neurosphere on MEA chip (A). Activity pattern shows single spikes as well as bursts (B).
Figure 2: Electrophysiological response of NS to neuroactive substances presented in a raster plot. Small bars represent spikes and large bars bursts. Application of the GABA receptor antagonist bicuculline resulted in increased number of spikes (A). In contrast, treatment with the sodium channel blocker carbamazepine caused quiescence of the network activity.
Acknowledgements
This work was supported by the Federal Ministry of Education and Research, Bonn, Germany (02NUK025 and 02NUK034C).
Keywords:
3D,
human embryonic stem cells,
neurospheres,
MEA Chips
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Mayer
M,
Arrizabalaga
O,
Ritter
S and
Thielemann
C
(2016). Human Embryonic Stem Cell Derived Neurospheres - A Novel Three Dimensional Model For Neurotoxicological Studies.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00081
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Margot Mayer, University of Applied Sciences, BioMEMS Lab, Aschaffenburg, Germany, Margot.Mayer@h-ab.de