Effect of substrate coating material on spontaneous activity of human-induced pluripotent stem cell-derived neuronal stem cells
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1
George Mason University, Department of Electrical and Computer Engineering, United States
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2
Old Dominion University, Department of Electrical and Computer Engineering, United States
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3
Old Dominion University, Department of Medical Diagnostic and Translational Sciences, United States
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4
Sentara Norfolk General Hospital, Molecular Diagnostics Laboratory, United States
Motivation:
Human-induced pluripotent stem cells (hiPSCs) provide an excellent avenue in studying in vitro neuronal networks that more closely mimic human brain models. Tracking the differentiation of hiPSC-derived neuronal stem cells (NSCs) into mature neuronal networks can provide insights into developmental neurobiology as well as the progression of neurodegenerative disorders. Micro-electrode arrays (MEAs) are an excellent tool in characterizing the electrophysiology of a specific neuronal culture and allow for monitoring of evoked or spontaneous activity over long-term periods. MEAs are ideal for studying changes in electrophysiological properties of hiPSC-derived cultures as they mature throughout the differentiation process from NSCs to neuronal networks. Here, we aim to measure electrical activity within a population of NSCs as they differentiate into mature neurons using two different substrate coatings for adhering cells to the MEAs to compare against primary neuronal cells extracted from prefrontal cortex and spinal cords of mice.
Materials and Methods:
Human-induced pluripotent stem cells were reprogrammed from breast-derived adipose stromal cells collected as medical waste from an unidentifiable patient using the CytoTune™ -iPS 2.0 Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA) along with its protocol. The resulting hiPSCs were then used for the generation of neural stem cells using STEMdiff™ Neural Induction Medium (Stem Cell Technologies, Vancouver, Canada). NSCs were subsequently passaged onto microelectrode array dishes with 60 electrodes (10 um diameter each, coated with TiN, 200 um distance between neighboring electrodes). Since the substrate of the MEAs is glass, two different coatings were tested for adhering the NSCs to the substrate, namely, laminin (10ug) and Geltrex™ (1:100 dilution; LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix, Gibco). Cultured dishes of NCSs were kept in an incubator at 37 C (humidity near 100%, 5% CO2 atmosphere) and media replenished every other day with a full-media replacement (1.0 mL) of maintenance media (StemPro™ NSC SFM; Gibco). To induce differentiation of NSCs into neurons, the media was replaced with differentiation media (Neurobasal™ Medium, Gibco) supplemented with 2% B-27™ Supplement (50x), 0.5 mM GlutaMAX-I, 1% ABAM, 500 nM puromorphamine, and 50 nM retinoic acid. Subsequently, half-media replacement (0.5 mL) was conducted every other day. Electrical activity from NSCs was recorded using the MEA1060-Inv-BC (Multichannel Systems, Reutlingen, Germany) amplifier and filter system with 1,200 total gain and band pass filter from 300 to 3k Hz. A -5 standard deviation threshold was used for selecting spikes. Spontaneous electrical activity of NSCs were recorded for 5 minutes each day for 5 days at which point the media was exchanged to induce differentiation. Activity was recorded every other day as the NSCs differentiated. After recording, sampled waveforms were distinguished as spikes using Offline Sorter (Plexon Inc., Dallas, TX) and further analysis was conducted using a custom Python script.
Results:
Recording of NSCs while they differentiate into neurons adhered to MEAs using GelTrex produced minimal waveforms uncharacteristic of action potentials using a -5 standard-deviation threshold. The RMS noise level seen in the recording of these dishes was on the order of 1 V, and thus 5 standard deviations was near 5 V. This considerably low level of noise may have been caused from insulation between the neurons and the electrodes by the GelTrex. The NSCs that were adhered to the MEAs using laminin showed similar levels in noise with minimal selected waveforms. However, differentiating NSCs (22 days after differentiation induced and onwards) adhered via laminin showed a greater level of noise (~40 V) and yielded several selected spikes more characteristic of action potentials, consistent with previous experiments from our group using primary cell cultures from mouse cortex and spinal cord neurons [1,2].
Discussion:
Our results indicate that using GelTrex to adhere NSCs to the glass substrate of MEA dishes inhibits recording of electrical activity, presumably by insulating the electrodes. Using laminin to adhere NSCs resulted in a higher baseline noise in the system (consistent with previous recordings from our group using primary mouse cortex cultures) [1]. We also observed the recording of several spontaneous spike waveforms, indicating that the laminin was not substantially insulating the electrodes from recording electrical activity. Figure 1 depicts raster plots from single recording sessions comparing the sorted spikes from a previous primary cell culture of spinal cord neurons with three active channels to that of the first spikes seen in differentiating hiPSC-derived neuronal stem cells adhered via laminin (27 days in vitro, 22 days after use of differentiation media). It is clear that the primary cell culture has more spiking behavior, likely due to their mature state. The hiPSC-derived NSCs plated with laminin may begin exhibiting more spikes as they mature and differentiate more in later recordings.
Conclusion:
In this study, we recorded hiPSC-derived NSCs plated on MEAs using two different substrate coatings to adhere the cells. We find that using Geltrex as a substrate coating does not allow for recording of spontaneous activity in early differentiating NSCs. However, NSCs plated with laminin show minimal spontaneous activity 22 days after differentiation is induced. Using this information, it is our goal to capture and to further characterize the electrophysiological behavior of hiPSC-dervied NSCs to establish an understanding of the progression of electrical activity in differentiating NSCs. This information will ultimately lead us to comparative studies with disease models of neural stem cell lines (i.e. Huntington's disease, ALS) to underpin characteristic changes in the electrophysiology due to the diseases.
Figure 1:
Comparison between typical spiking activity of a primary spinal cord neuron culture with 3 active channels (top) and the onset of spiking activity of a differentiated hiPSC-derived neuronal culture (bottom).
References
[1] Hamilton, Franz, Robert Graham, Lydia Luu, and Nathalia Peixoto. "Time-Dependent Increase in Network Response to Stimulation." PLOS ONE 10, no. 11 (November 6, 2015): e0142399.
[2] Knaack, Gretchen L., Hamid Charkhkar, Franz W. Hamilton, Nathalia Peixoto, Thomas J. O'Shaughnessy, and Joseph J. Pancrazio. "Differential Responses to ω-Agatoxin IVA in Murine Frontal Cortex and Spinal Cord Derived Neuronal Networks." NeuroToxicology 37 (July 1, 2013): 19-25.
Keywords:
HiPSC-derived neurons,
Neural Stem Cells,
microelectrode arrays,
spontaneous activity,
neural differentiation
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Stem cell-derived applications
Citation:
LaFosse
PK,
Zamponi
M,
Mollica
PA,
Therrien
K,
Bruno
RD,
Sachs
PC and
Peixoto
N
(2019). Effect of substrate coating material on spontaneous activity of human-induced pluripotent stem cell-derived neuronal stem cells.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00115
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Received:
17 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mr. Paul K LaFosse, George Mason University, Department of Electrical and Computer Engineering, Fairfax, Virginia, 22030, United States, plafosse@masonlive.gmu.edu