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www.cellulardynamics.com Madison, WI USA (608) 310-5100
Measuring Connectivity in Human iPS Cell-derived
Neurons Using Multi-electrode Array (MEA)
Eugenia Jones, Kile P. Mangan, Coby Carlson, Rachel Llanas, Brian
Arnold, Arne Thompson, Vanessa Ott, and Susan DeLaura
Cellular Dynamics International, Inc., Madison, WI USA
With the dramatic rise in the incidence of neurological
diseases, there is a sense of urgency to develop
better therapies. However, a major challenge in
neuroscience research and drug development is
access to clinically-relevant cell models. Human
induced pluripotent stem cell (iPSC)-derived neurons
provide a novel cell type to facilitate a better
understanding of the mechanisms of neurological
diseases in a physiologically relevant environment.
Using the Axion Maestro multi-electrode array (MEA)
platform, we have assessed neuronal activity and
bursting behaviors in iPSC-derived neuronal cultures.
This highly pure population of neurons is derived
from a normal human donor. Herein, we describe the
culture conditions, assay protocol, and analytical
tools used to record activity from these cells. We
have demonstrated dose-dependent effects of
multiple glutamatergic- (AP5, DNQX, Kainic Acid), as
well as GABAergic- (GABAzine, L-655,708), selective
pharmacological agents on both over-all firing rates
and bursting behaviors. Moreover, the complete block
of glutamatergic activity (co-application of AP5 &
DNQX) abolished all neuronal activity, indicating a
dependence on synaptic connectivity. Our findings
highlight the differences from primary rodent cultures
on MEA and support the conclusion that human
iPSC-derived neuronal cultures display network
activity that can be measured and modulated within
one week in culture.
Abstract
Collectively, these data demonstrate that human iPSC-derived neurons can be cultured on MEA
plates for just over one week and they can be perturbed with known synaptic modulators, channel
blockers, and excitability agents. The iCell NeuroAnalyzer can be used to parse out these
pharmaco-influences. Additionally, this MATLAB-based tool offers simple outputs of complex
analysis methods – beyond just mean firing rate.
Summary
Cells: iCell Neurons
Measuring Pharmaco-Influences
MEA System: Axion Maestro
Purity
SYP / vGAT /
vGLUT2
Phenotype
MAP2 / GABA /
Hoechst
bIII Tubulin /
Nestin / Hoechst
Morphology
Analysis: iCell NeuroAnalyzer
1. Mean Firing Rate –
Influence on Inhibition (IoI)
2. Bursting Rate –
Influence on Excitation (IoE)
3. Intensity within the Bursts –
Influence on Connectivity (IoC)
Fre
qu
en
cy (
Hz)
Peak Frequency (red circles)
Threshold Frequency (blue dots)
Cryopreserved
iCell Neurons Human Donor
Terminally
differentiated
cell types
Induced
Pluripotent Stem
(iPS) Cells
Days in
Culture 1 -1 8
Thaw and plate
cells into MEA
0 5
Apply compounds;
record MEA activity
Complete
media change
Coat MEA
plate with PEI
Media
change
Neuronal activity (ie. # of action potentials over time) measured on the MEA generates data (ie. spk
files) that can be processed and analyzed offline by various approaches. Traditionally, the raw
voltage data is converted into a raster plot of action potential ticks for visualization (see below) and
the primary parameter extracted from the culture is mean firing rate (MFR). Here we further collapse
all the spikes recorded from one well together and subsequently bin the activity into 500 msec bins,
producing a velocity graph that enables the examination of network-level bursting properties.
Ele
ctr
od
e #
M
FR
(H
z)
50 Sec
Before After
We have generated a custom “peak detection”
algorithm in MATLAB – called the iCell Neuro-
Analyzer – that examines the running average
firing rate, and any punctate increases in
recorded signal are marked and captured. It is
these captured events that represent “bursts”
in neuronal activity. The point at which the
firing rate increases and the burst begins is
called the “threshold frequency” (blue dot). As
the signal rises immediately following this
point, it reaches a maximum – designated as
the peak frequency (red circle). The key
outputs derived using this analysis tool are 1)
mean firing rate (MFR), 2) bursting rate or
number of bursts per min (BPM), and 3)
intensity within the bursts (which is the
difference between the peak and threshold
frequency), measured in Hz.
The pattern of “ticks” on each of the 16 electrodes in a
well changes after treatment of cells with GABAzine. The
number of red circles and the size of population bursts
has increased.
iCell NeurolAnalyzer Outputs:
• 3D ribbon diagram
• Well raster plots
• Binned firing rates (velocity graph)
• Column or plate statistics
• Network fingerprint
• Excel table
We have utilized iPSC technology to reprogram adult cells from either normal
healthy or disease-specific donors back to an iPS cell line. Here in this state,
iPS cells have the ability to differentiate into virtually any cell type – including
previously inaccessible human neurons. Importantly, iCell Neurons are provided
as cryopreserved material that can be thawed an used any day of the week.
The key features of the cell sample highlighted are as follows. iCell Neurons
are a highly pure population (>95%) of human iPSC-derived cortical neurons,
based on positive βIII-tubulin and nestin-negative staining. They possess the
classical neuronal morphology, with bipolar or multi-polar neurite outgrowths
that begin right at Day 1 in culture. These cells have been determined to be a
mixture of both inhibitory (GABAergic) and excitatory (glutamatergic)
neurons, and they have been analyzed at the gene expression level (data
not shown) and by phenotypic analysis for characteristic molecular markers.
Multi-electrode array (MEA) technology has been around for several decades. MEAs are
grids of tightly spaced electrodes that are capable of directly sensing changes in voltage
that are propagated down the membranes of excitable cells. The Maestro MEA system
commercialized by Axion BioSystems offers a non-invasive, label-free, high-throughput
solution to address complex electrophysiologically-based questions. With 768 electrodes
per MEA plate (16 electrodes per well of a 48-well plate) and sampling rates above 10 kHz,
nearly 10 million data points can be collected per second.
50 m
350 m Control GABAzine
Axion Maestro MEA Zoomed-in view
of electrodes
Single unit recordings
of Action Potentials
High-density “dots” of neurons are placed at the center of each well, positioned
directly on top of electrodes. Cells are then cultured for >1 week to allow
functional networks to form. Maestro MEA performs single unit recordings from
every electrode to create a real-time trace of voltage as changes in ion flow
occur through neuronal channels. The system then parses these voltage traces
for action potential waveforms, time-stamps the waveforms, and calculates
action potential firing rates on-line for each electrode in all wells. This on-line
firing rate is represented by color-coded heat-maps that depict real-time and
simultaneous indications of cell health, network activity, and condition effects.
48-well MEA plate Single channel recordings Real-time plate heat map
AP5 (20 µM)
100 Seconds
Fre
qu
en
cy
(H
z)
DNQX (5 µM)
100 Seconds
Fre
qu
en
cy (
Hz)
B
To demonstrate application of the iCell NeuroAnalyzer to
measure the effects of pharmacological agents on the network
activity of iCell Neurons, cells were treated on Day 8 post-
plating. The workflow diagram below (A) depicts how the
assay was performed. When compounds are applied to the
cells, recordings are taken pre- and post-drug treatment and
the changes in influences are reported (B). IoI is shown in red,
IoE is illustrated in green, and IoC is colored blue.
The effect of drug treatment can also be
visualized through changes in the velocity graphs
(C). Dosing with AP5, which blocks NMDA
receptors, results in a significant decrease in
activity. Similarly, incubation with DNQX, which
inhibits AMPA receptor activity, essentially
minimizes all firing activity in the iCell Neurons.
Alternate outputs featured in this analysis tool
can also be used to characterize the network
level effects of other compounds, including the
pre-synaptic cannabinoid receptor agonist (D); or
the bi-phasic response observed with GABAA-
selective inverse antagonist, L-655,708 (E).
A
C
D
E