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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