Functional and phenotypic in vitro modeling of Parkinson's disease and seizurogenic effects using human iPSC-derived neurons grown on micro electrode arrays (MEAs).

1. Multi-parametric Effect Score
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MPP+
control
GDNF/MPP+
1 div 14 div
Time post-MPP+ treatment
Functional and phenotypic in vitro modeling of Parkinson's
disease and seizurogenic effects using human iPSC-derived
neurons grown on micro electrode arrays (MEAs).
1
Benjamin M. Bader, Anna-Maria Pielka, Corina Ehner , Konstantin
Jügelt, Olaf H.-U. Schröder, Alexandra Gramowski-Voss
NeuroProof GmbH, 18055 Rostock, Germany;
.*Benjamin.bader@neuroproof.com
Introduction
Primary cultures are widely used for phenotypic testing
of drug and test compounds. Therefore, the use of
human induced pluripotent stem cell-derived (hiPSC)
neurons is relevant to evaluate whether these models
can be applied to human cells with the goal to increase
predictability, sensitivity and specificity of test systems.
Our goal was to evaluate toxin-induced cell-based
models for Parkinson's disease and seizure using hiPSC
neuronsandtocomparethemwithprimaryneurons.
Methods Conclusions
“Rescue Index / Effect Score”
calculation: Projection of up to 204
(here: 98) parameters into a single
parameter allows ranking of rescue
efficacies at different time points (or
concentrations) based on the functional
finger print of significantly affected
functional parameters. We calculate an
optimized combination MPP+ affected
features for an optimal separation of
control effects from those of MPP+.
Control is set to “0”. MPP+ is set to “1”.
The “Effect Score” describes the relative
effectsizeoftestagents.
GDNF prevents fun-
ctional MPP+ effects on
primary mid brain/
cortex co-culture net-
works.
A) Example spike trains
for control and MPP+
treated neuronal net-
works.
Hypersynchronization in
MPP+ treated networks
is observed 14 div post-
MPP+treatment.
B) 9 selected functional
parameters show inital
reduction of activity and
strong effects on burst
structure as well as
regularity. Network
activity is more irregular
1-2 div after MPP+ treat-
ment and more regular
after 7-14 div post-MPP+
treatment. GDNF is able
to prevent various
parameters, interestingly
atdifferentDIVs.
We use cryo-preserved
neurons derived from
human iPS cell cultures:
M a j o r i t y o f Tu J +
neurons express TH
(>50-70 %) Ventral
mesencephalic markers
FoxA2 and PitX3 are
expressed.
Primary culture: primary mouse midbrain/cortex co-cultures E14.5 embryos (NMRI)
wereculturedonMEAsfor3weeks.ApulseofMPP+wasperformedfor24hoursatday
7.GDNFwasappliedday5.
hiPSC culture: We cultured Dopa.4U Neurons (Axiogenesis AG, Germany) on multiwell
MEAs (Axion Biosystems) for 3 weeks. The treatment paradigm is the same as for the
primaryneurons.
Data analysis: multi-parametric data analysis of 204 spike train parameters was per-
formed using NPWaveX Software (NeuroProof). “Effect Score” calculation: Projection
ofupto204parametersintoasinglelayerparameterbasedonZ’factor.
TH TuJ Hoechst, 5 div Cell-aggregate on electrode, Axion12
0
0.5
1
1.5
2
2.5
3
3.5
4
vehicle MMP+
cellsurvival
(MTTODa.u.)±SD
0
2
4
6
8
10
12
vehicle MPP+
%TH+cells±SEM
0
0.2
0.4
0.6
0.8
1
1.2
vehicle MPP+
rel.THblotbandintensitiy
±SEM
*
Small Structural/morphological MPP+ effects
Strong Functional MPP+ Effects which can be prevented
No global cyto-toxicity
Rescue Index
P i a y idbra n C l r sr m r M i u tu e
MEA-active human dopaminergic neurons
GDNF-mediated prevention of functional MPP+ effects
uman i SC e i e u o sH P -d r v d Dopa Ne r n
25
17
50
75
FoxA2
PitX3
SNAP25
28 div
TH
SNAP-25
MTT assay Cell counting Western blotting FoxA2, PitX3 Western blot
Dopaminergicneurons(TH),
Neurites(ß3Tub),nuclei(Hoechst)
Dopaminergicneurons(TH),neurites
(ß3Tub),nuclei(Hoechst)
Thyrosine hydroxylase band
decreased by 20 µM MPP+
MTT intensity decreased by
50 µM but not by 20 µM MPP+
1A) 1B) 1C)
Culture
start
5 8 21 days in vitro
1x +GDNF+MPP+ (1 day)
7 day
Recording daysactivity 1 14 days after MPP+
Experimental scheme
Culture
start
5 8 21 days in vitro
1x +GDNF+MPP+ (1 day)
7 day
Recording daysactivity 1 14 days after MPP+
We demonstrate that the activity of both primary and hiPSC neu-
rons is affected by MPP+ which can be prevented by treatment
with compounds. Seizure-inducing compounds affect hiPSC neu-
ron activity, partly more potently than in primary neurons. In con-
clusion, despite our limited understanding of the maturation sta-
tus and correlation to the in vivo developmental stage, hiPSC-
derived neurons can be used for functional in vitro screening of
compounds and exhibit comparable response patterns compared
toknownprimarymouseneurons.
Decrease of TH
protein levels
No significant loss
of TH+ cells as intended
Summary of up to
204 parameters
into one readout
Effects of pro-convulsants on hiPSC-derived neurons
Concentration response effects on hiPSC neurons (Dopa.4U,
Axiogenensis) network activity of picrotoxin (14 Div, 240 sec, 5
neurons). Synchronized bursts occur within minutes after picrotoxin
application.
In human stem cell-derived neuronal networks picrotoxin induced a concentration-dependent increase
in spike and burst rate activity and a prolongation of the burst duration, which is comparable to quality
and sensitivity of activity changes in primary murine frontal cortex. Treatment of primary frontal cortex
andDopa.4Uneurons (Axiogenesis,Germany)ondayinvitro28.
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Spikerate[%]
0
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200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Spikerate[%]
0
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100
150
200
250
300
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Burstrate[%]
*
0
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100
150
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250
300
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Burstrate[%]
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Spikecontrast[%]
*
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Spikecontrast[%]
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
%spikesinburst[%]
*
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
%spikesinbursts[%]
0
100
200
300
400
500
600
700
800
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
BurstIBISD[%]
0
100
200
300
400
500
600
700
800
1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
BurstIBISD[%]
**
*
***
*
*
0
20
40
60
80
100
120
140
160
180
200
1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Spikerate[%]
***
*
*
***
0
50
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200
250
300
1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Burstrate[%]
***
*
**
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Spikecontrast[%]
***
*
**
**
***
***
***
0
20
40
60
80
100
120
1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
%spikesinbursts[%]
*
*
0
500
1000
1500
2000
2500
1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
BurstIBISD[%]
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05
NMDA [M]
Spikerate[%]
0
50
100
150
200
250
300
1E-08 1E-07 1E-06 1E-05
NMDA [M]
Burstrate[%]
0
50
100
150
200
1E-08 1E-07 1E-06 1E-05
NMDA [M]
Spikecontrast[%]
*
0
20
40
60
80
100
120
140
1E-08 1E-07 1E-06 1E-05
NMDA [M]
%spikesinburst[%]
*
0
100
200
300
400
500
600
700
800
1E-08 1E-07 1E-06 1E-05
NMDA [M]
BurstIBISD[%]
FrontalCortexPCneuonsHiSralFrontCortexPSC-nernHiuos
Concentration response effects on primary cortex
neurons (mouse) network activity of picrotoxin (28
Div, 60sec, 6 neurons). Picrotoxin induces stronger
populationburstswhichmaintainovertime.
Spikerate Burstrate Spikecontrast %Spikesinbursts BurstIBISD
Spikerate Burstrate Spikecontrast %Spikesinbursts BurstIBISD
Picrotoxin induced in human iPSC-derived neuronal networks a concentration-dependend increase in
spike and burst rate activity and a prolongation of the burst duration, which is comparable to quality and
sensitivityofactivitychangesinprimarymurinefrontalcortex.
Concentration-response-curves for NMDA-effects on primary murine frontal cortex, and hiPSC-neurons
(Dopa4.U,Axiogenesis)culture.EC SR:FC:3µM;Dopa.4U:300nM.50
native
100 nM
500 nM
1 µM
10 µM
50 µM
native
100 nM
300 nM
1 µM
10 µM
30 µM
PhenotypicScreeningwithMEA-Neurochips
Sychnztionroian
Burst
Full disinhibition (with bicuculline, strychnine, NBQX)
Native activity 30 s
Oscillation
2 Burst Structure
e.g. number, frequency and ISI of spikes in
bursts; burst duration, amplitude, area,
plateau position, plateau duration
1 General Activity
e.g. spike rate, burst rate,
burst period, percent of
spikes in burst
Read out:
Extracellular action potentials on a single neuron and network activity level
Spatio-temporal activity changes as well as synchronicity and oscillation in time scales
of spikes and bursts
!
!
Each specific spike train is described by 200 parameters in 4 categories:
3 Oscillation
Variation over time as an indicator for
the strength of the oscillation; in
addition e.g. Gabor function
parameters fitted to autocorrelograms
4 Synchronization
Variation within the network as an
indicator for the strength of the
synchronization; in addition e.g. simplex
synchronization, percent of units in
synchronized burst
Supportedby
Experimental scheme
MultiparametricCharacterizationofNeuronalNetworkActivity
Neuronal
Cell Culture
Phenotypic
Multichannel Recording
Multiparametric
Data Analysis
Pattern
Recognition
Primary murine cell culture:
- Frontal Cortex
- Hippocampus
- Midbrain
- Spinal Cord/DRG
Neuronal human Stem Cells
Network spike trains and
single neuron action
potential
Over 200 descriptors at
baseline and drug treatment
- General activity
- Synchronization
- Oscillation
- Burst structure
Data base with functional
fingerprints of over 100
basic and clinically
compounds
MAESTRORecordingSystem
Axion Maestro MEA recording Station
12-well MEA
(64 electrodes per well, optial-grade)
48-well MEA
(16 electrodes per well)
Neuronal network on electode field
Close-up showing electrodes
NeuroProofTechnology
Results
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Multi-parametric Effect Score
MPP+
control
1 div 14 div
Time post-MPP+ treatment
GDNF/MPP+
BDNF/MPP+
7 div
8 div
21 div
+ 5 µM MPP+
14 div
1 div
Post-MPP+ treatmentControl
G D N F p r e v e n t s
functional MPP+ effects
on human iPSC-derived
dopaminergic neuronal
networks. A) Selected
functional parameters
s h o w i n g i n c r e a s e d
activity and effects of
burststructureinducedby
a 24 hours-pulse of MPP+
at div 7. Treatment with
GDNF or BDNF partly
prevents MPP+ mediated
effects14daysafterMPP+
treatment.
B) Effect Score was
calculated on selected
parameters showing
MPP+effectsafter14div.
HiPSC neurons are
spontaneously active on
Axion 12 well MEAs
after 2 days, form
complex burst structures
within 7 days in vitro and
remain active for at least
28 div. Up to 60/64
electrodes active (12
well Axion MEA). >80 %
active Wells, increasing
number of bursting
neurons with culture
time. Show population
synchronization be-
tweenneurons.
Mean Spike Rate
0
1
2
3
4
5
6
7
8
7 div 14
div
21
div
Mean±SEM[1/s]
CVnet Spike Rate
0
20
40
60
80
100
120
7
div
14
div
21
div
Mean±SEM[%]
CVtime Spike Rate
0
5
10
15
20
25
30
35
7 div 14
div
21
div
Mean±SEM[%]
Mean Burst Rate
0
5
10
15
20
25
7 div 14
div
21
div
Mean±SEM[1/min]
CVnet Burst Rate
0
10
20
30
40
50
60
70
80
7 div 14
div
21
div
Mean±SEM[%]
CVtime Burst Rate
0
5
10
15
20
25
30
35
40
45
7 div 14
div
21
div
Mean±SEM[%]
Mean % of Spikes in
Bursts
0
20
40
60
80
100
7
div
14
div
21
div
Mean±SEM[%]
Mean Burst
Duration
0
0.1
0.2
0.3
0.4
0.5
0.6
7
div
14
div
21
div
Mean±SEM[s]
Mean Spikes in
Burst
0
5
10
15
20
25
7 div 14
div
21
div
Mean±SEM
Mean Peak Freq. in
Burst
0
50
100
150
200
250
300
350
7
div
14
div
21
div
Mean±SEM[Hz]
Mean Interburst
Interval
0
2
4
6
8
10
12
14
7 div 14
div
21
div
Mean±SEM[s]
Number Of Bursting
Units
0
10
20
30
40
50
60
70
7 div 14
div
21
div
Mean±SEM
Burst
Amplitude
Burst Duration
Burst Period
Burst IBI
Burst Plateau
Burst Area
Burst ISI
Burst Duration
F on a rter t l Co xHi S ne ronsP C u
Strong Functional MPP+ Effects on hiPSC dopa neurons
Mean Burst Rate
0
20
40
60
80
100
120
140
160
7 div
activity
1d 14d
Mean±SEM[%]
post-MPP+ pulse
*
CVnet % of Spikes in Bursts
0
20
40
60
80
100
120
7 div
activity
1d 14d
Mean±SEM[%]
*
*
post-MPP+ pulse
Mean Burst Duration
0
50
100
150
200
250
300
7 div
activity
1d 14d
Mean±SEM[%]
**
*
*
post-MPP+ pulse
CVnet Burst Duration
0
20
40
60
80
100
120
140
7 div
activity
1d 14d
Mean±SEM[%]
* **
post-MPP+ pulse
Mean Peak Freq. in Burst
0
100
200
300
400
500
600
7 div
activity
1d 14d
Mean±SEM[%]
post-MPP+ pulse
**
*
Mean Interburst Interval
0
20
40
60
80
100
120
140
160
7 div
activity
1d 14d
Mean±SEM[%]
post-MPP+ pulse
*
*
*
Number Of Bursting Units
0
20
40
60
80
100
120
140
7 div
activity
1d 14d
Mean±SEM[%]
post-MPP+ pulse
Mean Spike Rate
0
50
100
150
200
250
300
350
400
450
500
7 div
activity
1d 14d
Mean±SEM[%]
control
MPP+
GDNF/MPP+
BDNF/MPP+
*
post-MPP+ pulse
Summary of up to
204 parameters
into one readout
99%
23%
(p=0.022)
97%
21%
(p=0.017)
Increased synchronicity
Increased regularityStronger bursting
Increased activity
Functional development of hiPSC-neurons
Rescue Index
EeffctScore
EfefctScore
2.3 %
(p=0.038)
27%
(p=0.049)
control MPP+ GDNF/MPP+
vehicle GDNFvehicle5 div
8 div
7 div activity
1-2 div
post MPP+
14 div
post MPP+
Control MPP+ GDNF/MPP+
120 sec
Burst Amplitude
0
20
40
60
80
100
120
140
160
180
7 div activity 1-2 div 14 div
Mean±SEM[%]
***
*
Burst Spike Max Rate
0
50
100
150
200
250
7 div activity 1-2 div 14 div
Mean±SEM[%]
***
*
*
*
Burst Spike Rate
0
20
40
60
80
100
120
140
160
7 div activity 1-2 div 14 div
Mean±SEM[%]
*** *
*
*
Burst Spike Max Rate SD
0
20
40
60
80
100
120
140
160
180
200
7 div activity 1-2 div 14 div
Mean±SEM[%]
***
**
*
**
Spike Rate
0
20
40
60
80
100
120
140
160
7 div activity 1-2 div 14 div
Mean±SEM[%]
control
MPP+
GDNF/MPP+
***
Burst Rate
0
20
40
60
80
100
120
7 div activity 1-2 div 14 div
Mean±SEM[%]
**
Burst Spike Number
0
50
100
150
200
250
7 div activity 1-2 div 14 div
Mean±SEM[%]
***
*
*
Burst Plateau SD
0
50
100
150
200
250
300
350
7 div activity 1-2 div 14 div
Mean±SEM[%]
*
*
A)
B)
A)
B)
Control MPP+
7 div
1 div
14 div
8 div