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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).
1Benjamin M. Bader, Anna-Maria Pielka, Corina Ehner , Konstantin Jügelt, Olaf H.-U. Schröder, Alexandra Gramowski-VossNeuroProof GmbH, 18055 Rostock, Germany; .*[email protected]
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
neurons and to compare them with primary neurons.
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 effect size of test agents.
GDNF prevents fun-ctional MPP+ effects on p r i m a r y m i d b r a i n / 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 re g u l a r i t y. N e t wo r k activity is more irregular 1-2 div after MPP+ treat-ment and more regular after 7-14 div post-MPP+ treatment. GDNF is able t o p r e v e n t v a r i o u s parameters, interestingly at different DIVs.
We use cryo-preserved neurons derived from human iPS cell cultures: M a j o r i t y o f T u 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)
were cultured on MEAs for 3 weeks. A pulse of MPP+ was performed for 24 hours at day
7. GDNF was applied day 5.
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
primary neurons.
Data analysis: multi-parametric data analysis of 204 spike train parameters was per-
formed using NPWaveX Software (NeuroProof). “Effect Score” calculation: Projection
of up to 204 parameters into a single layer parameter based on Z’ factor.
TH TuJ Hoechst, 5 div Cell -aggregate on electrode, Axion12
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vehicle MMP+
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surv
ival
(MT
TO
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.)±
SD
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vehicle MPP+
%T
H+
cells
±S
EM
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rel.
TH
blo
tband
inte
nsi
tiy
±S
EM
*
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
FoxA2PitX3
SNAP25
28 div
TH
SNAP-25
MTT assay Cell counting Western blotting FoxA2, PitX3 Western blotDopaminergic neurons (TH), Neurites (ß3Tub), nuclei (Hoechst)
Dopaminergic neurons (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)
Culturestart
5 8 21 days in vitro
1x +GDNF+MPP+ (1 day)
7 day
Recording daysactivity 1 14 days after MPP+
Experimental scheme
Culturestart
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
to known primary mouse neurons.
Decrease of TH protein levels
No significant loss of TH+ cells as intended
Summary of up to 204 parametersinto 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 and Dopa.4U neurons (Axiogenesis, Germany) on day in vitro 28.
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Picrotoxin [M]
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[%]
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1E-08 1E-07 1E-06 1E-05 1E-04
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Picrotoxin [M]
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Picrotoxin [M]
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1E-08 1E-07 1E-06 1E-05 1E-04
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t[%
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1E-08 1E-07 1E-06 1E-05 1E-04
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t[%
]
*
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1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
%s
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]
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1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
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IBIS
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]
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1E-08 1E-07 1E-06 1E-05 1E-04
Picrotoxin [M]
Bu
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IBIS
D[%
]
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****
*
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1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Sp
ike
rate
[%]
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***
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1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Bu
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[%]
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1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
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t[%
]
***
***
**
***
******
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1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
%s
pik
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inb
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ts[%
]
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1E-08 1E-07 1E-06 1E-05 1E-04
NMDA [M]
Bu
rst
IBIS
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]
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1E-08 1E-07 1E-06 1E-05
NMDA [M]
Sp
ike
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[%]
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NMDA [M]
Bu
rst
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[%]
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1E-08 1E-07 1E-06 1E-05
NMDA [M]
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pik
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t[%
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1E-08 1E-07 1E-06 1E-05
NMDA [M]
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IBIS
D[%
]
Fro
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rtex
PC
ne
uo
ns
Hi
Sr
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Fro
nt
Co
rtex
PSC
-ne
rn
Hi
uo
s
Concentration response effects on primary cortex neurons (mouse) network activity of picrotoxin (28 Div, 60sec, 6 neurons). Picrotoxin induces stronger population bursts which maintain over time.
Spike rate Burst rate Spike contrast % Spikes in bursts Burst IBI SD
Spike rate Burst rate Spike contrast % Spikes in bursts Burst IBI SD
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 sensitivity of activity changes in primary murine frontal cortex.
Concentration-response-curves for NMDA-effects on primary murine frontal cortex, and hiPSC-neurons (Dopa4.U, Axiogenesis) culture. EC SR: FC: 3 µM; Dopa.4U: 300 nM.50
native
100 nM
500 nM
1 µM
10 µM
50 µM
native
100 nM
300 nM
1 µM
10 µM
30 µM
Phenotypic Screening with MEA-Neurochips
Sych
nz
tion
roi
an
Burst
Full disinhibition (with bicuculline, strychnine, NBQX)
Native activity 30 s
Oscillation
2 Burst Structuree.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 OscillationVariation over time as an indicator for the strength of the oscillation; in addition e.g. Gabor function parameters fitted to autocorrelograms
4 SynchronizationVariation within the network as an indicator for the strength of the synchronization; in addition e.g. simplex synchronization, percent of units in synchronized burst
Supported by
Experimental scheme
Multiparametric Characterization of Neuronal Network ActivityNeuronal
Cell Culture
Phenotypic
Multichannel Recording
Multiparametric
Data Analysis
Pattern
Recognition
Primary murine cell culture:- Frontal Cortex- Hippocampus- Midbrain- Spinal Cord/DRGNeuronal 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
MAESTRO Recording System
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
NeuroProof Technology
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 burst structure induced by a 24 hours-pulse of MPP+ at div 7. Treatment with GDNF or BDNF partly prevents MPP+ mediated effects 14 days after MPP+ treatment. B) Ef fect Score was calculated on selected pa ra m ete rs s h o w i n g MPP+ effects after 14 div.
HiPSC neurons are spontaneously active on Axion 12 well MEAs a f ter 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 synchronizat ion be-tween neurons.
Mean Spike Rate
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7 div 14
div
21
div
Me
an±
SEM
[1/s
]
CVnet Spike Rate
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div
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div
Mea
n±
SEM
[%]
CVtime Spike Rate
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Me
an±
SEM
[%]
Mean Burst Rate
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Me
an±
SEM
[1/m
in]
CVnet Burst Rate
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div
21
div
Me
an±
SEM
[%]
CVtime Burst Rate
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1015202530354045
7 div 14
div
21
div
Mea
n±
SEM
[%]
Mean % of Spikes in
Bursts
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Me
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[%]
Mean Burst
Duration
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Mean Spikes in
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Mea
n±
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Mean Peak Freq. in
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an±
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[Hz]
Mean Interburst
Interval
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div
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Me
an±
SEM
[s]
Number Of Bursting
Units
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Me
an±
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
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7 div
activity
1d 14d
Me
an
±S
EM
[%]
post-MPP+ pulse
*
CVnet % of Spikes in Bursts
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activity
1d 14d
Me
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±S
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[%]
**
post-MPP+ pulse
Mean Burst Duration
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Me
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±S
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[%]
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CVnet Burst Duration
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* **
post-MPP+ pulse
Mean Peak Freq. in Burst
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[%]
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***
Mean Interburst Interval
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[%]
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**
*
Number Of Bursting Units
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activity
1d 14d
Me
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±S
EM
[%]
post-MPP+ pulse
Mean Spike Rate
050
100150200250300350400450500
7 div
activity
1d 14d
Me
an
±S
EM
[%]
control
MPP+GDNF/MPP+
BDNF/MPP+
*
post-MPP+ pulse
Summary of up to204 parametersinto one readout
99%
23%(p=0.022)
97%
21%(p=0.017)
Increased synchronicity
Increased regularity Stronger bursting
Increased activity
Functional development of hiPSC-neurons
Rescue Index
Ee
ffct
Sco
re
Efe
f
ctSc
ore
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
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7 div activity 1-2 div 14 div
Me
an
±S
EM
[%]
****
Burst Spike Max Rate
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7 div activity 1-2 div 14 div
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an
±S
EM
[%]
***
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*
Burst Spike Rate
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Me
an
±S
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[%] *** *
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*
Burst Spike Max Rate SD
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7 div activity 1-2 div 14 div
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an
±S
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[%]
*****
*
**
Spike Rate
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7 div activity 1-2 div 14 div
Me
an
±S
EM
[%]
controlMPP+GDNF/MPP+
***
Burst Rate
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7 div activity 1-2 div 14 div
Me
an
±S
EM
[%]
**
Burst Spike Number
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7 div activity 1-2 div 14 div
Me
an
±S
EM
[%]
***
*
*
Burst Plateau SD
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350
7 div activity 1-2 div 14 div
Me
an
±S
EM
[%]
*
*
A)
B)
A)
B)
Control MPP+
7 div
1 div
14 div
8 div