phenotypic screening using meas
TRANSCRIPT
Multi-parametric Effect Score
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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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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
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FoxA2PitX3
SNAP25
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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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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
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Phenotypic Screening with MEA-Neurochips
Sych
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roi
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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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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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F on a rter t l Co xHi S ne ronsP C u
Strong Functional MPP+ Effects on hiPSC dopa neurons
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Summary of up to204 parametersinto one readout
99%
23%(p=0.022)
97%
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Increased synchronicity
Increased regularity Stronger bursting
Increased activity
Functional development of hiPSC-neurons
Rescue Index
Ee
ffct
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2.3 %(p=0.038)
27%(p=0.049)
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