Pharmacological Responses in Cultured Human iPSC … · 2017-10-11 · Cook et al., Nat. Rev. Drug...
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Pharmacological Responses in Cultured Human iPSC derived Cortical Neurons using Multi-electrode Array Department of electronics , Tohoku institute of Technology Ikuro Suzuki Pharmacological evaluation in human iPSC-derived cortical and sensory neurons using high-throughput MEA system ELRIG Drug Discovery 2017 Oct 3 - 4 Liverpool UK
Transcript of Pharmacological Responses in Cultured Human iPSC … · 2017-10-11 · Cook et al., Nat. Rev. Drug...
Pharmacological Responses in Cultured Human iPSC
derived Cortical Neurons using Multi-electrode Array
Department of electronics, Tohoku institute of Technology
Ikuro Suzuki
Pharmacological evaluation
in human iPSC-derived cortical and sensory neurons
using high-throughput MEA system
ELRIG
Drug Discovery 2017
Oct 3-4 Liverpool UK
Background
Application of these cells to toxicological assay and drug discovery
Normal neuron Disease model
Glutamate
GABA
Dopamine
Acetylcholine
・・・
Alzheimer’s
Parkinson’s
Huntington’s
Rett Syndrome
・・・
Many neuronal phenotypes have been derived from human iPSCs
Epilepsy
Human iPS cell Neuron
Cook et al., Nat. Rev. Drug Descov., 13, 419‐431, 2014
Human iPSC-derived neurons are expected as a new toxicological evaluation assay to replace animal experiments in preclinical studies.
Improvement of risk accuracy in preclinical studies is important issue.
Background
Purpose
Toxicological evaluation assay in human iPSC-derived neurons
■ MEA assay in CNS to predict the seizure risks
Today’s topics
■ MEA assay in PNS to predict the pain risks
Methods
Electrophysiological methods
High-throuput multi-electrode array system (Presto)
Low impedance electrode
■Real time■Non invasive ■Multi-point measurement of activity of cultured neurons
Features for MEAExtracellular signals of action potentials
5 sec
0.1
mV
High-sensitivity16 electrodes/well Presto System384 electrodes
24 wells (384 electrodes)
Methods
human iPSC-derived cortical neurons
Human iPSC-Derived Neural Stem Cells
Co-culture neurons with astrocyte
Human iPSC-Derived Mature Astrocyte
+
Hoechst 33258
Synaptophysin
β-tubulin Ⅲ
PSD95 PSD95/Synaptophysin
Merge
Colocalization of pre- and postsynaptic components
Colocalization was confirmed, and synapses had mainly formed around thick dendrites and the soma
50mm
Odawara A, et.al, Sci Rep 6, 26181 (2016).
β-tubulin ⅢSynaptophysinHoechst 33258
β-tubulin ⅢSynaptophysin
Morphology (Pyramidal-like morphology)
50mm50mm
These neurons were similar in appearance to cerebral cortical neurons in vivo
Odawara A, et.al, Sci Rep 6, 26181 (2016).
Pyramidal-like morphology with thick apical and basal dendrites
Pharmacological responses
Before
Bicuculline
Kainic acid
AP5
CNQX
5 sec0
.1 m
V
Before
Bicuculline
Kainic acid
AP5
CNQX
10 msec
CNQX
AP-5
Kainic acid
Bicuculline
Before
CNQX
AP-5
Kainic acid
Bicuculline
Before
5 sec0
.1 m
V10 sec
Before
Bicuculline
Kainic acid
AP5
CNQX
5 sec
0.1
mV
Before
Bicuculline
Kainic acid
AP5
CNQX
10 msec
CNQX
AP-5
Kainic acid
Bicuculline
Before
CNQX
AP-5
Kainic acid
Bicuculline
Before
5 sec
0.1
mV
10 sec
SynaptophysinPSD95
β-tubulin Ⅲ GABA-A
receptor
NMDA
Kainate
AMPA
Pharmacological responseshiPSC-derived neurons on the MEA
We confirmed electrophysiological responses of typical GABA and glutamate receptorsOdawara A, et.al, Sci Rep 6, 26181 (2016).
0
50
100
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250
0 5 10 15 20 25 30 35 40
No
. o
f sp
ike
s( %
)
Time (min)
before
after
HFS
0 15 30 45 60 75 90 105 120
LTP(b) (c)
(d) (e)
Time (ms)
Spik
es/
bin Before
After
(a)
34ch
54ch
34ch
54ch
Before
After
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Change rate (%)
Co
un
ts
LTP
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50 75 100 125
-10
-8
-6
-4
-2
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0 5 10 15 20 25 30 35 40
No
. o
f sp
ike
s (%
)
Time (min)
before
after
-10
-8
-6
-4
-2
0
2
4
6
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10
LTD
LTD
Change rate (%)
Co
un
ts
HFS
0 15 30 45 60 75 90 105 120
Time (ms)
Spik
es/
bin
BeforeAfter
0
400
800
1200
1600
0 50 100 150 200 250 300 350 400 450
(b) (c)
(d) (e)
(a)
45ch
57ch
45ch
57ch
Before
After
LTP
LTD
Plasticity in hiPSC-derived neurons
We reported that human iPSC-derived cortical neurons also have the plasticity.
Odawara A, et.al, BBRC 496, 856 (2016).
■4-Aminopyridine (4-AP): K+ channel antagonist
■Phenytoin: Na + channel antagonist
■Pentylenetetrazole (PTZ): GABA-A antagonist
Induction of epileptiform activity
Major toxicity of new drugs in CNS is seizure-like firings
Convulsant drugs
Anti-convulsant drug
■Pilocarpine : Muscarine receptor agonist
■Acetaminophen: analgesic effectNegative control
■Chlorpromazine : D2 receptor antagonist
Results：Raster plots in 4-AP administration
The number of synchronized bursts were increased in a concentration-dependent manner.
4-Aminopryridine
0 µM
0.3 µM
1 µM
3 µM
10 µM
30 µM
1 min
1s
Movie at before and 4-AP 10 μM administration
Results：Raster plots in pilocarpine administration
The number of synchronized bursts were also increased in a concentration-dependent manner at pilocarpine administration
Pilocarpine
0 µM
0.3 µM
1 µM
3 µM
10 µM
30 µM
1 min
Results：Raster plots in Chlorpromazine administration
The number of synchronized bursts were also increased up to 3 μMand disappeared at 10 μM administration.
Chlorpromazine
0 µM
0.1 µM
0.3 µM
1 µM
3 µM
10 µM
1 min
Results：Raster plots in PTZ administration
The number of synchronized bursts were not changed.The duration of syncoronized burst were increased in a concentration-dependent manner.
Pentylenetetrazole
0 µM
1 µM
10 µM
100 µM
1000 µM
1 min
Results：Firing analysis in 4-AP, pilocarpine, chlorpromazine, and PTZ administration
Number of synchronized burst Total spikes
No
. o
f b
urs
t v.
s. 0
µM
(%
)
0
50
100
150
200
250
4-AP Pilocarpine Chlorpromazine PTZ
No
. o
f b
urs
t v.
s. 0
µM
(%
)
400
700
4-AP Pilocarpine Chlorpromazine PTZ
No
. of
bu
rst
v.s.
0µ
M (
%)
0
20
40
60
80
100
120
4-AP Pilocarpine Chlorpromazine PTZ
No
. o
f to
tal s
pik
es
v.s.
0µ
M (
%)
4-AP Pilocarpine Chlorpromazine PTZ
No
. of
tota
l sp
ike
s v.
s. 0
µM
(%
)
Number of synchronized bursts were increased at 4-AP, pilocarpine and chlorpromazine administration.
On the other hand, number of synchronized bursts were decreased at PTZ administration.
4-AP Pilocarpine Chlorpromazine PTZ 4-AP Pilocarpine Chlorpromazine PTZ
Duration of synchronized burst Spikes in a synchronized burst
0
20
40
60
80
100
120
140
160
4-AP Pilocarpine Chlorpromazine PTZ
Du
rati
on
v.s
. 0
µM
(%
)
4-AP Pilocarpine Chlorpromazine PTZ
Du
rati
on
v.s
. 0µ
M (
%)
0
20
40
60
80
100
120
140
4-AP Pilocarpine Chlorpromazine PTZ
No
. o
f sp
ike
s in
a b
urs
t v.
s. 0
µM
(%
)
4-AP Pilocarpine Chlorpromazine PTZ
No
. of
spik
es
in a
bu
rst
v.s.
0µ
M (
%)
Results：Firing analysis in 4-AP, pilocarpine, chlorpromazine, and PTZ administration
We detected the effects of four convulsant durgs in human iPSC-derived neurons and found that the responses of PTZ is different from 4-AP, pilocarpine and chlorpromazine.
4-AP Pilocarpine Chlorpromazine PTZ 4-AP Pilocarpine PTZChlorpromazine
Results：Raster plots in phenytoin administration
The number of synchronized bursts disappeared at 100 μM phenytoin administration.We confirmed the responses of Na+ channel blocker.
Before
0μM
1μM
3μM
10μM
30μM
100μM
Phenytoin
0
20
40
60
80
100
120
before 0uM 1uM 3uM 10uM 30uM 100uM
Tota
l sp
ike
s v.
s. b
efo
re (
%)
**
**
Total spikes
Results：Raster plots in acetaminophen administration
The number of synchronized bursts and total spikes were not changed in a concentration-dependent manner at acetaminophen administration
Acetaminophen
0 µM
1 µM
3 µM
10 µM
30 µM
100 µM
1 min
0
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40
60
80
100
120
140
160
180
0uM 1uM 3uM 10uM 30uM 100uM
Tota
l sp
ike
s v.
s. b
efo
re (
%)
Total spikes
Summary ①
High-throughput MEA system in cultured hiPSC-derived neurons and our analysis tools are useful for toxicological evaluation in human CNS.
The induction of epileptiform activity by 4-AP, pilocarpine, chlorpromazine and PTZ and the suppressive effects by phenytoin were detected in hiPSC-derived cortical neurons made by Axol bioscience.
■
Peripheral nervous system
Waxman and Zamponi, 2014, Nat Neurosci, modyfied
Human iPSC-derived sensory neurons are suitable to toxicological assay for pain
Sensory neuron
Methods
human iPSC-derived sensory neurons
Human iPSC-Derived sensory neuron progenitors
Dr Edward Emery (University College London).
Results：Sensory marker expression
TRPA1
TRPV1
Nav 1.7 Hoechst 33258 Mergeβ-Tubulin Ⅲ
We confirmed the typical human sensory marker.
8 weeks
Results：Capsaicin
hiPSC-derived Sensory neuronson the MEA
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-5 -4 -3 -2 -1 0 1 2 3 4 5
Ele
ctro
de
nu
mb
er
time (s)
DMSO 0.001%, Tween80 0.001% Capsaicin 100 nM
2 3 4 5 6 7
Time (min)
We confirmed that TRPV1 channels was working electrophysiologically.
Capsaicin
Results：Temperature change
38 40 42 44 4637
37℃ 43℃
It was consistent with the activation temperature of TRPV1 channels
From 37 to 46℃
The responses against temperature change
Results：Menthol and AITC(wasabi)
40m
V
200ms
Menthol 100μM
0
1
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3
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-5 -4 -3 -2 -1 0 1 2 3 4 5
Ele
ctro
de
nu
mb
er
Time (s)
Before
0
1
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5
6
-5 -4 -3 -2 -1 0 1 2 3 4 5
Ele
ctro
de
nu
mb
er
Time (s)
Before
Menthol AITC（wasabi）
TRPM8 TRPA1
We also confirmed TRPM8 and TRPA1 channel was working electrophysiologically.
Results：Classification by electrophysiological responses
0
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+ + + + + + + + + - - - - - - - - - ± ± ± ± ± ± ± ± ±
+ + + - - - ± ± ± + + + - - - ± ± ± + + + - - - ± ± ±
+ - ± + - ± + - ± + - ± + - ± + - ± + - ± + - ± + - ±
Pe
rce
nta
ge o
f n
eu
ron
s (%
)
CapMenAIT
Human iPSC-derived sensory neurons (n=790)
Rat sensory neurons (n=345)
■Percentage of positive responses against capsaicin were high both hiPSC-derived neurons and rat DRG neurons.
Human iPSC-derived sensory neurons were classified into 27 types depending on physiological responses against 3 compounds (Capsaicin, Menthol, and AITC).
■hiPSC-derived sensory neurons have a variety of physiological properties and resemble to rat DRG neurons.
Results：Anti-cancer drug（Oxaliplatin and Vincristine)
Oxaliplatin
0 nM 1000 nM
1 min
1 nM 10 nM 100 nM
Ele
ctro
de
s
1
16
8
AW
SD
R
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No
rma
lized
firing ra
te(%
) Tota
l sp
ike
s v.
s. B
efo
re (%
) n=4
MEA measurement in hiPSC-derived sensory neurons is useful to pain evaluation of anti-cancer drugs
Vincristine
1
16
8
AWSD
REl
ectr
odes
0 nM 10 nM 100 nM 1000 nM
1 min1 min
Results：Pain relief drug
0 µM 300 μM1 μM 30 μM 100 μM 1000 μM
Ele
ctro
de
s
1
16
8
1 min
AW
SD
R
Acetylsalicyclic Acid (Aspirin)
We also confirmed no responses to pain relief asprinin cultured hiPSC-derived sensory neurons.
Summary ②
MEA measurement in cultured hiPSC derived sensory neurons are suitable to toxicological assay for pain in peripheral nervous system.
We detected the physiological responses to temperature change, capsaicin, menthol,and AITC.
■ Humen iPSC-derived sensory neurons (Axol Bioscience) show the expression of typical sensory neural marker Nav1.7, TRPV1, and TRPA1.
Human iPSC-derived sensory neurons were classified into 27 types depending on physiological responses against 3 compounds.
We detected the responses to anti-cancer drugs oxaliplatin and vincristinein cultured hiPSC-derived sensory neurons. We will investigate the mechanism of actionin the responses at anti-cancer drug administration.
■
■
■
Acknowledgement
■Tohoku Institute of technology Suzuki lab
A. Odawara, N. Matsuda, Y. Ishibashi,R. Yokoi, C. Iemura
■Alpha Med Scientific (Japan).
R. Yamazaki, H. Jiko, M.FukudaM. Trujillo, R.Arant, G. Cheng
■Axol Bioscience (UK).
Zoe Nilsson, Priyanka Dutta, Nick Clare,Sanj Kumar
