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iPSC-derived cardiomyocytes and neurons: Production, electrophysiological characterization
Hamamatsu Workshop, 10.06.2015, Cologne Dr. Gesa Rascher-Eggstein, Axiogenesis AG
Axiogenesis AG Company Background
Axiogenesis core cell production technology: Selection and Purification
MLC2 promotor CorV.4U
Available
to purchase
Available for
Co-development
Ventricular cells
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Cor.4U@ Production
Axiogenesis core cell production technology: Selection and Purification
Selection of 100% pure human Cardiomyocytes
Excitation-Contraction Coupling
Time Am
plitu
de
Currents
Action Potential
Na+
Ca2+ L-type
Ca2+ T-type
Na+/Ca2+-exchanger
K+ Ito
K+ IKs
K+ IKr
K+ IKur
Calcium is the messenger that integrates the electrochemcial signals of the action potential with the molecular signaling pathways that regulate contraction
Many Channel Types contribute to the Action Potential
Action Potential Assays - Cardiomyocytes
Action Potential Recording:
Manual Patch Clamp – Cor.4U@ Cardiomyocytes
Ventricular-like cell action potential stability over 30 minutes
Action Potential Recording:
Manual Patch Clamp – Cor.4U@ Cardiomyocytes
ATX II Ranolazine Terfenadine
E4031 Dofetilide
Parameters measured
The fAPD mirrors effects on re-polarization of
cardiomyocytes and is measured as the duration from
10% of the fAP minimum (fAPmin) to the fAP maximum
(fAPmax). The fAPDc is calculated from the fAPD and
corrected for frequency according to Fridericia 1920*.
Microelectrode-Array (MEA)
Principle of the Micro-Electrode Array (MEA)
Current clamp
recording
phase 1
Na+ ↓
phase 0
K+ ↓
Na+↑
phase 2
Ca2+↑
phase 3
Ca2+ ↓
K+ ↑
MEA
recording phase 1
phase 0 phase 2
phase 3
Correlation with Action Potential
fAPmin: field potential minimum
fAPmax: field potential maximum
fAPmax
10% fAPmin
fAPmin
fAPD
*L. S. Fridericia: Die Systolendauer im
Elektrokardiogramm bei normalen
Menschen und bei Herzkranken. Acta
Med Scand 1920; 53:469-86 Cardiomyocytes on MEA
Effect of the hERG Blocker E-4031 on Cor.4U® Cardiomyocytes
Induction of EAD-like events at 250 nM
10 sec
Effect on fAP
Demonstration of Major Mechanisms of Cardiac
Electrophysiology in Cor.4U® Cardiomyocytes
Compound class Drug (application)
Effect conc. (µM)
hSC-CM
IKr blocker E-4031
(Class III antiarhythmic)
10 nM; fAPD+
250 nM; IB
Cisapride
(Prokinetic)
100 nM; fAPD+
1 µM; IB
Dofetilide
(Class III antiarhythmic)
10 nM, fAPD+
INa blocker Tetrodotoxin (TTX)
(Neurotoxin)
1 - 10 µM; NC and SD+
Lidocaine
(local anaesthetic
100 µM; SD+, NC
1000 µM; BA
INa activator Aconitine 100nM ; PC
ICa,L blocker Nifedipine
(Anti-hypertensive)
100 nM; fAPD-
1000 nM; BA
Verapamil
(Anti-hypertensive)
0.1 µM fAPD-
1- 10 µM BA
ß-adrenergic receptor agonist Isoproterenol
(Bronchodilator)
10 nM; PC
-adrenergic
receptor agonist
Phenylephrine
(Vasoconstrictor)
100 nM; PC
Ryanodine receptor agonist Ryanodine 10 nM; NC, BA
Abbreviations:
NC - negative chronotropic
PC - positive chronortropic
fAPD+ - fAPD prolongation
fAPD- -fAPD shortening
IB - irregular beating
BA - beating arrest
SD+ - slope duration prolongation
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Dopa.4U
Dopaminergic Neurons
Derivation of Dopaminergic Neurons from hiPSCs
Directed differentiation of human iPS-derived cells
into dopaminergic
neurons results in approx.
60% - 70% tyrosine
hydroxylase positive neurons.
- Tyrosine hydroxylase (the
enzyme is responsible for catalyzing the
conversion of the amino acid L-
tyrosine to L-DOPA, which is a precursor
for dopamine)
Beta-3-tubulin (neuron-specific microtubuli)
Tyrosine hydroxylase (dopaminergic neuron-specific protein)
MAP2 (microtubule-associated protein 2,
dendrite-specific protein)
Tyrosine hydroxylase
Beta-3-tubulin
Synaptophysin (presynaptic protein)
Synaptophysin
MAP2
-> Dopa.4U express typical neuronal markers
Patch Clamp of hiPS-derived Dopamineric Neurons
Manual current clamp recording of spontaneous action potentials
Manual voltage clamp recording of sodium/ potassium currents
Data was kindly provided by PoreGenic GmbH, Rostock, Germany
Patch Clamp Measurements of Long-term Dopa.4U Cultures
-> Dopa.4U cells maintain sodium & potassium currents during long-term culture
Dopa.4U were seeded (30.000 cells/ cm2) onto cover slips and measured after 11 to 49 days in culture. The cells showed an adequate and comparable high quality, well suited for patch clamp measurements.
Dopa.4U:
15 days in culture
Dopa.4U:
49 days in culture
Dopa.4U: K & Na currents
Patch Clamp of Dopa.4U: Measurement of inhibitory postsynaptic potentials
Data was kindly provided by Anaxon
AG, Berne, Switzerland
Dopa.4U: GABAA receptor mIPSCs
Data was kindly provided by Poregenic
Rostock, Germany
Dopa.4U: Postsynaptic potentials
MEA Recordings of Dopa.4U Cultures
-> One vial Dopa.4U cells (2 million cells) is sufficient
for 12 wells on the Axion12 MEA system
Dopa.4U were seeded onto
the MEA chips of the Axion12 Multi Electrode Array device “Maestro” in
coculture with primary
astrocytes.
MEA Recordings of Dopa.4U Cultures
-> Dopa.4U: All 12 wells are active after 7 days in culture
Up to 50 of 64 electrodes active per well allow sophisticated NeuroProof data analysis (200 parameters describing general activity,
burst structure, regularity, synchrony)
Data was kindly provided by NeuroProof
GmbH, Rostock, Germany
Comparison of the Spontaneous Activity Patterns:
Dopa.4U vs. Primary Brain Region Specific Tissue Cultures
-> Dopa.4U show similar activity patterns compared to
primary neuronal Midbrain networks at 28 div
Data was kindly
provided by NeuroProof
GmbH, Rostock, Germany
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Peri.4U
Peripheral Neurons
Peri.4U: Axiogenesis human iPSC-derived Peripheral Neurons
Immunohistochemical Staining of Peri.4U
anti-Peripherin (peripheral neuron-specific intermediate
filament)
anti-beta-3-tubulin (neuron-specific microtubili)
anti-MAP-2 (neuron-specific
microtubule-associated protein 2)
anti-VGLUT2 (Vesicular glutamate transporter 2)
corresponding
phase contrast
images (10x)
-> Peri.4U express typical marker for peripheral neurons
23
Manual Patch Clamp of Peri.4U
Buffer composition:
EC (mM): NaCl (140), KCl (4),
HEPES (10), MgCl2 (1), CaCl2 (1.8), glucose (10); pH adjusted to 7.4, Osmolality: 303
mOsmol/L;
IC (mM): K-Gluconate (117), HEPES (10), NaGTP
(0.4), EGTA (5), Na2ATP (5), MgCl2 (1); pH adjusted to 7.2, Osmolality: 280
mOsmol/L
Data were kindly provided by NMI-TT
GmbH
Peri.4U were measured in current clamp mode (whole cell configuration) Cover slips were coated with Polyethylenimine (PEI) and Laminin
-> Peri.4U cells exhibit action potentials and repetitive
firing after corresponding current impulses
Elicited Action potential Elicited repetitive firing
24
Manual Patch Clamp of Peri.4U
Buffer composition:
EC (mM): NaCl (140), KCl (4),
HEPES (10), MgCl2 (1), CaCl2 (1.8), glucose (10);
pH adjusted to 7.4, Osmolality: 303
mOsmol/L;
IC (mM): K-Gluconate (117), HEPES (10), NaGTP (0.4), EGTA (5), Na2ATP (5), MgCl2
(1); pH adjusted to 7.2, Osmolality: 280
mOsmol/L
Data were kindly provided by NMI-TT GmbH
Peri.4U were measured in current clamp mode (whole cell configuration) Cover slips were coated with Polyethylenimine (PEI) and Laminin
-Elicited action potentials: 100% (n=7); -Repetitive firing: 57% (n=7); -Spontaneous activity: 43% (n=7)
-> Peri.4U cells exhibit spontaneous action
potentials
Spontaneous activity
Calcium-Transient Measurements on Peri.4U Cells
-Peri.4U were thawed and plated at 10,000 cells per well on a 384-well plate.
-Cells were cultured for 4 days. -Calcium responses were analyzed on a Hamamatsu FDSS/μCELL kinetic plate reader using the FLIPR Calcium 5 Assay Kit (Molecular Devices).
-Substances were used at 10 µM final concentrations.
-> Peri.4U express typical functional receptors
- Histamine (ligand of H3 Receptor expressed in peripheral neurons)
- Glutamate (ligand for metabotropic and ionotroph glutamate receptors as the NMDA receptor in peripheral neurons)
- Serotonin (ligand for 5-HT3 receptor, specific for peripheral neurons)
- NMDA (ligand for NMDA receptor expressed in peripheral neurons)
- ATP (Ligand for purinergic receptors P2RX4 and P2RX7 expressed in peripheral neurons)
- PBS (control)
Patch Clamp of Dopa.4U & Peri.4U Neurons
Data was kindly provided by Anaxon AG, Berne, Switzerland
Dopa.4U
Recombinant a1b2g2 Peri.4U
MEA measurements on Peri.4U cells
-> Peri.4U show drug sensitivity in MEA measurements
Fipronil (1µM)
Fipronil
(10µM)
Control
- Rat cortical neurons
Decreased Neurite Outgrowth with Fipronil:
IC50 ≥ 10µM
- Other Neuron Supplier IC50 ≥ 50µM
- Peri.4U IC50=4.4 ± 1.6µM
Collaborators:
Thank you!