use our discoveries to advance yours · 2015. 7. 24. · use our discoveries to advance yours...

Use our discoveries to advance yours

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!