Nanion User Meeting USA

Next Level Functional Readouts on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

May 28, 2019

T.K. Feaster, PhD

June 26, 2019 2

Outline of Presentation

1. FUJIFILM Cellular Dynamics, Inc. and iPSC Technology

2. Sodium-Calcium Exchanger (NCX) Characterization

3. Late Sodium Current (late INA) Assessment

4. Modulating Inward Rectifier Current (IK1) using Dynamic Clamp

5. Summary

June 26, 2019 3

FUJIFILM Cellular Dynamics iPSC Technology

Applications Support

BasicScience

Healthy donor orPatient-derived

DrugDiscovery

CellTherapy

June 26, 2019 4

BRAIN & NERVOUS SYSTEMiCell Astrocytes

iCell DopaNeurons

iCell GABANeurons

iCell GlutaNeurons

iCell Motor Neurons

iCell Microglia

HEART & VASCULARiCell Cardiomyocytes

iCell Endothelial Cells

iCell Cardiac Progenitor Cells

LIVERiCell Hepatocytes 2.0

iCell Hepatoblasts

OCULARiCell Retinal Pigment Epithelial Cells

iCell Hematopoietic Progenitor Cells

iCell Macrophages

BLOOD & IMMUNE

iCell® Products: Unprecedented Access to Human Cells

June 26, 2019 5

Cardiovascular Product Portfolio

HCM - Hypertrophic Cardiomyopathy

DCM - Dilated Cardiomyopathy

BrS - Brugada Syndrome

CPVT - Catecholaminergic Polymorphic Ventricular Tachycardia

iCell Cardiomyocytes (30 Healthy Donors)

iCell Cardiac Progenitor Cells

June 26, 2019 6

iCell CardiomyocytesExcitation-Contraction Coupling

Electrical Activity Calcium Handling Mechanical Beating

iCell Cardiomyocytes are spontaneously beating cells that are ideal for interrogation of excitation-contraction coupling.

SURFE2R N11x sensor

SURFE2R 96SE96 well parallel

Electrophysiological method able to resolve the low amplitude currents generated by electrogenic transporter proteins. The general concept:

A 3mm sensor is coated with a sample (membrane, liposomes, cells).

The SURFE2R Instruments for Transporter Research

Accumulation of the signal from the full sensor area allows measurement of very low current amplitude.

The protein on the sensor is activated by fast application of substrate containing buffers, leading to changes in the membrane potential.

Instrumentation

SURFE2R

The Sodium-calcium-exchanger NCX1 Function: Extrusion of calcium in exchange for sodium Expression: Cardiac and skeletal muscle, brain, liver and

other organs, slice variants Physiology: Calcium homeostasis and regulation of

excitability

NCX: A Cardiac Transporter Involved in AP

Electrophysiology Stoichiometry of 3:1 leads to a net ion current Common approach: Patch clamp (~10-100 pA amplitude,

inhibition of several channels necessary)

Pharmacological Interest Inhibition of reverse mode might reduce infarct area

(import of calcium in pathological situations). Potential antiarrhythmic effect is under investigation. Safety aspect: Inhibition in a healthy individual might cause

side effects and vulnerability.

Modified from: Knollmann B., Ph.D. Dissertation. 1999

SURFE2R

After a rinse with a sodium containing buffer cardiac NCX was activated with 300 µM calcium. Calcium affinity of NCX1 recorded from HEK cells was comparable with iCell Cardiomyocytes2.

A

B

C

Functional Evidence of NCX in iCell Cardiomyocytes2

CMs were detached with trypsin and adhere to a sensor via a lipid layer.

NCX1 in HEK cells and iCell Cardiomyocytes2cells was blocked by KB-R7943 and the specific inhibitor SEA0400 with similar IC50 values in good agreement with the literature.

SURF2R

SURFE2R

SEA0400 = NCX BlockerKB-R7943 = NCX Blocker

Maria Barthmes PhD - Product Manager & Application Scientist

iCell Cardiomyocytes2 were seeded on the CardioExcyte96 well plate and the beat rate was determined via impedance recordings. Addition of SEA0400 significantly increased the beat rate in a dose dependent manner. A: Individual traces comparing the development of the beat rate in one well shortly before and 4h after addition of SEA0400. B: Time-dependent, long-term effect of SEA0400 on the beat rate of iCell Cardiomyocytes2 (average of all wells, SD).

A

B

NCX Inhibition Increases Beat Rate of iCell Cardiomyocytes2

SURFE2R

CardioExcyte96

SEA0400 = NCX Blocker

Krisztina Juhasz PhD - Application Scientist

The Patchliner enables Straightforward Cardiac Safety Research

Patchliner Safety Edition Ready to use safety pharmacology protocols and analysis

routines

Pre-loaded routines and SOPs for hERG, Na 1.5-peak, Na 1.5-late and Cav1.2

Installation and training focused on safety pharmacology experiments

Temperature control for measurements at physiological temperature

Patchliner Cooling Plate for viability of cells and stability of compounds

Dynamic clamp for action potential recordings of hiPSC-derived cardiomyocytes

Exceptional support from Nanion CiPA experts

Patchliner

Late Sodium Current (Nav) in iCell Cardiomyocytes2 – CiPA Protocol

n=6

Patchliner

Robust APC system enabling Nav late voltage clamp recording in hiPSC-CMs

ATXII = Sodium Channel ActivatorRanolazine = Sodium Channel Blocker

András Horváth PhD - Application Scientist

Combining Dynamic Clamp with Automated Patch Clamp (APC)

Block diagram showing connections between Patchliner, patch clamp amplifier and dynamic clamp system

Patchliner

Goversen et al. 2018 Frontiers in Physiology

Successful Voltage and Current Clamp Recordings on iCell Cardiomyocytes2

Kv Voltage Clamp8 parallel Action Potential

Recordings on 8-Channel Patchliner Nav 1.5 Voltage Clamp

András Horváth PhD - Application Scientist

Patchliner

Generate Full Concentration-Response Curves on iCell Cardiomyocytes2Nav1.5 Pharmacology

Patchliner

Workflow routine and stability of AP recordings enable full concentration response measurements on a single hiPSC-CMs

TTX = Sodium Channel Blocker

Control10 µM BayK10 µM Nifedipine

0

–50 mV

100 ms

Control 10 µM BayK 10 µM Nifedipine0

50

100

150

200*

*

APD

90(m

s)

n=11

0

–50 mV

100 ms

0.0

0.5

1.0

-9 -8 -7 -6CTL

n=13EC50=233 nM

log [BayK] (M)

Act

ivat

ion

%

Generate Full Concentration-Response Curves on iCell Cardiomyocytes2Cav1.2 Pharmacology

Patchliner

Workflow routine and stability of AP recordings enable full concentration response measurements on a single hiPSC-CMs

BayK = Calcium Channel ActivatorNifedipine = Calcium Channel Blocker

András Horváth PhD - Application Scientist

June 26, 2019 17

Summary

NCX is Present and Functional in hiPSC-CMs, and the SURFE2R Represents a High-Throughput Method for Investigation.

Late Sodium Current can be Measured and Modulated using Patchliner (APC) system in hiPSC-CMs.

Patchliner and Dynamic Clamp Enable Introduction of IK1 and robust APC measurements in hiPSC-CMs.

18June 26, 2019

Thank you

Slide Number 1Outline of PresentationFUJIFILM Cellular Dynamics iPSC TechnologyiCell® Products: Unprecedented Access to Human CellsCardiovascular Product PortfolioiCell Cardiomyocytes�Excitation-Contraction CouplingSlide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16SummarySlide Number 18