Nanion User Meeting USA · Summary NCX is Present and Functional in hiPSC-CMs, and the SURFE 2 R...
Transcript of Nanion User Meeting USA · Summary NCX is Present and Functional in hiPSC-CMs, and the SURFE 2 R...
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Nanion User Meeting USA
Next Level Functional Readouts on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes
May 28, 2019
T.K. Feaster, PhD
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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
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FUJIFILM Cellular Dynamics iPSC Technology
Applications Support
BasicScience
Healthy donor orPatient-derived
DrugDiscovery
CellTherapy
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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
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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
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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.
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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
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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
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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
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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).
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B
NCX Inhibition Increases Beat Rate of iCell Cardiomyocytes2
SURFE2R
CardioExcyte96
SEA0400 = NCX Blocker
Krisztina Juhasz PhD - Application Scientist
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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
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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
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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
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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
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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
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Control10 µM BayK10 µM Nifedipine
0
–50 mV
100 ms
Control 10 µM BayK 10 µM Nifedipine0
50
100
150
200*
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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
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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.
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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