Pluripotent Stem Cell-derived
Cardiomyocytes as Suitable Substrates For
Automated Ion Channel and
Electrophysiological Analysis Systems
Dr. Ralf Kettenhofen Axiogenesis AG 7th Ion Channel Retreat June 29th, Vancouver
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Automated Recording and Pharmacology of Cardiac Ion Currents
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Automated Recording and Pharmacology of Cardiac Ion Currents
Automated Recording and Pharmacology of Cardiac Action Potentials
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Automated Recording and Pharmacology of Cardiac Ion Currents
Automated Recording and Pharmacology of Cardiac Action Potentials
Automated Analyses and Pharmacology of Cardiac Na+/K+-ATPases
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Automated Recording and Pharmacology of Cardiac Ion Currents
Automated Recording and Pharmacology of Cardiac Action Potentials
Automated Analyses and Pharmacology of Cardiac Na+/K+-ATPases
Summary
Content
Introduction
Transgenic Pluripotent Stem Cells
Selection of Cardiomyocytes from Differentiated mouse ES Cells
Automated Recording and Pharmacology of Cardiac Ion Currents
Automated Recording and Pharmacology of Cardiac Action Potentials
Automated Analyses and Pharmacology of Cardiac Na+/K+-ATPases
Summary
Conclusion
The Optimal Cellular Model
Physiological properties Ready-to-use availability
Lot-to-lot reproducibility
The Optimal Cellular Model
Physiological properties Ready-to-use availability
Lot-to-lot reproducibility No inter lab-differences
The Optimal Cellular Model
Physiological properties Ready-to-use availability
Lot-to-lot reproducibility No inter lab-differences
Purity
The Optimal Cellular Model
Physiological properties Ready-to-use availability
Lot-to-lot reproducibility No inter lab-differences
Purity Relevant and predictive
The Optimal Cellular Model
Physiological properties Ready-to-use availability
Lot-to-lot reproducibility No inter lab-differences
Purity Relevant and predictive
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Differentiation of ES cells and selection of cardiomyocytes
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Differentiation of ES cells and selection of cardiomyocytes
9d
Puro 0d
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Differentiation of ES cells and selection of cardiomyocytes
9d 10d 12d
Puro 0d Puro 1d Puro 3d
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Differentiation of ES cells and selection of cardiomyocytes
9d 10d 12d
Puro 0d Puro 1d Puro 3d
Quality control Freezing Dissociation
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Differentiation of ES cells and selection of cardiomyocytes
9d 10d 12d
Puro 0d Puro 1d Puro 3d
Quality control Freezing Dissociation
ES Cell-derived, Genetically Selected and Purified Cardiomyocytes
17d in culture after thawing
Frozen ES cell-derived and purfied cardiomyocytes are viable and retain their autonomous contractile phenotype for at least 3 weeks
after thawing when cultured in monolayer.
Gene Expression Analysis
P =Present A = Absent
Cor.At Cardiomyocytes Cor.At Cardiomyocytes
Gene symbol days in culture after thawing Protein Gene symbol days in culture after thawing Protein
2d 20d 2d 20d
SCN5a P P Nav1.5 ABCC8 P P SUR1
CACNA1c P P Cav1.2 (!1c) Pias3 P P KChAP, PIAS3
CACNA1h P P Cav3.2 (!1h) AKAP6 P P AKAP 6
KCNA1 P P Kv1.1 AKAP9 P P Yotiao
KCNA4 P P Kv1.4 AKAP10 P P D-AKAP2
KCNA5 P A Kv1.5
AKAP12 P P Gravin
KCNA7 P P Kv1.7
AKAP7 P P AKAP 7
KCNB1 P P Kv2.1
Slc8a1 P P NCX1
KCND2 A P Kv4.2
Slc12a2 P P ENCC3, BSC2, NKCC1
KCND3 P P Kv4.3
Slc9a1 P P SLC9A1, APNH, NHE1
KCNG2 P P Kv subfamily G, member 2
ATP1A1 P P Na+/K+-ATPase !1
KCNV2 A P Kv8.2
ATP1A2 P P Na+/K+-ATPase !2
KCNH2 P P erg1 (LQT2), Kv11.1
KvLQT1 (Kv7.1, JLN-1) ATP1A3 P P Na+/K+-ATPase !3
KCNQ1 P P
ATP1B1 P P Na+/K+-ATPase "1
CLCN3 P P ClC-3
ATP1B2 P P Na+/K+-ATPase "2
clcn4-2 P P ClC-4
CLCN6 P P ClC-6 ATP1B3 P P Na+/K+-ATPase "3
CLCN7 P P ClC-7 ATP2A2 P P SERCA2, cardiac muscle, slow twitch 2
CLCA1 P P ClCa-1 RYR2 P P ryanodine receptor , RYR2
KCNJ12 P P Kir2.2 Ank2 P P Akyrin B (LQT-4)
KCNJ3 P P Kir3.1 Gja1 P P Connexin 43
KCNJ5 P P Kir3.4 Gja3 P P Connexin 46
KCNJ6 P P Kir3.2 Gja7 P P Connexin 45
KCNJ11 P P Kir6.2 Slc4a3 P P anion exchanger 3 brain + cardiac isoforms
KCNK6 P P TWIK-2 Vdac2 P P voltage-dependent anion channel 2
HCN1 P P HCN-1 Vdac1 P P voltage-dependent anion channel 1
HCN2 P P HCN-2 Pacsin2 P P Kv Shab-related subfamily, member 1
SCN1b P P SCN1B KCNN1 A P calcium-activated SK1
CACNB2 P P CACNB2 KCNN2 P P calcium-activated SK2
CACNA2d1 P P CACNA2d1 Slc24a3 P P Slc24a (Na/K/Ca exchanger), member 3
KCNE1 P P minK Slc24a6 P P Slc24a (Na/K/Ca exchanger), member 6
KCNIP2 A P KChIP2 Slc12a9 P P Slc12a (Na/K/Ca exchanger), member 9
Manual Voltage Clamp of Cardiac Ion Currents
INa
I/V Diagram
(Normalized to Maximum Amplitude)
0
-70 -50 -30 -10 10 30 50 70
-0,2
Normalized current
-0,4
-0,6
-0,8
-1
-1,2
voltage [mV]
Manual Voltage Clamp of Cardiac Ion Currents
INa ICa
I/V Diagram I/V Diagram
(Normalized to Maximum Amplitude) (Normalized to Maximum Amplitude)
0 0
-70 -50 -30 -10 10 30 50 70 -70 -50 -30 -10 10 30 50 70
-0,2 -0,2
Normalized current
Normalized current
-0,4 -0,4
-0,6
-0,6
-0,8
-0,8
-1
-1
-1,2
-1,2
voltage [mV] voltage [mV]
Manual Voltage Clamp of Cardiac Ion Currents
INa ICa IK
I/V Diagram I/V Diagram I/V Diagram
(Normalized to Maximum Amplitude) (Normalized to Maximum Amplitude) (Normalized to Maximum Amplitude)
1,2
0 0
-70 -50 -30 -10 10 30 50 70 -70 -50 -30 -10 10 30 50 70
1
Normalized current
-0,2 -0,2
Normalized current
Normalized current
0,8
-0,4 -0,4
0,6
-0,6
-0,6
0,4
-0,8
-0,8
0,2
-1
-1
0
-1,2
-1,2 -60 -40 -20 0 20 40 60 80
voltage [mV] voltage [mV] voltage [mV]
Manual vs. Automated Patch - Chip replaces Pipette
Pipette: “Aperture towards the
2 µm
Manual vs. Automated Patch - Chip replaces Pipette
Pipette: “Aperture towards the Chip: “Cell towards the
2 µm
20 µm
Prerequisites for Automated Patch Clamp
Homogeneous cell population
No contamination with other cell types (e.g. fibroblasts)
Prerequisites for Automated Patch Clamp
Homogeneous cell population
No contamination with other cell types (e.g. fibroblasts)
Round morphology
Prerequisites for Automated Patch Clamp
Homogeneous cell population
No contamination with other cell types (e.g. fibroblasts)
Round morphology
Relatively high amount of cells.
Statistics
Subject Number of cells Result
Viability after harvest procedure (%) 87± 3
Size of cell (pF) 112 17 ± 7
Cells with recordable INa amplitude (%) 15 of 22 68
Current density INa (pA/pF) 52 104 ± 129
Peak INa (pA) (at -30 mV) 52 1842 ± 2521
Cells with recordable ICa amplitude (%) 12 of 22 55
Current density ICa (pA/pF) 32 2 ± 1.5
Avergage of 4 QPlates 16
Peak ICa (nA) (at +10 mV) 32 0.035 ± 0.027
Automated Current Clamp Recording
Port-a-Patch®
Stimulation Protocol (500 ms stimuli at 0.2 Hz)
-40 pA
I(memb)
150 ms
20 mV
Automated Current Clamp Recording
Port-a-Patch® Patchliner ®
Stimulation Protocol (500 ms stimuli at 0.2 Hz) Screen Shot from a
4 channel Patchliner
-40 pA
I(memb)
150 ms
20 mV
Preliminary Statistical Data
Subject Result
Patched Cells in 2.5 days 45
Success rate for gigaseal and whole cell (%) about 80
Cells with recordable action potentials amplitude (%) 50
Cells with recordable INa amplitude (%) 50
Cells with recordable ICa amplitude (%) 100
Cells with recordable IK amplitude (%) 60
Why is it possible to identify
hERG blocker effects in
mouse ES cell-derived cardiomyocytes?
Developmental Changes of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
Developmental Changes of Mouse Cardiac Repolarization
Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization
Wang et al. Developmental changes in the delayed rectifier K+ channels in mouse heart. Circ Res. 1996 Jul;79(1):79-85.
+ +
Cardiac Na /K -ATPases as Important
Drug Targets
+ +
Gene Expression of Na /K ATPase Subunits
Cor.At Cardiomyocytes
days in culture after
Gene symbol thawing Protein
2d 20d
ATP1A1 P P Na+/K+-ATPase α1
ATP1A2 P P Na+/K+-ATPase α2
ATP1A3 P P Na+/K+-ATPase α3
ATP1A4 A A Na+/K+-ATPase α4
ATP1B1 P P Na+/K+-ATPase β1
ATP1B2 P P Na+/K+-ATPase β2
ATP1B3 P P Na+/K+-ATPase β3
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Port-a-Patch® and Patchliner® from Nanion
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Port-a-Patch® and Patchliner® from Nanion
I(Ca,L) beta-adrenergic stimulation was revealed
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Port-a-Patch® and Patchliner® from Nanion
I(Ca,L) beta-adrenergic stimulation was revealed
For the first time recording of action potentials from primary-like cardiomyocytes were
established in the the Port-a-Patch® and Patchliner® from Nanion.
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Port-a-Patch® and Patchliner® from Nanion
I(Ca,L) beta-adrenergic stimulation was revealed
For the first time recording of action potentials from primary-like cardiomyocytes were
established in the the Port-a-Patch® and Patchliner® from Nanion.
hERG and I(Na) blocker effects on action potentials have been revealed recorded with the
Port-a-Patch and Patchliner.
Summary
Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
three different companies to analyse cardiac ion current:
PatchXpress® 7000A from MDS-AT
QPatch® from Sophion
Port-a-Patch® and Patchliner® from Nanion
I(Ca,L) beta-adrenergic stimulation was revealed
For the first time recording of action potentials from primary-like cardiomyocytes were
established in the the Port-a-Patch® and Patchliner® from Nanion.
hERG and I(Na) blocker effects on action potentials have been revealed recorded with the
Port-a-Patch and Patchliner.
Cor.At® cells have been validated to be suitable for HTS applications for the analyses of
Na+/K+-ATPase activity and pharmacology in the ICR 8000 from Aurora.
Conclusion
®
Cor.At cells are primary-like cardiomyocytes predictive
and relevant for pharmacological studies.
Conclusion
®
Cor.At cells are primary-like cardiomyocytes predictive
and relevant for pharmacological studies.
®
Cor.AT cells are suitable for automated electrophysiology
and are validated on leading systems
Conclusion
®
Cor.At cells are primary-like cardiomyocytes predictive
and relevant for pharmacological studies.
®
Cor.AT cells are suitable for automated electrophysiology
and are validated on leading systems
®
Cor.At cells capable for scalable HTS and HCS applications
Conclusion
®
Cor.At cells are primary-like cardiomyocytes predictive
and relevant for pharmacological studies.
®
Cor.AT cells are suitable for automated electrophysiology
and are validated on leading systems
®
Cor.At cells capable for scalable HTS and HCS applications
One relevant cell for all information
Conclusion
®
Cor.At cells are primary-like cardiomyocytes predictive
and relevant for pharmacological studies.
®
Cor.AT cells are suitable for automated electrophysiology
and are validated on leading systems
®
Cor.At cells capable for scalable HTS and HCS applications
One relevant cell for all information
®
Cor.At cardiomyocytes are available now.
Acknowledgment
MDS Analytical Technologies:
Dr. Xin Jiang
Dr. Jan Dolzer
Dr. James Costantin
Dr. David Yamane
Sophion SA
Dr. Rikke Schrøder-Perrier
Dr. Morten Sunesen
Nanion Technologies:
Dr. Sonja Stölzle
Dr. Niels Fertig
Dr. Cecilia Farre
Dr. Claudia Haarmann
Acknowledgment
MDS Analytical Technologies: Aurora Biomed:
Dr. Xin Jiang Dr. Sikander Gill
Dr. Jan Dolzer Sophia Liang
Dr. James Costantin Saranna Brugger
Dr. David Yamane
Sophion SA
Dr. Rikke Schrøder-Perrier
Dr. Morten Sunesen
Nanion Technologies:
Dr. Sonja Stölzle
Dr. Niels Fertig
Dr. Cecilia Farre
Dr. Claudia Haarmann
Acknowledgment
MDS Analytical Technologies: Aurora Biomed:
Dr. Xin Jiang Dr. Sikander Gill
Dr. Jan Dolzer Sophia Liang
Dr. James Costantin Saranna Brugger
Dr. David Yamane
Sophion SA Institute for Neurophysiology,
Dr. Rikke Schrøder-Perrier University of Colone:
Dr. Morten Sunesen Alexey Kuzmenkin
Huamin Liang
Prof. Jürgen Hescheler
Nanion Technologies:
Dr. Sonja Stölzle
Dr. Niels Fertig
Dr. Cecilia Farre
Dr. Claudia Haarmann
Acknowledgment
MDS Analytical Technologies: Aurora Biomed:
Dr. Xin Jiang Dr. Sikander Gill
Dr. Jan Dolzer Sophia Liang
Dr. James Costantin Saranna Brugger
Dr. David Yamane
Sophion SA Institute for Neurophysiology,
Dr. Rikke Schrøder-Perrier University of Colone:
Dr. Morten Sunesen Alexey Kuzmenkin
Huamin Liang
Prof. Jürgen Hescheler
Axiogenesis AG:
Nanion Technologies:
Dr. Heribert Bohlen
Dr. Sonja Stölzle
Dr. Eugen Kolossov
Dr. Niels Fertig
Dr. Silke Schwengberg
Dr. Cecilia Farre
Dr. Andreas Ehlich
Dr. Claudia Haarmann
Josef Tenelsen
Peter Metzger
Acknowledgment
North America: Special thanks to:
Dr. Eric Atkinson
Lynn MacIntyre
www.reachbio.com
Japan: Dr. Junya Koda
Dr. Chie Kodama
www.veritastk.com