Pluripotent Stem Cell-derived
               Cardiomyocytes as Suitable Substrates For
                               Auto...
Content
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 Cell...
Content

 Introduction
  Transgenic Pluripotent Stem Cells
  Selection of Cardiomyocytes from Differentiated mouse ES Cell...
Content

 Introduction
  Transgenic Pluripotent Stem Cells
  Selection of Cardiomyocytes from Differentiated mouse ES Cell...
Content

 Introduction
  Transgenic Pluripotent Stem Cells
  Selection of Cardiomyocytes from Differentiated mouse ES Cell...
Content

 Introduction
  Transgenic Pluripotent Stem Cells
  Selection of Cardiomyocytes from Differentiated mouse ES Cell...
The Optimal Cellular Model
The Optimal Cellular Model


  Physiological properties
The Optimal Cellular Model


  Physiological properties   Ready-to-use availability
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   N...
The Optimal Cellular Model


  Physiological properties     Ready-to-use availability




  Lot-to-lot reproducibility   N...
The Optimal Cellular Model


  Physiological properties     Ready-to-use availability




  Lot-to-lot reproducibility   N...
The Optimal Cellular Model


  Physiological properties     Ready-to-use availability




  Lot-to-lot reproducibility   N...
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells




        Differentiation of ES c...
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells




         Differentiation of ES ...
Puromycin Selection of Cardiomyocytes from Genetically
Engineered Embryonic Stem Cells




         Differentiation of ES ...
Puromycin Selection of Cardiomyocytes from Genetically
   Engineered Embryonic Stem Cells




            Differentiation ...
Puromycin Selection of Cardiomyocytes from Genetically
   Engineered Embryonic Stem Cells




            Differentiation ...
Specificity and In Vivo Relevance?
Specificity and In Vivo Relevance?




Fleischmann M. et al. FEBS Lett.
    1998 Dec 4;440(3):370-6
Specificity and In Vivo Relevance?




                                       Kolossov E. J Exp Med. 2006 Oct
            ...
ES Cell-derived, Genetically Selected and Purified Cardiomyocytes

                           17d in culture after thawing
ES Cell-derived, Genetically Selected and Purified Cardiomyocytes

                                             17d in cul...
Gene Expression Analysis
               P =Present      A = Absent


               Cor.At Cardiomyocytes                 ...
Manual Voltage Clamp of Cardiac Ion Currents
Manual Voltage Clamp of Cardiac Ion Currents
                                               INa




                      ...
Manual Voltage Clamp of Cardiac Ion Currents
                                               INa                           ...
Manual Voltage Clamp of Cardiac Ion Currents
                                               INa                           ...
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




    ...
Prerequisites for Automated Patch Clamp
Prerequisites for Automated Patch Clamp


  Homogeneous cell population
Prerequisites for Automated Patch Clamp


  Homogeneous cell population

  No contamination with other cell types (e.g. fi...
Prerequisites for Automated Patch Clamp


  Homogeneous cell population

  No contamination with other cell types (e.g. fi...
Prerequisites for Automated Patch Clamp


  Homogeneous cell population

  No contamination with other cell types (e.g. fi...
®
Cor.At Cardiomyocytes
               and

 PatchXpress 7000A   ®

   MDS - Analytical Technologies
®
Cor.At Cardiomyocytes
                and

 PatchXpress 7000A           ®

   MDS - Analytical Technologies



         ...
®
PatchXpress 7000A - Cardiac Ion Currents
®
PatchXpress 7000A - Cardiac Ion Currents
     INa




                  1 nA


           2 ms




 I/V Diagram
®
PatchXpress 7000A - Cardiac Ion Currents
     INa                     ICa




                  1 nA                100 ...
®
PatchXpress 7000A - Cardiac Ion Currents
     INa                     ICa                   IK




                  1 n...
Statistics



                 Subject                Number of cells   Result ± SEM

   High resistance seals (> 1GΩ), (%...
®
PatchXpress 7000A: Potassium Current Pharmacology

                  4-Aminopyridine

      Control                     ...
®
PatchXpress 7000A: Potassium Current Pharmacology

                  4-Aminopyridine

      Control                     ...
®
PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L)
®
PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L)
                 Epinephrine (Adrenaline)
®
PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L)
                      Epinephrine (Adrenaline)


               Ep...
®
PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L)
                      Epinephrine (Adrenaline)


               Ep...
®
Cor.At Cardiomyocytes
             and

         QPatch     ®

    Sophion Bioscience A/S
®
Cor.At Cardiomyocytes
               and

         QPatch               ®

    Sophion Bioscience A/S


         Dr. Rik...
®
QPatch - Ion Currents
®
QPatch - Ion Currents
      INa




    I/V Diagram
®
QPatch - Ion Currents
      INa                  ICa




    I/V Diagram         I/V Diagram
®
QPatch - Ion Currents
      INa                  ICa            IK




    I/V Diagram         I/V Diagram   I/V Diagram
Statistics
Statistics




Avergage of 4 QPlates 16
Statistics


                                          Subject                    Number of cells     Result
             ...
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX




                                       A)...
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX




                                       B)...
®
QPatch - Sodium Current Pharmacology

                  INa block with TTX




                                       C)...
®
    QPatch - ß-adrenergic modulation of I(Ca,L)



Voltage protocol
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                     Isoproterenol
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                     Isoproterenol
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                     Isoproterenol




 1) baseline
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                     Isoproterenol




 1) baseline




 2) 1 µM Iso
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                     Isoproterenol




 1) baseline




 2) 1 µM Iso




 3)...
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                       Isoproterenol



 4) 10 µM Nifedipine




 1) baselin...
®
QPatch - ß-adrenergic modulation of I(Ca,L)
                       Isoproterenol



 4) 10 µM Nifedipine




 1) baselin...
®
  Cor.At Cardiomyocytes
                 and
             ®
Port-a-Patch and Patchliner   ®

        Nanion Technologies
®
  Cor.At Cardiomyocytes
                  and
              ®
Port-a-Patch and Patchliner      ®

        Nanion Technol...
Voltage Clamp
Voltage Clamp


       INa




   I/V Diagram
Voltage Clamp


       INa           ICa




   I/V Diagram   I/V Diagram
Voltage Clamp


       INa           ICa           IK




   I/V Diagram   I/V Diagram   I/V Diagram
Automated Current Clamp Recording
Automated Current Clamp Recording


                     Port-a-Patch®

   Stimulation Protocol (500 ms stimuli at 0.2 Hz)...
Automated Current Clamp Recording


                     Port-a-Patch®                             Patchliner      ®


   ...
Preliminary Statistical Data



                          Subject                           Result

    Patched Cells in 2...
®
Port-a-Patch - Sodium Channel Pharmacology
               Tetrodotoxin (TTX)
®
Port-a-Patch - Sodium Channel Pharmacology
                   Tetrodotoxin (TTX)


       control


           TTX 20 µM...
®
Patchliner - Sodium Channel Pharmacology
®
Patchliner - Sodium Channel Pharmacology




                           min
®
Patchliner - Sodium Channel Pharmacology




                           min
®
Port-a-Port - Identification of hERG blocker
®
Port-a-Port - Identification of hERG blocker

                      Dofetilide
®
Port-a-Port - Identification of hERG blocker

                               Dofetilide


               control



    ...
®
Patchliner - Potassium Channel Pharmacology
®
Patchliner - Potassium Channel Pharmacology
                    Quinidine
®
Patchliner - Potassium Channel Pharmacology
                    Quinidine




                          min



         ...
®
Patchliner - Potassium Channel Pharmacology
                    Quinidine




                          min



         ...
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+ ch...
Developmental Changes of Mouse Cardiac Repolarization




 Wang et al. Developmental changes in the delayed rectifier K+ ch...
Developmental Changes of Mouse Cardiac Repolarization




 Wang et al. Developmental changes in the delayed rectifier K+ ch...
Developmental Changes of Mouse Cardiac Repolarization




 Wang et al. Developmental changes in the delayed rectifier K+ ch...
Developmental Changes of Mouse Cardiac Repolarization




 Change of the Dofetilide Sensitivity of Mouse Cardiac Repolariz...
Developmental Changes of Mouse Cardiac Repolarization




 Change of the Dofetilide Sensitivity of Mouse Cardiac Repolariz...
Developmental Changes of Mouse Cardiac Repolarization




 Change of the Dofetilide Sensitivity of Mouse Cardiac Repolariz...
Developmental Changes of Mouse Cardiac Repolarization




 Change of the Dofetilide Sensitivity of Mouse Cardiac Repolariz...
Developmental Changes of Mouse Cardiac Repolarization




 Change of the Dofetilide Sensitivity of Mouse Cardiac Repolariz...
+    +
Cardiac Na /K -ATPases as Important
             Drug Targets
+   +
Gene Expression of Na /K ATPase Subunits
+   +
Gene Expression of Na /K ATPase Subunits



                   Cor.At Cardiomyocytes
                     days in cu...
+    +
Na /K ATPase Subunits
Homology between Mouse and Human Amino Acid Sequences

      Na+/K+ ATPase catalytic alpha su...
®
Cor.At Cardiomyocytes
 and Pharmacological Studies with the


         ICR 8000           ®

            Aurora Biomed
®
Cor.At Cardiomyocytes
 and Pharmacological Studies with the


         ICR 8000                 ®

            Aurora Bi...
+
ICR 8000 - Rb Uptake Assay
+
ICR 8000 - Rb Uptake Assay

           Monitoring of cardiac Na+/K+ ATPases
+
ICR 8000 - Rb Uptake Assay

            Monitoring of cardiac Na+/K+ ATPases

      Activity
+
ICR 8000 - Rb Uptake Assay

            Monitoring of cardiac Na+/K+ ATPases

      Activity                            ...
Summary
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Summary

 Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from
 three different compani...
Conclusion
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.

...
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.

...
Conclusion

       ®
 Cor.At cells are primary-like cardiomyocytes predictive
 and relevant for pharmacological studies.

...
Acknowledgment
Acknowledgment

MDS Analytical Technologies:
 Dr. Xin Jiang
 Dr. Jan Dolzer
 Dr. James Costantin
 Dr. David Yamane
Acknowledgment

MDS Analytical Technologies:
 Dr. Xin Jiang
 Dr. Jan Dolzer
 Dr. James Costantin
 Dr. David Yamane



Soph...
Acknowledgment

MDS Analytical Technologies:
 Dr. Xin Jiang
 Dr. Jan Dolzer
 Dr. James Costantin
 Dr. David Yamane



Soph...
Acknowledgment

MDS Analytical Technologies:   Aurora Biomed:
 Dr. Xin Jiang                  Dr. Sikander Gill
 Dr. Jan D...
Acknowledgment

MDS Analytical Technologies:   Aurora Biomed:
 Dr. Xin Jiang                  Dr. Sikander Gill
 Dr. Jan D...
Acknowledgment

MDS Analytical Technologies:   Aurora Biomed:
 Dr. Xin Jiang                  Dr. Sikander Gill
 Dr. Jan D...
Acknowledgment
Acknowledgment


 North America:




  www.reachbio.com
Acknowledgment


 North America:      Special thanks to:


                      Dr. Eric Atkinson
                      L...
Acknowledgment


 North America:      Special thanks to:


                      Dr. Eric Atkinson
                      L...
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090630 Ion Cannel Retreat

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  • 090630 Ion Cannel Retreat

    1. 1. 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
    2. 2. Content
    3. 3. Content Introduction Transgenic Pluripotent Stem Cells Selection of Cardiomyocytes from Differentiated mouse ES Cells
    4. 4. Content Introduction Transgenic Pluripotent Stem Cells Selection of Cardiomyocytes from Differentiated mouse ES Cells Automated Recording and Pharmacology of Cardiac Ion Currents
    5. 5. 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
    6. 6. 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
    7. 7. 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
    8. 8. 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
    9. 9. The Optimal Cellular Model
    10. 10. The Optimal Cellular Model Physiological properties
    11. 11. The Optimal Cellular Model Physiological properties Ready-to-use availability
    12. 12. The Optimal Cellular Model Physiological properties Ready-to-use availability Lot-to-lot reproducibility
    13. 13. The Optimal Cellular Model Physiological properties Ready-to-use availability Lot-to-lot reproducibility No inter lab-differences
    14. 14. The Optimal Cellular Model Physiological properties Ready-to-use availability Lot-to-lot reproducibility No inter lab-differences Purity
    15. 15. The Optimal Cellular Model Physiological properties Ready-to-use availability Lot-to-lot reproducibility No inter lab-differences Purity Relevant and predictive
    16. 16. The Optimal Cellular Model Physiological properties Ready-to-use availability Lot-to-lot reproducibility No inter lab-differences Purity Relevant and predictive
    17. 17. Puromycin Selection of Cardiomyocytes from Genetically Engineered Embryonic Stem Cells
    18. 18. Puromycin Selection of Cardiomyocytes from Genetically Engineered Embryonic Stem Cells
    19. 19. Puromycin Selection of Cardiomyocytes from Genetically Engineered Embryonic Stem Cells Differentiation of ES cells and selection of cardiomyocytes
    20. 20. Puromycin Selection of Cardiomyocytes from Genetically Engineered Embryonic Stem Cells Differentiation of ES cells and selection of cardiomyocytes 9d Puro 0d
    21. 21. 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
    22. 22. 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
    23. 23. 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
    24. 24. Specificity and In Vivo Relevance?
    25. 25. Specificity and In Vivo Relevance? Fleischmann M. et al. FEBS Lett. 1998 Dec 4;440(3):370-6
    26. 26. Specificity and In Vivo Relevance? Kolossov E. J Exp Med. 2006 Oct 2;203(10):2315-27. Fleischmann M. et al. FEBS Lett. 1998 Dec 4;440(3):370-6
    27. 27. ES Cell-derived, Genetically Selected and Purified Cardiomyocytes 17d in culture after thawing
    28. 28. 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.
    29. 29. 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
    30. 30. Manual Voltage Clamp of Cardiac Ion Currents
    31. 31. 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]
    32. 32. 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]
    33. 33. 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]
    34. 34. Manual vs. Automated Patch - Chip replaces Pipette Pipette: “Aperture towards the 2 µm
    35. 35. Manual vs. Automated Patch - Chip replaces Pipette Pipette: “Aperture towards the Chip: “Cell towards the 2 µm 20 µm
    36. 36. Prerequisites for Automated Patch Clamp
    37. 37. Prerequisites for Automated Patch Clamp Homogeneous cell population
    38. 38. Prerequisites for Automated Patch Clamp Homogeneous cell population No contamination with other cell types (e.g. fibroblasts)
    39. 39. Prerequisites for Automated Patch Clamp Homogeneous cell population No contamination with other cell types (e.g. fibroblasts) Round morphology
    40. 40. Prerequisites for Automated Patch Clamp Homogeneous cell population No contamination with other cell types (e.g. fibroblasts) Round morphology Relatively high amount of cells.
    41. 41. ® Cor.At Cardiomyocytes and PatchXpress 7000A ® MDS - Analytical Technologies
    42. 42. ® Cor.At Cardiomyocytes and PatchXpress 7000A ® MDS - Analytical Technologies Dr. Xin Jiang
    43. 43. ® PatchXpress 7000A - Cardiac Ion Currents
    44. 44. ® PatchXpress 7000A - Cardiac Ion Currents INa 1 nA 2 ms I/V Diagram
    45. 45. ® PatchXpress 7000A - Cardiac Ion Currents INa ICa 1 nA 100 pA 50 ms 2 ms I/V Diagram
    46. 46. ® PatchXpress 7000A - Cardiac Ion Currents INa ICa IK 1 nA 100 pA 50 ms 2 ms I/V Diagram I/V Diagram
    47. 47. Statistics Subject Number of cells Result ± SEM High resistance seals (> 1GΩ), (%) 96 45 ± 12 Successful whole cell (%) 96 49 ± 16 Peak INa (nA) (at -20 mV) 6 1.52 ± 0.23 Peak ICa (nA) (at +10 mV) 11 0.067 ± 0.008 Peak IK (nA) (at +20 mV) 21 0.49 ± 0.11
    48. 48. ® PatchXpress 7000A: Potassium Current Pharmacology 4-Aminopyridine Control 10 µM
    49. 49. ® PatchXpress 7000A: Potassium Current Pharmacology 4-Aminopyridine Control 10 µM
    50. 50. ® PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L)
    51. 51. ® PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L) Epinephrine (Adrenaline)
    52. 52. ® PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L) Epinephrine (Adrenaline) Epi, 10 µM
    53. 53. ® PatchXpress 7000A: ß-adrenergic modulation of I(Ca,L) Epinephrine (Adrenaline) Epi, 10 µM
    54. 54. ® Cor.At Cardiomyocytes and QPatch ® Sophion Bioscience A/S
    55. 55. ® Cor.At Cardiomyocytes and QPatch ® Sophion Bioscience A/S Dr. Rikke Schrøder
    56. 56. ® QPatch - Ion Currents
    57. 57. ® QPatch - Ion Currents INa I/V Diagram
    58. 58. ® QPatch - Ion Currents INa ICa I/V Diagram I/V Diagram
    59. 59. ® QPatch - Ion Currents INa ICa IK I/V Diagram I/V Diagram I/V Diagram
    60. 60. Statistics
    61. 61. Statistics Avergage of 4 QPlates 16
    62. 62. 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
    63. 63. ® QPatch - Sodium Current Pharmacology INa block with TTX
    64. 64. ® QPatch - Sodium Current Pharmacology INa block with TTX
    65. 65. ® QPatch - Sodium Current Pharmacology INa block with TTX
    66. 66. ® QPatch - Sodium Current Pharmacology INa block with TTX A) baseline
    67. 67. ® QPatch - Sodium Current Pharmacology INa block with TTX B) 50 nM A) baseline
    68. 68. ® QPatch - Sodium Current Pharmacology INa block with TTX C) 5 µM B) 50 nM A) baseline
    69. 69. ® QPatch - ß-adrenergic modulation of I(Ca,L) Voltage protocol
    70. 70. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol
    71. 71. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol
    72. 72. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol 1) baseline
    73. 73. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol 1) baseline 2) 1 µM Iso
    74. 74. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol 1) baseline 2) 1 µM Iso 3) 10 µM Iso
    75. 75. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol 4) 10 µM Nifedipine 1) baseline 2) 1 µM Iso 3) 10 µM Iso
    76. 76. ® QPatch - ß-adrenergic modulation of I(Ca,L) Isoproterenol 4) 10 µM Nifedipine 1) baseline 2) 1 µM Iso 3) 10 µM Iso
    77. 77. ® Cor.At Cardiomyocytes and ® Port-a-Patch and Patchliner ® Nanion Technologies
    78. 78. ® Cor.At Cardiomyocytes and ® Port-a-Patch and Patchliner ® Nanion Technologies Dr. Sonja Stölzle
    79. 79. Voltage Clamp
    80. 80. Voltage Clamp INa I/V Diagram
    81. 81. Voltage Clamp INa ICa I/V Diagram I/V Diagram
    82. 82. Voltage Clamp INa ICa IK I/V Diagram I/V Diagram I/V Diagram
    83. 83. Automated Current Clamp Recording
    84. 84. Automated Current Clamp Recording Port-a-Patch® Stimulation Protocol (500 ms stimuli at 0.2 Hz) -40 pA I(memb) 150 ms 20 mV
    85. 85. 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
    86. 86. 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
    87. 87. ® Port-a-Patch - Sodium Channel Pharmacology Tetrodotoxin (TTX)
    88. 88. ® Port-a-Patch - Sodium Channel Pharmacology Tetrodotoxin (TTX) control TTX 20 µM 150 ms 20 mV washout
    89. 89. ® Patchliner - Sodium Channel Pharmacology
    90. 90. ® Patchliner - Sodium Channel Pharmacology min
    91. 91. ® Patchliner - Sodium Channel Pharmacology min
    92. 92. ® Port-a-Port - Identification of hERG blocker
    93. 93. ® Port-a-Port - Identification of hERG blocker Dofetilide
    94. 94. ® Port-a-Port - Identification of hERG blocker Dofetilide control Dofetilide, 1µM 150 ms 20 mV 150 ms 20 mV washout
    95. 95. ® Patchliner - Potassium Channel Pharmacology
    96. 96. ® Patchliner - Potassium Channel Pharmacology Quinidine
    97. 97. ® Patchliner - Potassium Channel Pharmacology Quinidine min stimulation interval: 0.1 Hz
    98. 98. ® Patchliner - Potassium Channel Pharmacology Quinidine min stimulation interval: 0.1 Hz 150 ms
    99. 99. Why is it possible to identify hERG blocker effects in mouse ES cell-derived cardiomyocytes?
    100. 100. 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.
    101. 101. 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.
    102. 102. 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.
    103. 103. 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.
    104. 104. 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.
    105. 105. 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.
    106. 106. 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.
    107. 107. 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.
    108. 108. 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.
    109. 109. + + Cardiac Na /K -ATPases as Important Drug Targets
    110. 110. + + Gene Expression of Na /K ATPase Subunits
    111. 111. + + 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
    112. 112. + + Na /K ATPase Subunits Homology between Mouse and Human Amino Acid Sequences Na+/K+ ATPase catalytic alpha subunits: ATP1A1 Identities = 963/992 (97%), Positives = 979/992 (98%), Gaps = 0/992 (0%) Identities = 991/1023 (96%), Positives = 1008/1023 (98%), Gaps = 0/1023 (0%) ATP1A2 Identities = 1011/1020 (99%), Positives = 1018/1020 (99%), Gaps = 0/1020 (0%) ATP1A3 Identities = 1001/1005 (99%), Positives = 1003/1005 (99%), Gaps = 0/1005 (0%) Na+/K+ ATPase regulatory beta subunits: ATP1B1 Identities = 285/304 (93%), Positives = 298/304 (98%), Gaps = 1/304 (0%) Identities = 282/302 (93%), Positives = 295/302 (97%), Gaps = 1/302 (0%) ATP1B2 Identities = 282/290 (97%), Positives = 287/290 (98%), Gaps = 0/290 (0%)
    113. 113. ® Cor.At Cardiomyocytes and Pharmacological Studies with the ICR 8000 ® Aurora Biomed
    114. 114. ® Cor.At Cardiomyocytes and Pharmacological Studies with the ICR 8000 ® Aurora Biomed Dr. Sikander Gill
    115. 115. + ICR 8000 - Rb Uptake Assay
    116. 116. + ICR 8000 - Rb Uptake Assay Monitoring of cardiac Na+/K+ ATPases
    117. 117. + ICR 8000 - Rb Uptake Assay Monitoring of cardiac Na+/K+ ATPases Activity
    118. 118. + ICR 8000 - Rb Uptake Assay Monitoring of cardiac Na+/K+ ATPases Activity Pharmacology
    119. 119. Summary
    120. 120. Summary Cor.At® cardiomyocytes were successsfully applied to automated patch clamp systems from three different companies to analyse cardiac ion current:
    121. 121. 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
    122. 122. 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
    123. 123. 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
    124. 124. 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
    125. 125. 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.
    126. 126. 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.
    127. 127. 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.
    128. 128. Conclusion
    129. 129. Conclusion ® Cor.At cells are primary-like cardiomyocytes predictive and relevant for pharmacological studies.
    130. 130. 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
    131. 131. 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
    132. 132. 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
    133. 133. 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.
    134. 134. Acknowledgment
    135. 135. Acknowledgment MDS Analytical Technologies: Dr. Xin Jiang Dr. Jan Dolzer Dr. James Costantin Dr. David Yamane
    136. 136. 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
    137. 137. 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
    138. 138. 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
    139. 139. 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
    140. 140. 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
    141. 141. Acknowledgment
    142. 142. Acknowledgment North America: www.reachbio.com
    143. 143. Acknowledgment North America: Special thanks to: Dr. Eric Atkinson Lynn MacIntyre www.reachbio.com
    144. 144. Acknowledgment North America: Special thanks to: Dr. Eric Atkinson Lynn MacIntyre www.reachbio.com Japan: Dr. Junya Koda Dr. Chie Kodama www.veritastk.com

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