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Use our discoveries to advance yours HTS of Ca 2+ Transients in Human iPS-derived Cardiomyocytes as a Predictive and Cost Effective Assay Early on in Drug Development Dr. Ralf Kettenhofen Hamamatsu 10th European Functional

  1. Use ¡our ¡discoveries ¡to ¡advance ¡yours HTS of Ca 2+ Transients in Human iPS-derived Cardiomyocytes as a Predictive and Cost Effective Assay Early on in Drug Development Dr. Ralf Kettenhofen � Hamamatsu 10th European Functional Drug Screening Symposium � Axiogenesis AG, Cologne, Germany Actelion, Allschwil, Switzerland

  2. Content • Introduction into the production of Cor.4U cardiomyocytes • Overview of established applications with Cor.4U cardiomyocytes • HTS calcium transient assay in the Hamamatsu FDSS/ µ Cell • Compound screening • Data analysis • Outlook 2

  3. Generation of Cor.4U human iPS derived Cardiomyocytes • iPS generated according to Yamanaka (licensed from iPS Academia) • Production system by Axiogenesis enables large lot sizes • Tightly controlled differentiation results in minimal lot to lot variations • Selection based purification technology results in pure cardiomyocytes • Cryopreserved in various formats • GFP positive and colourless varieties will be available • Complete FTO for commercial use on the limited use label license • Cryopreserved • Fresh cultures on different substrates (cell culture flasks, 96/384 well plates) 3

  4. 4

  5. Use ¡our ¡discoveries ¡to ¡advance ¡yours Introduction Recording of Calcium Transients in Cor.4U Cardiomyocytes

  6. Excitation-Contraction Coupling Action Potential Currents Na + Amplitude Ca 2+ L-type Ca 2+ T-type Na + /Ca 2+ -exchanger K + I to K + IK s K + IK r Time K + IK ur Many Channel Types contribute to the Calcium is the messenger that integrates the Action Potential electrochemcial signals of the action potential with the molecular signaling pathways that regulate contraction

  7. Hamamatsu FDSS/ µ Cell

  8. Plating Efficiency on 384 Well Plates Fluo-4 Assay Data was kindly provided by Dr. Thomas Licher, Sanofi Germany

  9. FDSS Parameters Analysed by the CalDio/Wave Checker Software (3) P-P duration time (1)Peak Number (2)P rate(BPM) (6) A M P (5) RMP=Fluorescence Bottom intensity at the bottom peak (7) Max_slope[Fluorescence counts/ms] same as upstroke slope AMP= Peak Fluorescence count- RMP (but bottom to peak) (8) MaxNeg_slope[Fluorescence (4)RATIO = AMP/RMP counts/ms] same as downstroke slope (but peak to bottom) (9) APD 10 to 90 (1) Peak number (total, BPM) APD10 (2) P-P time [ms] (Ave, Std, Max, Min) APD50(Peak Width, FWHM) (3) Ratio (Ave, Std) � Ratio = (AMP+RMP)/RMP A M (4) AMP (Ave, Std) P (5) RMP (Ave, Std) (6) Slope (Ave, Std) � Rising Slope: Slope from bottom to peak � Falling Slope: Slope from peak to bottom � APD90 PWD 90 0% - 10%, 10% - 90%, 20% - 80%, 30% - 70% (7) Integration (Ave, Std) (8) - (16) PWD (10% - 90%) [ms] (Ave, Std)

  10. Use ¡our ¡discoveries ¡to ¡advance ¡yours hERG/I Kr blocker

  11. Use our discoveries to advance yours Astemizole hERG Blocker

  12. 5 min FDSS µ Cell - Astemizole vehicle control 41 nM 370 nM

  13. 30 min FDSS µ Cell - Astemizole vehicle control 4.57 nM 13.7 nM

  14. 30 min FDSS µ Cell - Astemizole 41 nM 123 nM 370 nM

  15. Use our discoveries to advance yours Data Analysis with the Wave Checker Module Astemizole

  16. FDSS µ Cell - Astemizole Wave Checker Module

  17. FDSS µ Cell - Astemizole

  18. FDSS µ Cell - Astemizole

  19. FDSS µ Cell - Astemizole export of text file format

  20. FDSS µ Cell - Astemizole

  21. FDSS µ Cell - Astemizole

  22. FDSS µ Cell - Astemizole

  23. FDSS µ Cell - Astemizole 45,00# 14,00# 40,00# average&falling&slope&[RFU/ms]& 12,00# bea$ng'frequency'[bpm]' 35,00# baseline# 10,00# baseline# 30,00# 5#min# 5#min# 8,00# 25,00# 10#min# 10#min# 20,00# 6,00# 15#min# 15#min# 15,00# 4,00# 20#min# 20#min# 10,00# 2,00# 25#min# 25#min# 5,00# 30#min# 30#min# 0,00# 0,00# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# 35#min# 10000# 3333# 1111# 370# 123# 41# 14# 5# 2# 1# Control# 35#min# concentra$on'[nM]' concentra7on&[nM]&

  24. FDSS µ Cell - Astemizole 18000,00# 14000,00# 16000,00# average&peak&width&60%&[ms]& 12000,00# 14000,00# average&peak&width&90%&[ms]& 10000,00# baseline# 12000,00# baseline# 5#min# 8000,00# 10000,00# 5#min# 10#min# 6000,00# 10#min# 8000,00# 15#min# 15#min# 4000,00# 6000,00# 20#min# 20#min# 4000,00# 2000,00# 25#min# 25#min# 30#min# 2000,00# 0,00# 30#min# 35#min# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# 0,00# 35#min# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# concentra8on&[nM]& concentra8on&[nM]& 14000,00# 14000,00# average&peak&width&80%&[ms]& 12000,00# 12000,00# average&peak&width&70%&[ms]& 10000,00# 10000,00# baseline# baseline# 5#min# 8000,00# 8000,00# 5#min# 10#min# 10#min# 6000,00# 6000,00# 15#min# 15#min# 4000,00# 4000,00# 20#min# 20#min# 2000,00# 25#min# 25#min# 2000,00# 30#min# 0,00# 30#min# # # # # # # # # # # # 35#min# 0 3 1 7 6 5 2 7 2 1 l o 0,00# 0 3 1 3 4 1 7 5 5 5 r , , , , , , , , , , t 35#min# 0 3 1 0 3 1 4 1 0 3 n 0 3 1 7 2 4 1 # # # # # # # # # # # o 0 3 1 7 6 5 2 7 2 1 l 0 3 1 3 1 o 0 3 1 3 4 1 7 5 5 5 C r 0 3 1 , , , , , , , , , , t 0 3 1 0 3 1 3 4 1 0 n 1 0 3 1 7 2 4 1 o 0 3 1 3 1 C 0 3 1 1 concentra8on&[nM]& concentra8on&[nM]&

  25. Use our discoveries to advance yours Dofetilide hERG Blocker

  26. 5 min FDSS µ Cell - Dofetilide vehicle control 24.7 nM 222 nM

  27. 30 min FDSS µ Cell - Dofetilide vehicle control 24.7 nM

  28. FDSS µ Cell - Dofetilide 70.00# 60.00# bea$ng'frequency'[bpm]' 50.00# baseline# 40.00# 5#min# 10#min# 30.00# 15#min# 20#min# 20.00# 25#min# 10.00# 30#min# 35#min# 0.00# # # # # # # # # # # # # l 3 0 0 1 4 3 9 7 2 7 0 o 0 1 3 9 7 2 6 0 2 6 0 r t . . . . . . . . . . . 0 0 0 0 2 8 4 4 2 6 n 0 2 7 2 6 0 o 2 6 0 C 2 concentra$on'[nM]' 10.00# 35.00# 9.00# average&falling&slope&[RFU/ms]& 30.00# 8.00# average&rising&slope&[RFU/ms]& 7.00# 25.00# baseline# 6.00# baseline# 20.00# 5#min# 5.00# 5#min# 10#min# 15.00# 10#min# 4.00# 15#min# 15#min# 3.00# 10.00# 20#min# 20#min# 2.00# 25#min# 5.00# 1.00# 25#min# 30#min# 0.00# 0.00# 30#min# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# # # # # # # # # # # # # l 3 0 0 1 4 3 9 7 2 7 0 o 0 1 3 9 7 2 6 0 2 6 0 r . . . . . . . . . . . t 0 0 0 0 2 8 4 4 2 6 0 n 2 7 2 6 0 o 2 6 0 C 2 concentra6on&[nM]& concentra7on&[nM]&

  29. FDSS µ Cell - Dofetilide 800.00# 1600.00# 700.00# 1400.00# average&peak&width&30%&[ms]& average&peak&width&50%&[ms]& 600.00# 1200.00# baseline# baseline# 500.00# 1000.00# 5#min# 5#min# 400.00# 800.00# 10#min# 10#min# 300.00# 600.00# 15#min# 15#min# 200.00# 400.00# 20#min# 20#min# 100.00# 200.00# 25#min# 25#min# 0.00# 0.00# 30#min# 30#min# Control# 0.91# 24.69# 74.07# 222.22# 666.67# 2000.00# 0.03# 0.10# 0.30# 2.74# 8.23# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# concentra8on&[nM]& concentra8on&[nM]& 14000.00# 5000.00# 4500.00# average&peak&width&70%&[ms]& 12000.00# average&peak&width&90%&[ms]& 4000.00# 10000.00# 3500.00# baseline# baseline# 3000.00# 8000.00# 5#min# 5#min# 2500.00# 10#min# 6000.00# 10#min# 2000.00# 15#min# 15#min# 1500.00# 4000.00# 1000.00# 20#min# 20#min# 2000.00# 500.00# 25#min# 25#min# 0.00# 0.00# 30#min# 30#min# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# concentra8on&[nM]& concentra8on&[nM]&

  30. FDSS µCell - Clustering of Compounds Confidential

  31. Outlook

  32. Outlook - Hamamatsu FDSS 7000EX Camera for fast recording of fluorescence and luminescence.

  33. Outlook Evaluation of different kinds of fluorescent calcium and membrane 
 potential dyes for the Cor.4U Cardiomyocytes Establishment of cardiac cytotoxicity/viability assay Combination of different luminescence assays viability cell death detection apoptosis 


  34. Dopaminergic and Sensory Neurons

  35. Patch Clamp of hiPS-derived Dopamineric Neurons Manual current clamp recording Manual voltage clamp recording of spontaneous action potentials of sodium currents 0.06 0.04 0.02 0.00 U[V] -0.02 -0.04 -0.06 -0.08 0 2 4 6 8 10 12 T[s] Data was kindly provided by PoreGenic GmbH, Rostock, Germany

  36. Patch Clamp of hiPS-derived Peripheral Neurons Manual voltage clamp recording of sodium currents raw data I/V diagram 1.00E-009 0.00E+000 -1.00E-009 I[A] -2.00E-009 -3.00E-009 -4.00E-009 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 U[V] Data was kindly provided by PoreGenic GmbH, Rostock, Germany

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