HTS of Ca 2+ Transients in Human iPS-derived Cardiomyocytes as a - - PowerPoint PPT Presentation

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HTS of Ca 2+ Transients in Human iPS-derived Cardiomyocytes as a - - PowerPoint PPT Presentation

Sep 29, 2023 451 likes •871 views

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

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Use ¡our ¡discoveries ¡to ¡advance ¡yours

HTS of Ca2+ Transients in Human iPS-derived Cardiomyocytes as a Predictive and Cost Effective Assay Early on in Drug Development

  • Dr. Ralf Kettenhofen

Axiogenesis AG, Cologne, Germany Hamamatsu 10th European Functional Drug Screening Symposium Actelion, Allschwil, Switzerland

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SLIDE 2

Content

2

  • Introduction into the production of Cor.4U cardiomyocytes
  • Overview of established applications with Cor.4U

cardiomyocytes

  • HTS calcium transient assay in the Hamamatsu FDSS/

µCell

  • Compound screening
  • Data analysis
  • Outlook
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SLIDE 3

Generation of Cor.4U human iPS derived Cardiomyocytes

3

  • 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)

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SLIDE 4

4

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Use ¡our ¡discoveries ¡to ¡advance ¡yours

Introduction Recording of Calcium Transients in Cor.4U Cardiomyocytes

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SLIDE 6

Excitation-Contraction Coupling

Time Amplitude

Currents Action Potential

Na+ Ca2+ L-type Ca2+ T-type Na+/Ca2+-exchanger K+ Ito K+ IKs K+ IKr K+ IKur

Calcium is the messenger that integrates the electrochemcial signals of the action potential with the molecular signaling pathways that regulate contraction Many Channel Types contribute to the Action Potential

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SLIDE 7

Hamamatsu FDSS/µCell

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SLIDE 8

Plating Efficiency on 384 Well Plates Fluo-4 Assay

Data was kindly provided by Dr. Thomas Licher, Sanofi Germany

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SLIDE 9

FDSS Parameters Analysed by the CalDio/Wave Checker Software

(6) A M P (5) RMP=Fluorescence intensity at the bottom peak (1)Peak Number Bottom (3) P-P duration time

(7)Max_slope[Fluorescence counts/ms]

same as upstroke slope (but bottom to peak)

(8) MaxNeg_slope[Fluorescence

counts/ms] same as downstroke slope (but peak to bottom)

AMP= Peak Fluorescence count- RMP (4)RATIO = AMP/RMP (2)P rate(BPM) A M P APD10 APD50(Peak Width, FWHM) APD90 (9) APD 10 to 90

PWD 90

(1) Peak number (total, BPM) (2) P-P time [ms] (Ave, Std, Max, Min) (3) Ratio (Ave, Std) Ratio = (AMP+RMP)/RMP (4) AMP (Ave, Std) (5) RMP (Ave, Std) (6) Slope (Ave, Std) Rising Slope: Slope from bottom to peak Falling Slope: Slope from peak to bottom 0% - 10%, 10% - 90%, 20% - 80%, 30% - 70% (7) Integration (Ave, Std) (8) - (16) PWD (10% - 90%) [ms] (Ave, Std)

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SLIDE 10

Use ¡our ¡discoveries ¡to ¡advance ¡yours

hERG/IKr blocker

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SLIDE 11

Use our discoveries to advance yours

Astemizole hERG Blocker

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FDSS µCell - Astemizole

vehicle control 370 nM 41 nM

5 min

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FDSS µCell - Astemizole

30 min

vehicle control 4.57 nM 13.7 nM

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SLIDE 14

FDSS µCell - Astemizole

41 nM 123 nM 370 nM

30 min

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SLIDE 15

Use our discoveries to advance yours

Data Analysis with the Wave Checker Module Astemizole

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FDSS µCell - Astemizole

Wave Checker Module

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FDSS µCell - Astemizole

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FDSS µCell - Astemizole

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FDSS µCell - Astemizole

export of text file format

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FDSS µCell - Astemizole

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FDSS µCell - Astemizole

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FDSS µCell - Astemizole

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0,00# 5,00# 10,00# 15,00# 20,00# 25,00# 30,00# 35,00# 40,00# 45,00# 10000# 3333# 1111# 370# 123# 41# 14# 5# 2# 1# Control# bea$ng'frequency'[bpm]' concentra$on'[nM]' baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

0,00# 2,00# 4,00# 6,00# 8,00# 10,00# 12,00# 14,00# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# average&falling&slope&[RFU/ms]& concentra7on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

FDSS µCell - Astemizole

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SLIDE 24

0,00# 2000,00# 4000,00# 6000,00# 8000,00# 10000,00# 12000,00# 14000,00# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# average&peak&width&60%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

0,00# 2000,00# 4000,00# 6000,00# 8000,00# 10000,00# 12000,00# 14000,00# 1 , # 3 3 3 3 , 3 3 # 1 1 1 1 , 1 1 # 3 7 , 3 7 # 1 2 3 , 4 6 # 4 1 , 1 5 # 1 3 , 7 2 # 4 , 5 7 # 1 , 5 2 # , 5 1 # C

  • n

t r

  • l

# average&peak&width&70%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

0,00# 2000,00# 4000,00# 6000,00# 8000,00# 10000,00# 12000,00# 14000,00# 1 , # 3 3 3 3 , 3 3 # 1 1 1 1 , 1 1 # 3 7 , 3 7 # 1 2 3 , 4 6 # 4 1 , 1 5 # 1 3 , 7 2 # 4 , 5 7 # 1 , 5 2 # , 5 1 # C

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t r

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# average&peak&width&80%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

0,00# 2000,00# 4000,00# 6000,00# 8000,00# 10000,00# 12000,00# 14000,00# 16000,00# 18000,00# 10000,00# 3333,33# 1111,11# 370,37# 123,46# 41,15# 13,72# 4,57# 1,52# 0,51# Control# average&peak&width&90%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

FDSS µCell - Astemizole

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Use our discoveries to advance yours

Dofetilide hERG Blocker

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FDSS µCell - Dofetilide

5 min

vehicle control 24.7 nM 222 nM

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FDSS µCell - Dofetilide

30 min

vehicle control 24.7 nM

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FDSS µCell - Dofetilide

0.00# 10.00# 20.00# 30.00# 40.00# 50.00# 60.00# 70.00# C

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t r

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# . 3 # . 1 # . 3 # . 9 1 # 2 . 7 4 # 8 . 2 3 # 2 4 . 6 9 # 7 4 . 7 # 2 2 2 . 2 2 # 6 6 6 . 6 7 # 2 . # bea$ng'frequency'[bpm]' concentra$on'[nM]' baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 35#min#

0.00# 5.00# 10.00# 15.00# 20.00# 25.00# 30.00# 35.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# average&rising&slope&[RFU/ms]& concentra6on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min#

0.00# 1.00# 2.00# 3.00# 4.00# 5.00# 6.00# 7.00# 8.00# 9.00# 10.00# C

  • n

t r

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# . 3 # . 1 # . 3 # . 9 1 # 2 . 7 4 # 8 . 2 3 # 2 4 . 6 9 # 7 4 . 7 # 2 2 2 . 2 2 # 6 6 6 . 6 7 # 2 . # average&falling&slope&[RFU/ms]& concentra7on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min#

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SLIDE 29

FDSS µCell - Dofetilide

0.00# 100.00# 200.00# 300.00# 400.00# 500.00# 600.00# 700.00# 800.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# average&peak&width&30%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 0.00# 200.00# 400.00# 600.00# 800.00# 1000.00# 1200.00# 1400.00# 1600.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# average&peak&width&50%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min#

0.00# 500.00# 1000.00# 1500.00# 2000.00# 2500.00# 3000.00# 3500.00# 4000.00# 4500.00# 5000.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# average&peak&width&70%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min# 0.00# 2000.00# 4000.00# 6000.00# 8000.00# 10000.00# 12000.00# 14000.00# Control# 0.03# 0.10# 0.30# 0.91# 2.74# 8.23# 24.69# 74.07# 222.22# 666.67# 2000.00# average&peak&width&90%&[ms]& concentra8on&[nM]& baseline# 5#min# 10#min# 15#min# 20#min# 25#min# 30#min#

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Confidential

FDSS µCell - Clustering of Compounds

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Outlook

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

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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


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Dopaminergic and Sensory Neurons

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Patch Clamp of hiPS-derived Dopamineric Neurons

2 4 6 8 10 12

  • 0.08
  • 0.06
  • 0.04
  • 0.02

0.00 0.02 0.04 0.06

U[V] T[s]

Manual current clamp recording

  • f spontaneous action potentials

Manual voltage clamp recording

  • f sodium currents

Data was kindly provided by PoreGenic GmbH, Rostock, Germany

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SLIDE 36

Patch Clamp of hiPS-derived Peripheral Neurons Manual voltage clamp recording

  • f sodium currents

Data was kindly provided by PoreGenic GmbH, Rostock, Germany

  • 0.06
  • 0.04
  • 0.02

0.00 0.02 0.04 0.06 0.08

  • 4.00E-009
  • 3.00E-009
  • 2.00E-009
  • 1.00E-009

0.00E+000 1.00E-009

I[A] U[V]

raw data I/V diagram

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SLIDE 37

Use our discoveries to advance yours

Health and Environmental Science Institute (HESI) Initiative

  • CiPA Initiative

(Comprehensive in vitro Pro-Arrhythmia Assay)

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SLIDE 38
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HESI - Initiative Stem Cell Working Groups - Newly Initiated Following the workshop, the Cardiac Stem Cell Working Group formed and three subteams were created: Cytotoxicity Contractility, Electrophysiology 
 
 to explore issues of sensitivity, reproducibility, and predictivity for safety endpoints.

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SLIDE 40

4Q ¡2013 1-­‑2Q ¡2014 2-­‑3Q ¡2014 2015

  • Subteam ¡goals ¡

and ¡objec1ves ¡ dra4ed ¡

  • Data ¡collec1on ¡

ini1ated ¡

  • Link ¡to ¡CIPA ¡

project

  • Manuscript(s) ¡

dra4ing ¡and ¡ submission

  • Laboratory ¡study ¡

ini1ated ¡

  • Manuscript(s) ¡

dra4ing

  • Literature ¡search ¡

conducted ¡

  • Laboratory ¡study ¡

protocol ¡planning

Cardiac Stem Cell Working Group: Timeline

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SLIDE 41

Thanks to:

  • Axiogenesis AG


Anika Duenbostell

  • Hamamatsu


Jean Marc D'Angelo
 Thomas Niedereichholz
 Cyril Guerinot
 Thibault Poissinger
 Hirofumi Horai


  • Sanofi, Frankfurt

  • Dr. Thomas Licher