Welcome to the Exhibitor Tutorial presented by

Transcription

1 Welcome to the Exhibitor Tutorial presented by February 1, 217 1

2 Calcium Handling Assays with Human ipsc-derived Cell Types Coby Carlson, Ph.D. 7 February 217

3 Transformative Potential of ipsc Technology Differentiation Reprogramming ips Cell Lines Blood Sample Genome Editing Human Donor(s) Terminally-differentiated Cell Types Enabling for Drug Discovery Toxicology and Regenerative Medicine February 1, 217 3

4 Why Choose ipsc? Industrialized Processes Multiple Citations in Peer- Reviewed Publications Transformed Cell Lines Quantity CDI Disease Modeling Animal Cell Models Drug Screening Primary Human Cells Understanding Mechanism Functional cell types provide translatable answers!! February 1, 217 4

5 Human ipsc-derived Cardiomyocytes for Safety / Toxicity Drugs & Arrhythmia Human Heart Ion Channels icell Cardiomyocytes 1. Electrophysiology / MEA (voltage) 3. Calcium Transients 2. Mechanical / Contraction (impedance) February 1, 217 5

6 FDSS/µCell Functional Drug Screening System Whole Plate Dispensing and Imaging Compact and Simple to Use Enabling for Multiple Applications Electrical Field Stimulation (EFS) February 1, 217 6

7 FDSS/µCell Ca 2+ Handling Assay to Assess Cardiac Beating Activity Cryopreserved Cells 96- or 384-well plates Calcium Dye Protocol developed by CDI Technical Support and Application Development February 1, 217 7

8 RFU Example Pharmacology Baseline Isoproterenol (1 µm) E-431 (1 µm) 5 1, Time (sec) Time (sec) Time (sec) Beats Per Minute (BPM) Baseline ISO E-431 BPM EC 5 = 7.9 nm log [Isoproterenol] (M) log [E-431] (M) February 1, BPM EC 5 = 11.7 nm Cells respond to known ligands with expected responses

9 Example Pharmacology (continued) [ISO] 5 Beats Per Min 3 Peak-to-Peak Time 15 Peak Width P rate [ /min] (BPM) P-P time (msec) PWD9 (msec) log [ISO] (M) log [ISO] (M) log [ISO] (M) Many different ways to analyze the data February 1, 217 9

10 Analysis of the Cardiac Calcium Waveform (1) Peak number (total, BPM) (2) P-P time (msec) (3) Ratio (4) AMP (amplitude) (5) RMP (6) Slope (rising and falling) (7) Integration (area under curve) (8) (16) PWD (peak width duration; msec) February 1, 217 1

11 Assay Development and Optimization ECM / Plate Coating Calcium Dyes Geltrex Baseline Comparison of ECM on Baseline Beat Rate 4 Gelatin Fibronectin Beats Per Minute (BPM) Geltrex Gelatin Fibronectin Various conditions affect assay performance P Rate [ /min] (BPM) Codex Codex EarlyTox AMP (RFU) Codex EarlyTox EarlyTox February 1,

12 Robust Assay Performance with icell Cardiomyocytes 2 Thaw and plate cells according to App Protocol icmm media change Record Days in Culture 4 h 2 No Weekend Work icpm to icmm icmm media change Beat Rate (baseline) Amplitude (baseline) Beat Rate CV (baseline) sec P rate [ /min] (BPM) Amplitude (RFU) Coefficient of Variation A B C A B C. A B C Consistency from plate-to-plate, day-to-day, and lot-to-lot February 1,

13 Hamamatsu Data SLAS217 Poster # Test Compounds Calcium Transients Membrane Potential February 1,

14 Ca 2+ Assays in Action Sofosbuvir Case Study DMSO control Sofosbuvir [3.3 µm] Amiodarone [25 µm] Sofosbuvir [3.3 µm] + 3 Amiodarone [5 nm] Sofosbuvir (HCV Drug) Amiodarone (Arrhythmia) 3 Area Under Curve (AUC) Axion / CDI Merck Area Under Curve (AUC) Ctrl Log [Sofosbuvir] (M) DMSO Control Sofosbuvir only Sofosbuvir + Amiodarone (.5 um) Interaction of sofosbuvir + amiodarone result in decreased Ca 2+ flux Ctrl Log [Sofosbuvir] (M) February 1,

15 icell Cardiomyocytes not just for Safety / Toxicity assessment, but also for Disease Modeling and Drug Discovery 15

16 Disease Modeling with ipsc Disease-in-a-Dish INNATE ENGINEERED INDUCED Healthy Donor Donor with Genetic Disease Healthy Donor Donor with Genetic Disease Healthy Donor Reprogramming to ipsc ipsc Differentiation Genome Engineering Healthy Conditions Disease-inducing Conditions Controls Phenotypic and Functional Analysis

17 Phenotypic Screening Diabetic Cardiomyopathy (DCM) Disease Modeling Induced Disease Model of DCM Baseline (Control) Drawnel et al. Cell Reports 214 9(3): Healthy State Healthy Cellular stress Diabetic Medium Hits from ~5 compound library phenotypic screen Diseased State Diseased α-actinin DAPI February 1,

18 Can a diabetic phenotype be observed on the FDSS? February 1,

19 icell CM Show Altered Ca 2+ Transients in Diabetic Medium Healthy State Baseline (Control) P rate [ /min] (BPM) Coefficient of Variation Baseline Diabetic. Baseline Diabetic 45 Diseased State Diabetic Medium 6 sec Baseline Diabetic PWD2 Coefficient of Variation Coefficient of Variation PWD 2 (msec) Baseline 1 Diabetic February 1,

20 Diabetic Phenotype is Rescued with Small Molecules 5 Diabetic Medium P rate [ /min] (BPM) Topotecan U Log [ligand] (M) +Lycorine Calcium transients from icell Cardiomyocytes in Diabetic Medium ± 1 µm of each compound for 48 hours February 1, 217 2

21 Innate Disease Modeling Hypertrophic Cardiomyopathy icell CM control Healthy Donor Donor with Genetic Disease 8 Gene Expression icell CM control 7 * Reprogramming to ipsc ipsc Differentiation / MHC Ratio MYH7 R43Q MYH7 R43Q icell CM MYH7 R43Q BNP / DAPI MyCell MYH7 R43Q Cardiomyocytes Patient-derived ipsc sample xcelligence impedance assay February 1,

22 CM with MYH7 Mutations have Irregular Ca 2+ Handling Publications w/ human ipsc-cm (missense R442G and R663H) Lan et. al. Cell Stem Cell (213) CLARIOstar plate reader (BMG Labtech) can measure Ca 2+ flux 1X per sec for 1 seconds using fluorescent Ca 2+ dye (Fluo-4) [but only a single well at a time] February 1,

23 MYH7 R43Q is Less Sensitive to Ca 2+ Channel Blocking 3 25 Ca 2+ Handling Assay: icell Cardiomyocytes (control) RFU RFU icell control-baseline Nifedipine 5nM icell Cardiomocytes respond to Nifedipine BPM Lan et. al. Cell Stem Cell (213) [Nifedipine] ( M) R43Q-Baseline Time (sec) Nifedipine 5nM 2 15 Beating activity of R43Q CM is less sensitive Time (sec) February 1,

24 R43Q Ca 2+ Transients are Slower and More Irregular icmm (standard medium) icell CM control MYH7 R43Q CM 3 sec P Rate [ /min] (BPM) Beat Rate P Rate CV (%) Beat Rate Irregularity icell CM R43Q icell CM R43Q February 1,

25 Continuous Verapamil Treatment Improves Ca 2+ Handling icmm (standard medium) icell CM control MYH7 R43Q CM 3 sec P Rate [ /min] (BPM) Beat Rate P Rate CV (%) Beat Rate Irregularity icell CM R43Q icell CM R43Q icmm + Verapamil (5 nm) Beat Rate Beat Rate Irregularity icmm + Verap (5 nm) icmm + Verap (5 nm) February 1, P rate [ /min] (BPM) P Rate CV (%) icmm + Verap (5 nm) icmm + Verap (5 nm)

26 Electrical Field Stimulation (EFS) February 1,

27 Verapamil also Rescues R43Q from Mechanical Overload icell CM control MYH7 R43Q CM 3 sec R43Q +Verapamil (5 nm) no EFS.5 Hz cardiac pacing no EFS 1 Hz no EFS 1.5 Hz no EFS 2 Hz February 1,

28 Media Optimization for Phenotypic Screening [Ca 2+ ] in medium icell Cardiomyocytes icell CM control MYH7 R43Q CM MyCell DDP MYH7 R43Q icell Maintenance Medium + 1 mm Glucose with.9 mm CaCl 2 [Ca 2+ ] in medium February 1,

29 Next Generation Assays with FDSS/µCell Ca 2+ response evoked by EFS Membrane Potential Voltage (FluoVolt dye) 3D Cell Culture / Co-Culture Calcium (Fluo-4 dye) (Data from cells on same plate) February 1,

30 Several Different Human Neural Cell Types Available... icell Neurons icell DopaNeurons icell GlutaNeurons icell Motor Neurons icell Induced Neurons icell Astrocytes icell Macrophages with Numerous Functional Applications February 1, 217 3

31 Assay Development with icell GlutaNeurons on FDSS Synchronous Bursting Baseline + 2% FBS +.2 mm Mg K Astro February 1,

32 Final Thoughts ipsc technology Lego building blocks Platform provider; Data analysis Highly pure, Cryopreserved cells icell Operating System Safety/Tox, Drug Discovery Disease modeling February 1,

33 Thank You! February 1,