Diversity in a dish: A human population-based organotypic in vitro - - PowerPoint PPT Presentation

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Diversity in a dish: A human population-based organotypic in vitro - - PowerPoint PPT Presentation

Aug 21, 2023 22 likes •183 views

Society of Toxicology In Vitro & Alternative Methods Section Webinar, May 18 th 2017 Cardiotoxicity Adverse Outcome Pathways C-AOP STAR Center Diversity in a dish: A human population-based organotypic in vitro model for cardiotoxicity

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Diversity in a dish:

A human population-based organotypic in vitro model for cardiotoxicity testing

Fabian Grimm Department of Veterinary Integrative Biosciences Texas A&M University E-mail: fgrimm@cvm.tamu.edu

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Cardiotoxicity Adverse Outcome Pathways C-AOP STAR Center Society of Toxicology In Vitro & Alternative Methods Section Webinar, May 18th 2017

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Raymond Tice, PhD Kristen Ryan, PhD Mamta Behl, PhD Frederick Parham, PhD U.S. Environmental Protection Agency: STAR RD83516602 / RD83580201 Society of Toxicology: 2015-2016 Colgate-Palmolive Fellowship 2017-2018 Syngenta Fellowship Ivan Rusyn, MD PhD Weihsueh Chiu, PhD David Threadgill, PhD William Klaren, PhD Yasuhiro Iwata, DVM Sarah Burnett Alec Wright

Funding

Fred Wright, PhD David Reif, PhD John House, PhD

Texas A&M University North Carolina State University National Toxicology Program/ NIEHS

Blake Anson, PhD

Cellular Dynamics International

Oksana Sirenko, PhD

Molecular Devices LLC

Acknowledgements Society of Toxicology – In Vitro and Alternative Methods Specialty Section

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SLIDE 3

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Introduction: Bridging Emerging Technologies And Chemical Safety Assessments

The implementation of organotypic culture models in human health safety assessments is impeded by the lack of multidimensional high-throughput testing strategies that are:

  • Functionally and physiologically-relevant1,2,3
  • Medium- to high-throughput applicable format1,2,3
  • Amenable for in vitro-to-in vivo extrapolation3
  • Capable of estimating inter-individual susceptibilities to adverse chemical effects

Advances in stem cell technologies and organotypic culture methods have the potential to overcome major limitations in contemporary risk assessment:

  • Limited interpretability of animal model-derived data
  • Low-throughput asssociated with in vivo testing (“Chemical Data Gap”)
  • Standardized, rather than chemical-specific population-level adjustment factors

Goal: Demonstrate the potential of organotypic culture systems to fill crucial needs in chemical risk assessment using a population-based in vitro cardiotoxicity model

1. Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol. (2015) 13: 529-46 2. Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem. (2016) 18: 4407-19 3. Sirenko O, Grimm FA, Ryan KR, Iwata Y, Behl M, Wignall JA, Parham F, Anson B, Cromwell EF, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128

Adverse Outcome Pathway

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Uncertainty in extrapolating from animals to humans:

Dichloromethane IRIS: “The use of PBPK models to extrapolate internal doses from rats to humans reduces toxicokinetic uncertainty in extrapolating from the rat liver lesion data but does not account for the possibility that humans may be more sensitive than rats to dichloromethane due to toxicodynamic differences.”

Uncertainty in human variation (from average to sensitive):

Dichloromethane IRIS: “The probabilistic human PBPK model used in this assessment incorporates the best available information about variability in toxicokinetic disposition of dichloromethane in humans but does not account for humans who may be sensitive due to toxicodynamic factors.”

Advances in Induced Pluripotent Stem Cell (iPSC) Technologies Enable Population-wide studies

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Inter-Species Variability (10-fold) Intra-Species Variability (10-fold)

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Cardiotoxicity Adverse Outcome Pathways – A Three-Tiered Approach

100 µM 10 µM 1 µM 0.1 µM

Cardiotox Screening

Cytotoxicity [HC-Imaging] Mechanistic [Transcriptomics]

1: Assay Multiplexing and Quality Assessment 2: Bioactivity Profiling in iCell Cardiomyocytes 3: Population Variability Assessment

100 “healthy” donors [no known cardiovascular disease]

Functional, Cytotox & Mechanistic Panels

Functional [Ca2+-Flux]

Dose-Response Profiling QC Variability in chemical treated cardiomyocytes Inherent Biological Variability In untreated cardiomyocytes

“Point-of-Departure”

Bioactivity Profiling In a Single Individual Population Variability Assessment [phenotypic effects modeling and IVIVE]

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  • 1. Assay Multiplexing in iCell Cardiomyocytes

[Calcium-Flux, High-Content Cell Imaging, High-Throughput Transcriptomics]

Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol. (2015) 13: 529-546 Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem. (2016) 18: 4407-19

Functional: Ca2+-Flux Cytotoxicity: Live Cell Imaging

Hoechst MitoTracker Calcein AM

Mechanistic: Targeted Transcriptomics

FLIPR™ tetra ImageXpress™ Micro Confocal HiSeq 2500

8 cardiophysiologic descriptors, 10 cytotoxicity parameters Pathway analysis for mechanistic evaluation All output files are quantitative Concentration-response assessment 0- 90 min 24 hours

~ 3000 selected, “toxicologically relevant” transcripts

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  • 1. Assay Multiplexing in iCell Cardiomyocytes

[Cardiophysiologic Phenotyping and Reproducibilty Assessment]

RFU RFU Phenotypic Resemblance of In Vivo Drug Effects Reproducibility of Baseline Cardiophysiology Reproducibility of Chemical Effects in iCell Cardiomyocytes Positive Inotrope Negative Inotrope K+ channel antagonist

r2=0.97

Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol. (2015) 13: 529-546 Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem. (2016) 18: 4407-19

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  • 1. Assay Multiplexing in iCell Cardiomyocytes

[Targeted Transcriptomics using the TempO-seq platform]

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  • 2. Environmental Cardiotoxicity Profiling in iCell Cardiomyocytes

[69 Chemicals: Drugs, Pesticides, Flame Retardants, PAHs, Others]

Data Acquisition Quantitative Assessment

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Sirenko O, Grimm FA, Ryan KR, Iwata Y, Parham F, Wignall JA, Anson B, Cromwell EF, Behl M, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128

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1. Sirenko O, Grimm FA, Ryan KR, Iwata Y, Parham F, Wignall JA, Anson B, Cromwell EF, Behl M, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128 2. Pearce RG, Setzer RW, Strope CL, Sipes NS, Wambaugh JF J. Stat. Softw. (2017) In press. 3. Wetmore BA, Wambaugh JF, Ferguson SS, Sochaski MA, Rotroff DM, Freeman K, Clewell 3rd HJ, Dix DJ, Andersen ME, Houck KA, Allen B, Judson RS, Singh R, Kavlock RJ, Richard AM, Thomas RS. Toxicol. Sci. (2012) 125, 157–174. 4. Wetmore BA, Wambaugh JF, Ferguson SS, Li L, Clewell 3rd HJ, Judson RS, Freeman K, Bao W, Sochaski MA, Chu TM, Black MB, Healy E, Allen B, Andersen ME, Wolfinger RD, Thomas RS. Toxicol. Sci. (2013) 132, 327–346.

  • 2. Environmental Cardiotoxicity Profiling in iCell Cardiomyocytes

[In Vitro-to-In Vivo Extrapolation Using Reverse Toxicokinetics Data]

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margin of exposure

Plasma protein binding Liver clearance Human donor pool Human donor pool

Population based IVIVE model

Steady State Concentration (Css) Upper 95% percentile among healthy adults

In Vitro Pharmacokinetics

Cellular assays

  • Oral Equivalent Dose

(mg/Kg-day) Reverse Dosimetry Chemical

Comparison between Css and POD values

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  • 3. Population Variability Assessment in iPSC Cardiomyocytes:

[Study Design and Data Integration]

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Diversity in a Dish Concept for Cardiotoxicity Testing Experimental Design

iPSC reprogramming and differentiation

Population Variability Screening Donor Pool 100 individual, “healthy” donors

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  • 3. Population Variability Assessment in iPSC Cardiomyocytes:

[Inter-Indervidual Variability in Cardiophysiology of Untreated Cardiomyocytes]

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  • 3. Population Variability Assessment in iPSC Cardiomyocytes:

[Inter-Individual Variability in Cardiomyocytes after Chemical Treatment]

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  • 3. Population Variability Assessment in iPSC Cardiomyocytes:

[Inter-Individual Variability in Cardiomyocytes after Chemical Treatment]

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  • 3. Population Variability Assessment in iPSC Cardiomyocytes

[Population-Level Concentration-Response Assessment]

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  • 1. Collected Ca2+ flux and HC-Imaging data plus cell lysates for HT-transcriptomics in

concentration-response for ~140 chemicals in iPSC-derived cardiomyocytes

  • Data acquisition is complete for first batch of cells from 27 donors
  • Identical data sets will be generated for cells from an additional 70 donors
  • HT-transcriptomics currently underway
  • 2. Observable variability in baseline cardiophysiological parameters
  • not attributable to technical variation in plating efficiencies
  • is an important factor to be considered for evaluation of chemical effects
  • 3. Chemical treatments qualitatively reflect the anticipated phenotypic responses
  • Qualitative characteristics remain consistent for the vast majority of chemicals
  • 4. Quantitative variation in responses to chemical treatments is observable
  • Differences in chemical-associated potencies are an indicator of biol. variability

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Summary