2018 Progress Report: Linking in vitro-to-in vivoToxicity Testing Using Genetically-matchedOrganoids and Mice from a Novel Genetic Reference Population

EPA Grant Number: R835802C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R835802
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Organotypic Culture Models For Predictive Toxicology Center
Center Director: Rusyn, Ivan
Title: Linking in vitro-to-in vivoToxicity Testing Using Genetically-matchedOrganoids and Mice from a Novel Genetic Reference Population
Investigators: Threadgill, David W.
Institution: University of North Carolina at Chapel Hill , Texas A & M University
EPA Project Officer: Klieforth, Barbara I
Project Period: June 1, 2015 through May 31, 2019 (Extended to May 31, 2020)
Project Period Covered by this Report: June 1, 2018 through May 31,2019
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Human Health , Health , Safer Chemicals

Objective:

Important target areas for addressing data gaps through in vitro screening include evaluations of cardiotoxicity. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative environmental agents remain largely uncharacterized. Moreover, cardiotoxicity remains among the most pronounced reasons, comparable to those associated with hepatotoxicity, for drug attrition during clinical trials and post-marketing. Current pre-clinical approaches for cardiophysiologic evaluations of chemicals rely almost exclusively on large animal models and do not consider underlying genetic variation in evaluation of risk. Use of large animals has significant limitations in terms of cost and complexity of the studies, especially for the inclusion of genetic diversity. Hence, there is a pressing demand for the development of comprehensive, multi-parametric screening strategies that provide improved predictability of cardiotoxic effects.

The central hypotheses of this project are that: (i) genetic variation influences how mouse iPSC-derived embryoid body (EB)-OCM respond to insults; (ii) analyses using the CC population can be used to uncover genetic and transcriptional networks that control cardiotoxic responses within AOP; and (iii) discoveries using in vitro OCMs are predictive of in vivo cardiotoxic responses. We are testing these hypotheses by pursuing the following specific objectives.

Specific Objective 1: To perform in vitro screening of ToxCast chemicals using iPSC-derived EB-OCM CC genetic reference population.

Specific Objective 2: To collect RNA-seq data from EB-OCM to inform molecular and cellular events in the AOP for cardiotoxicity.

Specific Objective 3: To validate the IVIVE paradigm and risk prediction using genetically matched and exposure-naïve CC mice, respectively.

Progress Summary:

Most of the work in the past year has focused on the activities under proposed Specific Objective 1 and 3. To date, we have accomplished the following:

  • Despite early success in generating robust embryoid bodies from CC derived iPSC, during year 3 most lines began to lose totipotency, and did not form reproducible embryoid bodies. We have spent the last year investigating the source of the culture issues. Surprisingly, a collaborator at the University of Washington whom we had sent several iPSC was not having the same issue, and was getting robust differentiation. After discussing their protocols, which were from out lab, and testing multiple sources of reagents and tracking changes to reagent production that may have contributed, we were changed sources from early successful embryoid body generation to the present, the functionality of the serum had changed and caused the iSPC cultures to differentiate into a metastable epiblast stem cell (EpiSC) state that do not readily form embryoid bodies. These cells can be readily reversed to an iPSC state under proper conditions. We have found that by changing how the serum is prepared allows the iPSC to remain totipotent and to revert EpiES back to an iSPC state. Although delayed, the iPSC are back on track to make substantial project over the remaining time of the project.
  • To investigate the in vitro-to-in vivo predictive ability of OCM, we acquired two ECGenie instruments for recording electrocardiograms in vivo. These instruments, along with echocardiography (Echo) using a Vevo High-Frequency Ultrasound have been used to generate ECG and detailed baseline cardiac phenotypes for 31 CC lines using both male and female mice. This is an important foundation for Aim 3. These Echo measures were performed in both conscience and unconscious mice, with substantial strain-specific differences observed in many measurements that are far greater than anticipated.
  • We have built a web app called gEKGo that allows ECG tracings to be processed to extract relevant phenotypic data that overcomes variation in inter-strain differences in ECG that were challenging for the current commercial software based on C57BL/6 ECG patterns.
  • To evaluate the utility of mouse genetic diversity to model human diversity, two cardiotoxicants from Project 1 (chloroquine and isoproterenol) were evaluated in vivo using 8 CC lines. Exposed mice were evaluated for ECGs using the ECGenie and cardiac function using Echo followed by Spectral Tracking analysis for detailed cardiovascular function.

Future Activities:

We will use existing iPS lines with the new media preparation to confirm the reproducibility of embryoid body formation. The eight lines used to screen chloroquine and isoproterenol in vivo will be tested for in vitro effects of cardiomyocyte function in collaboration with Project 1. The graduate student working on this project recently started a summer externship with Amgen in their iPSC program and will be focusing on cardiotoxicity. This opportunity should enhance moving the project forward over the remaining time. Additional staff have also been recruited to focus on iPSC and embryoid body culture studies. This should be completed by the end of summer, after which 8 cell lines, corresponding to the CC lines used in vivo, will be used to screen putative cardiotoxicants identified in Project 1.

  • An additional six chemicals beyond the two already screened will also be screened in vivo using the same 8 CC lines already used.
  • Analysis of baseline cardiac function using both EKG an Echo (conscience and unconscious) will be completed by the fall.
  • By conclusion of the project, we anticipate having generated detailed baseline cardiac phenotypes for 31 CC lines, performed genetic analysis to identify the genetic architecture underlying cardiac phenotypic variation, tested 8 iPSC and corresponding CC lines for 8 chemicals identified in Project 1 as candidate cardiotoxicants. Although not of the scale original envisioned, the results will still address the original primary goal of the project, are mice predictive of human cardiotoxicity and do in vitro OCM accurately recapitulate in vivo phenotypes.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 3 publications 2 publications in selected types All 2 journal articles
Other center views: All 192 publications 35 publications in selected types All 35 journal articles
Type Citation Sub Project Document Sources
Journal Article Garbutt TA, Konneker TI, Konganti K, Hillhouse AE, Swift-Haire F, Jones A, Phelps D, Aylor DL, Threadgill D. Permissiveness to form pluripotent stem cells may be an evolutionarily derived characteristic in Mus musculus. Scientific Reports 2018;8:14706. R835802 (2018)
R835802 (Final)
R835802C002 (2018)
  • Full-text from PubMed
  • Abstract from PubMed
  • Full-text: Scientific Reports - Full Text HTML and PDF
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  • Abstract: Abstract
  • Journal Article Konganti K, Ehrlich A, Rusyn I, Threadgill DW. gQTL:a web application for QTL analysis using the collaborative cross mouse genetic reference population. G3:Genes, Genomes, Genetics 2018;8(8):2559-2562 R835802 (2017)
    R835802 (2018)
    R835802 (Final)
    R835802C001 (2018)
    R835802C002 (2018)
  • Abstract from PubMed
  • Full-text: G3-Full Text PDF
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  • Abstract: G3-Abstract
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  • Supplemental Keywords:

    cardiovascular, stem cells, toxicity pathway, variability, pharmacokinetic model.

    Relevant Websites:

    gQTL: web-based genetic analysis of CC data Exit
    gEKGo: web-based automated automatic PQRST detection and analysis with manual override capability Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016
  • 2017

  • Main Center Abstract and Reports:

    R835802    Organotypic Culture Models For Predictive Toxicology Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835802C001 High-throughput Hazard,Dose-responseandPopulationVariabilityAssessmentof Cardiotoxicity in aHumanInducedPluripotentStem Cell(iPSC)-derivedin vitro Culture Model
    R835802C002 Linking in vitro-to-in vivoToxicity Testing Using Genetically-matchedOrganoids and Mice from a Novel Genetic Reference Population
    R835802C003 A Pipeline for in vitro-to-in vivo Extrapolation, Population Modeling, & Prioritization