Grantee Research Project Results
2015 Progress Report: Linking in vitro-to-in vivoToxicity Testing Using Genetically-matchedOrganoids and Mice from a Novel Genetic Reference Population
EPA Grant Number: R835802C002Subproject: 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: Aja, Hayley
Project Period: June 1, 2015 through May 31, 2019 (Extended to May 31, 2020)
Project Period Covered by this Report: June 1, 2015 through May 31,2016
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
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 up to 200 ToxCast chemicals using iPSC-derived EB-OCM from both sexes of 96 genomes of the 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. To date, we have accomplished the following:
- We completed development of mouse embryonic fibroblast (MEF) lines from all available Collaborative Cross (CC) mouse strains. We are not converting these lines into induced pluripotent stem (iPS) cell lines. We have completed generation of eight lines from the CC parental strains and have begun induction of batches the CC lines in parallel after bringing on a new graduate student to work on the project (using a approached described below).
- At the OCM Center’s meeting in New Orleans, LA, discussions were held with Dr. Jamie Thomson (the University of Wisconsin-Morgridge Institute OCM center) about optimal cell differentiation approaches, which lead to the substitution of a Sendai Virus-based approach for our original adenovirus-based approach. This allows a one-step conversion of MEFs to iPSs since we no longer need to excise the adenoviral vector before inducing EBs. We had observed that if the transforming vector remained, EBs could form but not terminally differential into function cardiomyocytes. The Sendai Virus is non-integrating and degraded within a few iPS cell passages.
- To investigate the in vitro-to-in vivo predictive ability of OCM, we have been exploring cardiac defects including early endocardial cushion morphogenesis that results from exposure to dioxin in a related project. We have collected exposure data from 16 strains in vivo and will be comparing the response of those same stains in vitro.
- We have acquired (via institution funding sources) advanced cardiac image processing software (VivoStrain) that can quantify strain measures using speckle-tracking analysis, which are far more sensitive for detecting cardiotoxicity in vivo. This will be an enormous benefit for Objective 3. We have already tested the approach and submitted a manuscript documenting our capability.
- With Project 1, we have finalized the list of 140 compounds to be used in screening. Chemical selection was coordinated with FDA, NTP and EPA-NCCT. All compounds have been procured, dilutions prepared, and chemical master plates created for screening.
Progress to date has set us up to make substantial advances on OCM exposure in the next year.
Future Activities:
- We will develop iPS from the entire CC MEF population.
- We will perform screening studies on the first 30 iPS lines from CC mice using the identified 140 compound set.
- We will work with Project 1 staff to conduct screening of mouse-derived EBs.
- We plan to conduct targeted transcriptomic analyses using TempO-seq high-throughput transcriptomics as the mouse panel is developed.
- We will work closely with Project 3 staff to analyze the data from high-content screening and high-throughput transcriptomics.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this subprojectSupplemental Keywords:
cardiovascular, stem cells, toxicity pathway, variability, pharmacokinetic modelProgress and Final Reports:
Original AbstractMain 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-responseandPopulationVariabilityAssessmentofCardiotoxicity in aHumanInducedPluripotentStem Cell(iPSC)-derivedinvitro 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
2 journal articles for this subproject
Main Center: R835802
149 publications for this center
44 journal articles for this center