Organotypic Culture Model to Analyze Developmental Limb Malformations Resulting from Toxicant/Teratogen Exposure

EPA Grant Number: R835736C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R835736
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Vanderbilt–Pittsburgh Resource for Organotypic Models for Predictive Toxicology (VPROMPT)
Center Director: Hutson, Michael Shane
Title: Organotypic Culture Model to Analyze Developmental Limb Malformations Resulting from Toxicant/Teratogen Exposure
Investigators: Tuan, Rocky
Institution: University of Pittsburgh - Main Campus , Vanderbilt University
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018
Project Amount: Refer to main center abstract for funding details.
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

Our objective is to develop robust three-dimensional (3D) organotypic culture models (OCMs) using human mesenchymal stem cells (MSCs) to examine critical phenomena of embryonic limb development and its susceptibility to perturbation by known teratogens and environmental toxicants. Our specific aims are: (1) to develop OCMs that recapitulate chondrogenic differentiation, subsequent chondrocyte maturation/hypertrophy, and finally cartilage segmentation; (2) validate these OCMs using their responses to known teratogens; (3) use these OCMs to evaluate chemicals from the TSCA inventory with suspected limb teratogenic activity; (4) couple our limb development OCMs to an upstream liver OCM to evaluate the role of hepatic metabolism and metabolic activation of xenobiotics on limb teratogenesis; and (5) assess species concordance at the OCM level by comparing results of toxicant exposure in OCMs using human MSCs versus rat embryonic limb bud mesenchymal cells. 

Approach:

We will develop two limb development OCMs: one using a precartilage disc (CD) of MSCs to recapitulate chondrogenic differentiation and subsequent chondrocyte maturation/hypertrophy; and a second using a hyaline cartilage rod of MSCs that is subjected to mechanical activation to model the initiation of cartilage segmentation. The OCMs will be maintained in microbioreactors, custom designed by our VPROMPT collaborators in Project 5 to enable both perfusion-controlled exposure to candidate toxicants/teratogens and in situ mechano-activation. The spatiotemporal and dosimetric responses of OCMs will be monitored for specific cellular and morphogenetic activities using non-invasive live imaging of OCMs carrying AAV6 promoter-reporter constructs. Reporters will be chosen to match bioactivities associated with limb development, including cell adhesion, differentiation, matrix production, hypertrophy, matrix turnover, apoptosis, and cartilage segmentation. We will also assess OCM production of specific growth factors, cytokines, and/or metabolites using ion mobility mass spectrometry (IM-MS) and immunochemical measurements. Finally, at the end of the culture period, the phenotype of the OCMs will be assessed by histological, biochemical, and gene expression (RT-PCR) analyses

Expected Results:

We expect to develop and validate two classes of OCMs using human MSCs for investigating toxicant exposure effects on chondrogenic steps in limb development. We expect these OCMs to be sufficiently robust to be useful for medium throughput toxicity screening and experimentally accessible to enable elucidation of Adverse Outcome Pathways. 

Publications and Presentations:

Publications have been submitted on this subproject: View all 3 publications for this subprojectView all 64 publications for this center

Journal Articles:

Journal Articles have been submitted on this subproject: View all 1 journal articles for this subprojectView all 15 journal articles for this center

Supplemental Keywords:

biology, genetics, infants, risk assessment, human health effects, reproductive effects, sensitive populations, pathophysiology, metabolism

Progress and Final Reports:

2015 Progress Report
2016 Progress Report


Main Center Abstract and Reports:

R835736    Vanderbilt–Pittsburgh Resource for Organotypic Models for Predictive Toxicology (VPROMPT)

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835736C001 Mammosphere Bioreactor For Life-Stage Specific Toxicology
R835736C002 Organotypic Culture Model to Analyze Developmental Limb Malformations Resulting from Toxicant/Teratogen Exposure
R835736C003 Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks
R835736C004 Organotypic Liver Model for Predictive Human Toxicology and Metabolism
R835736C005 Systems Engineering & Analysis for Organotypic Culture Models