Dynamic In Vitro Models for Reproductive and Developmental Toxicology

Abstract

As in vitro models play an increasingly important role in evaluation of potential developmental and reproductive toxicants, it is essential that we define how well these models can capture developmental stages and processes that are vulnerable to toxicant perturbation. In this research we evaluate two dynamic in vitro models: a primary co-culture of neonatal rat testis and a human progenitor cell model of neuronal differentiation. Longterm viability, morphological characteristics, and expression of cell type- and stage-specific proteins in in vitro cultures were characterized by live/dead cell staining, immunofluorescent imaging, and western blotting. We then defined a framework for quantifying dynamic changes in pathways through time in normal development in vivo. Gene expression and pathway dynamics distilled from microarray data were used to anchor the in vitro models to in vivo developmental processes. Striking similarities were found between in vivo and in vitro models in gene expression for key pathways associated with each developmental process. Basic characterization revealed that testicular co-cultures survive up to 21 days in culture and contain spermatogonial germ cells, Sertoli cells and Leydig cells, form tube-like structures in vitro and actively produce testosterone through the first week in culture. Pathway analysis of gene expression data through time reveals that the testicular co-culture captures key developmental processes that can be targeted by male reproductive toxicants, including Sertoli cell and Leydig cell differentiation, testosterone production, immune activity, signal transduction, tube formation, and tight junction formation. Basic characterization of differentiating neural progenitor cells indicates that the cells survive and differentiate through 21 days in culture, increasingly expressing protein associated with differentiated neurons, adopting neuronal morphology and forming complex neural networks. Pathway analysis reveals that differentiating neural progenitor cells successfully capture differentiation towards a forebrain identity, expressing receptors associated with specific neuronal subtypes. In addition to identifying processes that are clearly captured in vitro, we also define the limits of these models by identifying gene expression dynamics found only in vitro or only in vivo. By further defining the domain of applicability for each of these in vitro models, we can help to ensure that these models are used effectively and appropriately for identification of potential chemical hazards.