2018 Progress Report: Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks

EPA Grant Number: R835736C003
Subproject: this is subproject number 003 , 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
Center Director: Hutson, Michael Shane
Title: Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks
Investigators: Osteen, Kevin G. , Aronoff, David , Bruner-Tran, Kaylon L.
Institution: University of Pittsburgh Main Campus , Vanderbilt University
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2017 through November 30,2018
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

Fetal membrane inflammation due to infection, known as chorioamnionitis (CAM), is a leading cause of preterm birth (PTB). Although numerous infectious agents have been associated with CAM; many women with microbial contamination of the amniotic cavity carry their pregnancies to term. Equally significant, women without an identifiable infection may also exhibit signs of CAM and deliver preterm. These clinical observations strongly suggest that, in addition to infection, host factors must also influence the risk for CAM-associated PTB. Supporting this concept, in a murine model, we identified a doubling in the occurrence of infection-mediated PTB in animals with a previous exposure to an environmental toxicant (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD). This two-hit model of CAM is further supported by our in vitro studies using isolated human endometrial cells, which, following TCDD exposure, also exhibit an enhanced response to an inflammatory challenge related to an infection (i.e., LPS, IL-1β or TNF-α). Taken together, our data suggest the novel hypothesis that environmental toxicants prime the gravid uterus for exaggerated inflammatory responses to microbial invasion. More than 80,000 chemicals have been released into our environment since the Toxic Substances Control Act of 1976; however, few have undergone controlled experimental examination related to reproductive health. Therefore, we have developed unique platforms for the establishment and analysis of organ-on-chip systems amenable to medium throughput screening.

The Objective of Project 3 is to develop and validate an instrumented fetal membrane on a chip (IFMOC) system in order to specifically identify toxicants which negatively impact the maintenance of pregnancy.

Progress Summary:

Specific Aim 1: Define the temporal and concentration-dependent effects of TCDD on native fetal membrane immune responses to infection. As noted in our previous reports, native fetal membranes from non-laboring women at delivery exhibit an enhanced responsiveness to an infection mimic (lipopolysaccharides (LPS, 10 ng/ml)) following pretreatments with TCDD (10 nM). These findings support our central hypothesis that environmental toxicants prime the gravid uterus for exaggerated inflammatory responses. Our studies further identified inflammatory biomarkers that are influenced by TCDD plus LPS treatment (IL- 1 β, TNF-α, IL-6 and IL-8). More recently, we have examined the potential influence of TCDD metabolism by the liver on altering the downstream tissue response. Our initial studies demonstrated the IFMOC cells are not adversely affected by the liver media diluted 50% with fresh media. Next, we treated human endometrial stromal cells with liver conditioned media (CM) collected following exposure to TCDD. Importantly, treatment of stromal cells with liver CM containing estradiol and progesterone (EP) induces decidualization, as expected. However, liver CM that additionally contains TCDD fails to induce stromal cell differentiation, indicating that liver metabolism of TCDD does not significantly reduce the adverse effects of this compound on the endometrium. Studies examining additional toxicants are currently underway.

Specific Aim 2: Implement an in vitro, microscaled, living, instrumented fetal membrane on-a-chip (IFMOC) that recapitulates the physiological properties of the human tissue. A major goal of the current application is to determine the utility of the IFMOC compared to traditional cultures for screening of individual toxicants. However, humans are not exposed to single toxicants, but rather combinations of agents. Therefore, in order to assess the impact of combinatorial exposures, we directly compared the two chamber organ-on-chip (OoC) model of the human fetal membrane containing human amnion epithelial cells and decidual cells to the transwell cultures of human fetal membranes. Both culture types were exposed to cigarette smoke extract versus TCDD alone and subsequently examined for membrane integrity/permeability and cellular senescence. Our results reveal CSE treatment of AEC produced higher membrane permeabilization (1.6 fold) than treatment on the decidual side (1.2 fold); whereas TCDD (dioxin) treatment on the decidual cell side produced higher permeabilization (1.1 fold) than treatment of the same on AEC side (0.7 fold). In contrast, this differential effect was minimal in corresponding transwell treatments. Next, to assess senescence associated with toxicant exposure, we utilized expression of the Senescence Associated β-galactosidase (SA-β-Gal), which is an independent biomarker of DNA synthesis and distinguishes senescent cells from normal cells. Compared to control cultures, CSE and dioxin treatments induced cellular senescence in AECs and decidual cells after 48 hours. Additionally, both CSE and dioxin treated sides in the FM-OO-C induced senescence and forced similar changes on the opposite chamber (i.e. decidua treatment of CSE induced AEC senescence 1.4 fold higher than controls, while AEC treatment with dioxin induced decidual senescence 1.4 fold higher than controls), suggesting transfer of biochemical signals between layers are capable of producing labor-associated changes. Transwell cultures compared to the corresponding FM-OO-C treatments did not allow propagation of such signals as indicated by minimal effect on SA-β-Gal.

Specific Aim 3: Validate the IFMOC as a model of environmental toxicant immunomodulation compared with primary human fetal membranes. As noted above, using IFMOC 3.0 we have now exposed the primary cell types that comprise the endocrine/immune sensitive cells of the human maternal-fetal interface to a variety of toxicants and/or inflammatory agents. Using identical treatments with estradiol (1nM) and medroxyprogesterone acetate (500 nM) during the decidualization phase within our device we have confirmed the cellular responses within the IFMOC relative to the responses observed in the transwell system. During the next year's studies each observation within the IFMOC related to disruption of progesterone action will be compared to the results obtained within the transwell system. These comparisons are necessary to validate the findings within the IFMOC 3.0 relative to intact membranes. However, an important limitation of the transwell is its 48-hour lifespan. In contrast, the utility of the IFMOC is its capacity to maintain cell viability for up to 30 days. Thus, the next step will utilize these extended cultures and are expected to reveal toxicant-mediated responses that cannot be duplicated within the time constraints of the transwells. Specifically, we have noted epigenetic modifications affecting progesterone receptor expression in the IFMOC, a toxicant mediated response that cannot be seen in the transwell cultures.


Journal Articles on this Report : 10 Displayed | Download in RIS Format

Other subproject views: All 21 publications 10 publications in selected types All 10 journal articles
Other center views: All 149 publications 39 publications in selected types All 39 journal articles
Type Citation Sub Project Document Sources
Journal Article Bruner-Tran KL, Duleba AJ, Taylor HS, Osteen KG. Developmental toxicant exposure is associated with transgenerational adenomyosis in a murine model. Biology of Reproduction 2016;95(4):73 (10 pp.). R835736 (2015)
R835736 (2016)
R835736 (2018)
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  • Journal Article Bruner-Tran KL, Gnecco J, Ding T, Glore DR, Pensabene V, Osteen KG. Exposure to the environmental endocrine disruptor TCDD and human reproductive dysfunction: translating lessons from murine models. Reproductive Toxicology 2017;68:59-71. R835736 (2015)
    R835736 (2016)
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  • Journal Article Bruner-Tran KL, Mokshagundam S, Herington JL, Osteen KG. Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets. Current Women's Health Reviews 2018;14(2):173–188 R835736C003 (2018)
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  • Journal Article Ding T, Lambert LA, Aronoff DM, Osteen KG, Bruner-Tran KL. Sex-dependent influence of developmental toxicant exposure on group B Streptococcus -mediated preterm birth in a murine model. Reproductive Sciences 2018;25(5):662-673. R835736 (2017)
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  • Journal Article Gnecco JS, Anders AP, Cliffel D, Pensabene V, Rogers LM, Osteen K, Aronoff DM. Instrumenting a fetal membrane on a chip as emerging technology for preterm birth research. Current Pharmaceutical Design 2017;23(40):6115-6124. R835736 (2016)
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  • Journal Article Gnecco JS, Pensabene V, Li DJ, Ding T, Hui EE, Bruner-Tran KL, Osteen KG. Compartmentalized culture of perivascular stroma and endothelial cells in a microfluidic model of the human endometrium. Annals of Biomedical Engineering 2017;45(7):1758-1769. R835736 (2015)
    R835736 (2016)
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  • Journal Article Gnecco JS, Ding T, Smith C, Lu J, Bruner-Tran KL, Osteen KG. Hemodynamic forces enhance decidualization via endothelial-derived prostaglandin E2 and prostacyclin in a microfluidic model of the human endometrium. Human Reproduction 2019;43(4):702-714 R835736C003 (2018)
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  • Journal Article Hutson MS, Alexander PG, Allwardt V, Aronoff DM, Bruner-Tran KL, Cliffel DE, Davidson JM, Gough A, Markov DA, McCawley LJ, McKenzie JR, McLean JA, Osteen KG, Pensabene V, Samson PC, Senutovitch NK, Sherrod SD, Shotwell MS, Taylor DL, Tetz LM, Tuan RS, Vernetti LA, Wikswo JP. Organs-on-chips as bridges for predictive toxicology. Applied In Vitro Toxicology 2016;2(2):97-102. R835736 (2015)
    R835736 (2016)
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  • Journal Article Rogers JM, Anders AP, Doster RS, Gill EA, Gnecco JS, Holley JM, Randis TM, Ratner AJ, Gaddy JA, Osteen K, Arnoff DM. Decidual stromal cell-derived PGE(2) regulates macrophage responses to microbial threat. American Journal of Reproductive Immunology 2018;80(4). R835736C003 (2018)
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  • Journal Article Ding T et al, 2018. Reproductive Sciences 25(5):662-673 Bruner-Tran KL et al, 2018. Current Women’s Health Rev Jun;14(2):173-188 Rogers et al, 2018. American Journal of Reproductive Immunology Oct;80(4):e13032. R835736 (2018)
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    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • 2017 Progress Report

  • Main Center Abstract and Reports:

    R835736    Vanderbilt Pittsburgh Resource for Organotypic Models for Predictive Toxicology

    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 LimbMalformationsResulting 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