High-throughput screening of toxicants that modulate extravillous trophoblast migration

Cassandra Meakin; Christine Kim; Thomas Lampert; Lauren M. Aleksunes

doi:10.1016/j.toxlet.2022.12.004

High-throughput screening of toxicants that modulate extravillous trophoblast migration

Cassandra Meakin, Christine Kim, Thomas Lampert, Lauren M. Aleksunes

Ernest Mario School of Pharmacy, Pharmacology & Toxicology

Rutgers, The State University

Research output: Contribution to journal › Article › peer-review

Abstract

Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris™ cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 h and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.

Original language	American English
Pages (from-to)	1-7
Number of pages	7
Journal	Toxicology Letters
Volume	375
DOIs	https://doi.org/10.1016/j.toxlet.2022.12.004
State	Published - Feb 15 2023

ASJC Scopus subject areas

Toxicology

Keywords

Bisphenol
Cadmium
Extravillous trophoblast
Migration
Placenta
Zearalenone

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10.1016/j.toxlet.2022.12.004

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title = "High-throughput screening of toxicants that modulate extravillous trophoblast migration",

abstract = "Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris{\texttrademark} cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 h and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.",

keywords = "Bisphenol, Cadmium, Extravillous trophoblast, Migration, Placenta, Zearalenone",

author = "Cassandra Meakin and Christine Kim and Thomas Lampert and Aleksunes, {Lauren M.}",

note = "Funding Information: This work was supported by the National Institute of Environmental Health Sciences [Grants R01ES029275 , T32ES007148 , and P30ES005022 ] and the National Center for Advancing Translational Sciences [Grant UL1TR003017 ]. Funding Information: This work was supported by the National Institute of Environmental Health Sciences [Grants R01ES029275, T32ES007148, and P30ES005022] and the National Center for Advancing Translational Sciences [Grant UL1TR003017]. The authors are able to make data available upon request. Supplemental Fig. 1. Immunofluorescent Characterization of HTR8/SVneo cells. Indirect immunofluorescent staining (green) against cytokeratin-7, a trophoblast marker, and vimentin, a fibroblast marker, was performed on fixed HTR8/SVneo cells. Nuclei were stained with Hoechst 33342 dye (blue). Images show no presence of fibroblasts indicating an enriched trophoblast population. Images were captured at 100X magnification. White bars represent 1000 µm. Supplemental Fig. 2. Concentration-dependent cytotoxicity in human trophoblasts following cadmium chloride treatment. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1–10 µM) of cadmium chloride for 48 h. Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p < 0.05) from vehicle control. Supplemental Fig. 3. Concentration-dependent cytotoxicity in human trophoblasts following treatment with environmental chemicals. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1–10 µM) of screening chemicals for 48 h. Environmental chemicals included organophosphate flame retardants (tricresyl phosphate (TCP), triphenyl phosphate (TPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP)), bisphenols (bisphenol A (BPA), bisphenol B (BPB), bisphenol S (BPS), bisphenol F (BPF)), and estrogenic mycotoxins including zearalenone (ZEN), its metabolites (α-ZOL and β-ZOL), and the synthetic form zeranol (ZER). Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p < 0.05) from vehicle control. Publisher Copyright: {\textcopyright} 2023 Elsevier B.V.",

year = "2023",

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day = "15",

doi = "10.1016/j.toxlet.2022.12.004",

language = "American English",

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T1 - High-throughput screening of toxicants that modulate extravillous trophoblast migration

AU - Meakin, Cassandra

AU - Kim, Christine

AU - Lampert, Thomas

AU - Aleksunes, Lauren M.

N1 - Funding Information: This work was supported by the National Institute of Environmental Health Sciences [Grants R01ES029275 , T32ES007148 , and P30ES005022 ] and the National Center for Advancing Translational Sciences [Grant UL1TR003017 ]. Funding Information: This work was supported by the National Institute of Environmental Health Sciences [Grants R01ES029275, T32ES007148, and P30ES005022] and the National Center for Advancing Translational Sciences [Grant UL1TR003017]. The authors are able to make data available upon request. Supplemental Fig. 1. Immunofluorescent Characterization of HTR8/SVneo cells. Indirect immunofluorescent staining (green) against cytokeratin-7, a trophoblast marker, and vimentin, a fibroblast marker, was performed on fixed HTR8/SVneo cells. Nuclei were stained with Hoechst 33342 dye (blue). Images show no presence of fibroblasts indicating an enriched trophoblast population. Images were captured at 100X magnification. White bars represent 1000 µm. Supplemental Fig. 2. Concentration-dependent cytotoxicity in human trophoblasts following cadmium chloride treatment. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1–10 µM) of cadmium chloride for 48 h. Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p < 0.05) from vehicle control. Supplemental Fig. 3. Concentration-dependent cytotoxicity in human trophoblasts following treatment with environmental chemicals. Apoptosis was detected using propidium iodide staining of HTR8/SVneo cells following treatment with increasing concentrations (0.1–10 µM) of screening chemicals for 48 h. Environmental chemicals included organophosphate flame retardants (tricresyl phosphate (TCP), triphenyl phosphate (TPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP)), bisphenols (bisphenol A (BPA), bisphenol B (BPB), bisphenol S (BPS), bisphenol F (BPF)), and estrogenic mycotoxins including zearalenone (ZEN), its metabolites (α-ZOL and β-ZOL), and the synthetic form zeranol (ZER). Total cell numbers were calculated using staining of nuclei with Hoechst 33342 dye. The percentage of apoptotic cells was calculated as a percentage of propidium iodide-stained cells divided by the total number of Hoechst-stained cells. Data are presented as % apoptotic cells ± SD. Asterisks (*) represent a statistically significant difference (p < 0.05) from vehicle control. Publisher Copyright: © 2023 Elsevier B.V.

PY - 2023/2/15

Y1 - 2023/2/15

N2 - Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris™ cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 h and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.

AB - Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris™ cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 h and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.

KW - Bisphenol

KW - Cadmium

KW - Extravillous trophoblast

KW - Migration

KW - Placenta

KW - Zearalenone

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High-throughput screening of toxicants that modulate extravillous trophoblast migration

Abstract

ASJC Scopus subject areas

Keywords

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