Characterization of Xenobiotic and Steroid Disposition Potential of Human Placental Tissue and Cell Lines (BeWo, JEG-3, JAR, and HTR-8/SVneo) by Quantitative Proteomics

Kruger, Laken; Lapehn, Samantha; Paquette, Alison G.; Singh, Dilip Kumar; MacDonald, James W.; Bammler, Theo K.; Enquobahrie, Daniel A.; Zhao, Qi; Mozhui, Khyobeni; Sathyanarayana, Sheela; Prasad, Bhagwat

doi:10.1124/dmd.123.001345

Cited by 3 publications

(1 citation statement)

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“…Examples of this work include: an evaluation of BeWo, JAR, JEG-3, and HTR-8/SVneo cells as a suitable model for preeclampsia drug screening [13], an assessment of BeWo, JAR, JEG-3, and ACH-3P as models for endocrine and transport studies [10], and DNA methylation profiling of JAR, BeWo, JEG-3, HTR-8/SVneo, AC1M-32, TEV-1, and SWAN71 [14]. Additional studies include an analysis of HTR-8/SVneo, JEG-3, JAR and BeWo for epithelial and mesenchymal cell markers [7], studies of invasive properties of HTR-8/SVneo and JEG-3 cells [15,16], a quantitative proteomic evaluation of xenobiotic and steroid metabolizing enzymes in placental tissue and BeWo, JEG-3, JAR, and HTR-8/SVneo cell lines [17], as well as studies of single genes or small gene subsets across multiple placental cell lines [18,19]. Overall, these studies reveal that in vitro models of the human placenta have individual strengths that should be considered during model selection; however, they have not directly compared these models to human placental tissue.…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic comparison of in vitro models of the human placenta

Lapehn,

Nair,

Firsick

et al. 2024

Preprint

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Studying the human placenta through in vitro cell culture methods is necessary due to limited access and amenability of human placental tissue to certain experimental methods as well as distinct anatomical and physiological differences between animal and human placentas. Selecting an in vitro culture model of the human placenta is challenging due to representation of different trophoblast cell types with distinct biological roles and limited comparative studies that define key characteristics of these models. Therefore, the aim of this research was to create a comprehensive transcriptomic comparison of common in vitro models of the human placenta compared to bulk placental tissue from the CANDLE and GAPPS cohorts (N=1083). We performed differential gene expression analysis on publicly available RNA sequencing data from 6 common in vitro models of the human placenta (HTR-8/SVneo, BeWo, JEG-3, JAR, Primary Trophoblasts, and Villous Explants) and compared to CANDLE and GAPPS bulk placental tissue or cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell types derived from bulk placental tissue. All in vitro placental models had a substantial number of differentially expressed genes (DEGs, FDR<0.01) compared to the CANDLE and GAPPS placentas (Average DEGs=10,873), and the individual trophoblast cell types (Average DEGs=5,346), indicating that there are vast differences in gene expression compared to bulk and cell-type specific human placental tissue. Hierarchical clustering identified 53 gene clusters with distinct expression profiles across placental models, with 22 clusters enriched for specific KEGG pathways, 7 clusters enriched for high-expression placental genes, and 7 clusters enriched for absorption, distribution, metabolism, and excretion genes. In vitro placental models were classified by fetal sex based on expression of Y-chromosome genes that identified HTR-8/SVneo cells as being of female origin, while JEG-3, JAR, and BeWo cells are of male origin. Overall, none of the models were a close approximation of the transcriptome of bulk human placental tissue, highlighting the challenges with model selection. To enable researchers to select appropriate models, we have compiled data on differential gene expression, clustering, and fetal sex into an accessible web application:“Comparative Transcriptomic Placental Model Atlas (CTPMA)”which can be utilized by researchers to make informed decisions about their selection of in vitro placental models.

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Section: Introductionmentioning

confidence: 99%

Transcriptomic comparison of in vitro models of the human placenta

Lapehn,

Nair,

Firsick

et al. 2024

Preprint

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Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Li,

Guo,

Zhou

et al. 2024

The American Journal of Pathology

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Decidual macrophages derived NO downregulates PD-L1 in trophoblasts leading to decreased Treg cells in recurrent miscarriage

et al. 2023

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IntroductionPlacental trophoblasts contribute to regulatory T (Treg) function via the programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway during normal pregnancy. Decreased expression of PD-L1 in trophoblasts was closely associated with Treg deficiency in the development of pregnancy failure. Thus, targeting PD-L1 might be a novel therapy to prevent pregnancy loss. However, the mechanisms for modulating the expression of PD-L1 in trophoblasts are an enigma.MethodsThe proportion of decidual Treg cells, and the profile of decidual macrophages (DMs) sampled from women with normal pregnancy (NP) and recurrent miscarriage (RM) were evaluated by flow cytometry. The expression of Yin and Yang 1 protein (YY1) and PD-L1 in human villous were measured by Immunohistochemistry (IHC), qRT-PCR and western blot. The determination of soluble PD-L1 (sPD-L1) in serum from NP and RM, and trophoblast conditioned media (TCM) was performed by the PD-L1 SimpleStep ELISA kit. Knockdown of YY1 was processed in the human trophoblast derived cell lines, HTR-8 and Bewo, with siYY1 transfection. Peripheral naïve CD4+ T cells were isolated from women with NP for the in vitro culture. The percentages of Treg cells differentiated from peripheral naïve CD4+ T cells were measured by flow cytometry. The interaction between YY1 and CD274 was proved by CHIP. The expression of inducible nitric oxide synthase (iNOS) in decidua was evaluated by IHC. The level of NO in serum from women with NP and RM was determined by the Griess reagent system. The effects of NO on YY1 were determined by the in vitro culture of HTR-8 cells with the NO donor, SNAP. The in vivo model comprising twelve pregnant mice and underwent different treatment. The percentages of Treg cells in murine uterus were measured by flow cytometry. Similarly, Western blot and IHC were performed to determine the expression of YY1 and PD-L1 in murine placenta.ResultsDecreased expression of YY1 and PD-L1 in trophoblasts and lower proportion of decidual Treg cells were observed in patients with RM. Knockdown of YY1 contributes to a lower expression of YY1 and PD-L1. Soluble PD-L1 in the supernatant from HTR-8 cells was also decreased with siYY1 administration. Lower Treg differentiation was observed in the presence of supernatant from HTR-8 cells treated with siYY1. CHIP analysis revealed that endogenous YY1 directly occupied the promoter region of the CD274 (PD-L1) gene. Accompanied with increased M1 DMs, higher NO was observed in serum sampled from patients with RM. In the presence of Reduced expression of YY1 and PD-L1 was observed in HTR-8 cells with the treatment of SNAP. Furthermore, less Treg differentiation was observed with SNAP treated TCM. Moreover, our in vivo data found that YY1 deficiency was associated with decreased PD-L1, which further resulting in less Treg differentiation and Treg deficiency at the maternal-fetal interface and increased embryo loss.DiscussionOur work found the modulatory capacity of YY1 on PD-L1 in trophoblasts during early pregnancy. Furthermore, reduced YY1 was supposed resulting from higher levels of NO produced from the M1 DMs in RM.

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Characterization of Xenobiotic and Steroid Disposition Potential of Human Placental Tissue and Cell Lines (BeWo, JEG-3, JAR, and HTR-8/SVneo) by Quantitative Proteomics

Abstract: Non-standard Abbreviations: -Iodoacetamide (IAA) -Bovine Serum Albumin (BSA) -Dithiothreitol (DTT) -Label free quantification (LFQ) -Di(2-ethylhexyl) (DEHP) -Diisononyl (DINP)

Cited by 3 publications

References 60 publications

Transcriptomic comparison of in vitro models of the human placenta

Transcriptomic comparison of in vitro models of the human placenta

Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Decidual macrophages derived NO downregulates PD-L1 in trophoblasts leading to decreased Treg cells in recurrent miscarriage

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