Research ArticleMICROBIOLOGY

A three-dimensional culture system recapitulates placental syncytiotrophoblast development and microbial resistance

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Science Advances  04 Mar 2016:
Vol. 2, no. 3, e1501462
DOI: 10.1126/sciadv.1501462
  • Fig. 1 Coculturing of human trophoblast JEG-3 cells with human microvascular cells promotes their ability to be cultured in 3D.

    (A) Schematic for the monotypic culturing of trophoblast cell lines in the RWV bioreactor. (B) Scanning electron micrograph of JEG-3 cells cultured in the RWV bioreactor for 21 days. Yellow arrows denote uncoated beads and white arrows denote unattached cells. (C) Schematic of the 3D coculture-based system of trophoblast cell lines using the RWV bioreactor. (D) Actin localization (green) of human brain microvascular endothelial cells (HBMECs) (top row) and human foreskin fibroblast (HFF) cells (bottom row) cultured in the RWV bioreactor for 5 days. 4′,6-Diamidino-2-phenylindole (DAPI)–stained nuclei are shown in blue. Cross section of beads is shown on the right. (E) Scanning electron micrographs of HBMECs (top) or RL95-2 cells cultured in the RWV bioreactor for 21 days. (F) (Top) Confocal microscopy for cytokeratin-19 (red) in JEG-3/HBMEC cocultured Cytodex beads cultured for 21 days. (Bottom) Scanning electron micrograph of JEG-3/HBMEC cocultured Cytodex beads cultured for 21 days.

  • Fig. 2 3D cultures of JEG-3 cells express syncytiotrophoblast-specific markers and fuse to form syncytia.

    (A) Enzyme-linked immunosorbent assays (ELISAs) for βhCG from supernatants of JEG-3 cells cultured in 2D (gray for 72 to 96 hours) or 3D (red for 21 days) or from PHT cells (blue, cultured ~72 hours). In all cases, medium was changed 24 hours before collection for ELISA. Levels of βhCG are presented as milli–international units per milliliter and were normalized to total protein from each cell condition to account for differences in cell number. Data are shown as means ± SD. **P < 0.01, *P < 0.05. (B) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) for human placental lactogen (hPL), hCG, syncytin, MFDS2, or placental protein 13 (PP13) from JEG-3 cells cultured in 2D (gray) or 3D (red) or from PHT cells (blue). Data are from three independent STLVs or PHT preparations, as indicated, and are shown as means ± SD. ***P < 0.001, **P < 0.01. n.s., not significant. (C) Confocal microscopy for ZO-1 (red) in JEG-3/HBMEC cocultured Cytodex beads cultured for 21 days (top row) or 2D cultures of JEG-3 cells (bottom row). DAPI-stained nuclei are shown in blue. (D) Fusion ratio of JEG-3 cells cultured in 2D and treated with the indicated conditioned medium (CM) for 7 to 10 days, from JEG-3 cells cultured in 3D, or from PHT cells. n.d., not detected. ***P < 0.001. (E) Scanning electron micrographs of JEG-3 cells cultured in 2D (top row) or 3D (bottom row).

  • Fig. 3 3D cultures of JEG-3 cells have transcriptional profiles with similarity to cultured primary human trophoblasts.

    (A) Hierarchical clustering heat map of genes expressed in two cultures of JEG-3 cells grown in 2D or 3D and a single preparation of PHT cells or HBMECs cultured in 3D. (B) Hierarchical clustering heat map of differentially expressed genes between two cultures of JEG-3 cells grown in 2D and PHT cells. Genes that were specifically up-regulated in PHT cells were used to create the “PHT-enriched library” used to identify the genes in (C). (C) Hierarchical clustering heat map of 55 genes specifically expressed in 3D JEG-3 cultures and PHT cells (and not in 2D JEG-3 cells or 3D HBMECs) after GSEA and Pavlidis template matching (PTM). The color intensity in (A) to (C) indicates the level of gene expression (yellow for up-regulation and blue for down-regulation), and gray indicates that no RNASeq reads were detected for that transcript in that sample. (D) RT-qPCR analysis of 2D JEG-3 (gray), 3D STLV (red), or PHT (blue) preparations for cyclin-dependent kinase inhibitor 1C/Kip2 (CDKN1C), Cystatin-M (CST6), pregnancy-specific glycoprotein 1 (PSG1), PSG5, and S100 calcium-binding protein A9 (S100A9). Data are from three independent STLVs or PHT preparations, as indicated, and are shown as means ± SD. ***P < 0.001, **P < 0.01.

  • Fig. 4 3D cultures of JEG-3 cells resists viral and T. gondii infection.

    (A) RT-qPCR analyses of viral RNA from 2D or 3D cultures of JEG-3 cells, and PHT cells, infected with either 0.1 or 1 particle-forming unit (PFU) per cell of VSV. Data are shown as percentage of infection normalized to 2D control cultures. Data are shown as means ± SD. ***P < 0.001. (B) Confocal microscopy for YFP–T. gondii (strain I, RH) and mitochondria (red) 48 hours following infection [multiplicity of infection (MOI) = 5] in U2OS or PHT cells. (Top) xy image. (Bottom) xz cross section. (C) Infection of T. gondii [type I (RH), type II (ME49B7), or type III (CEP)] ~48 hours following infection (MOI = 5) of U2OS or PHT cells. Data are shown as means ± SD. ***P < 0.001, **P < 0.01. (D) Infection of T. gondii (strain I, RH) ~48 hours following infection of JEG-3 cells grown in 2D (gray) and 3D (red) or in PHT cells (blue) at either MOI = 5 or MOI = 10. Data are shown as means ± SD. ***P < 0.001, **P < 0.01. (E) Confocal microscopy for YFP–T. gondii (strain I, RH) and mitochondria (red) 48 hours following infection (MOI = 1) in JEG-3 cells grown in 2D (left) or 3D (right). The white box denotes the zoomed area shown at the bottom. For the infections shown in (A) and (B), beads were removed from the STLV and infected immediately in 24-well plates for ~24 hours.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/3/e1501462/DC1

    Fig S1. Monotypica cultures of human trophoblast cells lines are not compatible with culturing in the RWV bioreactor.

    Fig S2. Coculturing of JEG-3 cells with human microvascular endothelial cells promotes their attachment to Cytodex beads in the RWV bioreactor.

    Fig S3. Human microvascular cells are removed from Cytodex beads by JEG-3 cells.

    Fig S4. Levels of pregnancy hormones increases during culturing of JEG-3 cells in 3D.

    Fig S5. JEG-3 cells cultured in 3D express high levels of syncytin, form brush borders, and can be transfected with siRNAs.

    Fig S6. GSEA plots of genes with higher or lower abundance in JEG-3 cells cultured in 2D or 3D or in primary human trophoblasts.

    Table S1. Thirteen “core” genes identified using GSEA gene clustering as being up-regulated in both 3D JEG-3 and PHT cells, while being of low abundance in both 2D JEG-3 cells and 3D HBMECs.

    Table S2. Spreadsheet of gene expression profiles from RNASeq in 2D and 3D cultures of JEG-3 cells, PHT cells, and 3D cultures of HBMECs.

    Data set S1. Spreadsheet from RNASeq studies of 2D and 3D cultures of JEG-3 cells, PHT cells, and 3D cultures of HBMECs. Shown are gene symbols, normalized expression values, and RPKM values from each condition.

    Data set S2. Spreadsheet from differential expression analyses using DESeq2 of 2D and 3D cultures of JEG-3 cells.

    Data set S3. Spreadsheet from differential expression analyses using DESeq2 of 2D and 3D cultures of HBMECs.

    Data set S4. Spreadsheet from differential expression analyses using DESeq2 of 2D cultures of JEG-3 cells and PHT cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig S1. Monotypica cultures of human trophoblast cells lines are not compatible with culturing in the RWV bioreactor.
    • Fig S2. Coculturing of JEG-3 cells with human microvascular endothelial cells promotes their attachment to Cytodex beads in the RWV bioreactor.
    • Fig S3. Human microvascular cells are removed from Cytodex beads by JEG-3 cells.
    • Fig S4. Levels of pregnancy hormones increases during culturing of JEG-3 cells in 3D.
    • Fig S5. JEG-3 cells cultured in 3D express high levels of syncytin, form brush borders, and can be transfected with siRNAs.
    • Fig S6. GSEA plots of genes with higher or lower abundance in JEG-3 cells cultured in 2D or 3D or in primary human trophoblasts.
    • Table S1. Thirteen “core” genes identified using GSEA gene clustering as being up-regulated in both 3D JEG-3 and PHT cells, while being of low abundance in both 2D JEG-3 cells and 3D HBMECs.
    • Table S2. Spreadsheet of gene expression profiles from RNASeq in 2D and 3D cultures of JEG-3 cells, PHT cells, and 3D cultures of HBMECs.
    • Legends for date set S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Data set S1 (Microsoft Excel format). Spreadsheet from RNASeq studies of 2D and 3D cultures of JEG-3 cells, PHT cells, and 3D cultures of HBMECs. Shown are gene symbols, normalized expression values, and RPKM values from each condition.
    • Data set S2 (Microsoft Excel format). Spreadsheet from differential expression analyses using DESeq2 of 2D and 3D cultures of JEG-3 cells.
    • Data set S3 (Microsoft Excel format). Spreadsheet from differential expression analyses using DESeq2 of 2D and 3D cultures of HBMECs.
    • Data set S4 (Microsoft Excel format). Spreadsheet from differential expression analyses using DESeq2 of 2D cultures of JEG-3 cells and PHT cells.

    Files in this Data Supplement:

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