Journal: bioRxiv

Article Title: Amyloid-β fibrils accumulated in preeclamptic placentas suppress syncytialization of cytotrophoblasts

doi: 10.1101/2025.03.06.641957

Figure Lengend Snippet: ( A ) BeWo cells were cultured in 15% MEM-α with roxadustat (0, 5, or 10 μM) for 16 hours. Protein levels of HIF-1α and BACE1 were analyzed by means of Western blotting with the anti-HIF-1α antibody and the anti-human BACE1 antibody, respectively. β-Actin served as the loading control. Graphs show quantification of HIF-1α and BACE1. Data are means ± SEM ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( B ) Hypoxia promoted Aβ production in BeWo cells. Aβ40/42 generated by BeWo cells was analyzed by means of Western blotting with the anti-Aβ (Nt) β001 antibody. BeWo cells were cultured in Opti-MEM medium containing 2% FBS with or without LY2886721 for 24 hours under normoxic conditions (20% O 2 ) or hypoxic conditions (2% O 2 ). Generated Aβ40/42 was analyzed. β-Actin served as the loading control. Graphs show quantification of Aβ40/42. Data are means ± SEM ( n = 6). *** P < 0.001; **** P < 0.0001.

Article Snippet: Human choriocarcinoma-derived BeWo cells that are widely used as a model of trophoblast syncytialization were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan).

Techniques: Cell Culture, Western Blot, Control, Generated

Journal: bioRxiv

Article Title: Amyloid-β fibrils accumulated in preeclamptic placentas suppress syncytialization of cytotrophoblasts

doi: 10.1101/2025.03.06.641957

Figure Lengend Snippet: Aβ42 fibrils inhibited syncytialization of BeWo cells by inducing loss of membrane localization of cell-cell adhesion proteins. ( A ) BeWo cells were pre-treated with Aβ42 fibrils (10 μM) in serum-free Opti-MEM for 12 hours and then were stimulated with Fsk (50 μM) for 48 hours. The effect of Aβ fibrils on syncytialization of BeWo cells was analyzed by means of Western blotting with the anti-hCG β antibody and the anti-syncytin-1 antibody. β-Actin served as the loading control. Graphs show quantification of β-hCG and syncytin-1. Data are means ± SEM ( n = 6). ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( B ) BeWo cells were cultured on cover glasses and treated with Aβ42 fibrils (10 μM) for 24 hours, after which they were fixed in 4% paraformaldehyde and stained with the anti-ZO-1 antibody or the anti-E-cadherin antibody. Arrowheads indicate loss of membrane localization of ZO-1 and E-cadherin in Aβ fibril-treated BeWo cells. Nuclei were counterstained with DAPI. Scale bars, 20 µm. ( C ) BeWo cells were treated with Aβ42 fibrils (10 μM) in serum-free Opti-MEM for 24 hours. Quantitative analysis of ZO-1 and E-cadherin of Aβ42 fibril-treated BeWo cells was performed by using Western blotting with the anti-ZO-1 antibody and the anti-E-cadherin antibody. Evaluation of the effect of Aβ42 fibrils on autophagy of BeWo cells was performed by using Western blotting with an anti-LC3 antibody. β-Actin served as the loading control. Graphs show quantification of ZO-1, E-cadherin, and LC3. Data are means ± SEM ( n = 6). ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human choriocarcinoma-derived BeWo cells that are widely used as a model of trophoblast syncytialization were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan).

Techniques: Membrane, Western Blot, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: Healthy and diseased placenta barrier on-a-chip models suitable for high-throughput studies

doi: 10.1101/2022.06.30.498115

Figure Lengend Snippet: ( A ) Bottom view of the OrganoPlate 3-lane, a 384-well plate format containing in its bottom 40 individual microfluidics chips. Zoomed-in image of one microfluidic chip. ( B ) Schematic representation of one chip consisting of three adjacent channels accessible by top (A1), bottom (C1) inlets and top (A3), bottom (C3) outlets wells. Perfusion channels are separated by small ridges (PhaseGuide™) which enabled the patterning of ECM gel in the middle lane and culture of cells in the perfusion channels in a membrane-free manner. Cultures are observed from the observation window (B2). ( C ) Schematic of the placenta barrier multilayered architecture. The interface is composed of syncytiotrophoblasts and cytotrophoblasts (maternal compartment) on top of their basement membrane. A thin connective tissue separates the maternal interface from the fetal capillaries (fetal compartment). ( D ) 3-dimensional schematic of the placenta barrier modeling in the OrganoPlate 3-lane, representing the maternal and fetal compartment in close proximity. Continuous flow through the maternal and fetal compartments is depicted by the arrows. ( E ) Schematic workflow depicting placenta barrier modeling set-up, BeWo b30 differentiation and controlled invasion over time.

Article Snippet: Human choriocarcinoma cell line BeWo b30 clone (#C0030002; AddexBio) was cultured in T75 flasks in Optimized-DMEM (#C0003-02; AddexBio) supplemented with 10% fetal bovine serum (FBS; F4135; Sigma) and 1% penicillin-streptomycin (#15070063; Thermo Fisher).

Techniques:

Journal: bioRxiv

Article Title: Healthy and diseased placenta barrier on-a-chip models suitable for high-throughput studies

doi: 10.1101/2022.06.30.498115

Figure Lengend Snippet: ( A ) Representative phase contrast images of (i) BeWo b30 monoculture (maternal compartment), or (ii) co-culture with HUVEC (fetal compartment) depicting cell culture growth from day 3 to day 9. Middle channel contains a collagen-I/IV ECM gel. Scale bar: 200 μm. ( B ) Maximum intensity z-projection of the placenta barrier co-culture after 9 days of culture. Staining for Actin (green) and DNA (blue) shows the maternal-fetal interface, with the distinct maternal compartment stained for ECAD (red). Scale bar: 200 μm. ( C ) 3-dimensionnal reconstruction of the placenta barrier stained for ZO-1 (red), Actin (green) and DNA (blue) showing a lumenized maternal and fetal compartment in close proximity. ( D ) View of 20 chips containing placenta barrier cultures after 9 days of culture, stained for Actin (yellow). Scale bar: 100 μm. ( E ) Maternal-fetal interface distance quantification obtained from placenta barrier Actin immunostaining. For every chip, the shortest, longest, and average interface distance between mother and fetal compartment was measured. Data are plotted as box and whiskers (N=3, n=16-20). The line and plus sign represent the median and mean, respectively.

Article Snippet: Human choriocarcinoma cell line BeWo b30 clone (#C0030002; AddexBio) was cultured in T75 flasks in Optimized-DMEM (#C0003-02; AddexBio) supplemented with 10% fetal bovine serum (FBS; F4135; Sigma) and 1% penicillin-streptomycin (#15070063; Thermo Fisher).

Techniques: Co-Culture Assay, Cell Culture, Staining, Immunostaining

Journal: bioRxiv

Article Title: Healthy and diseased placenta barrier on-a-chip models suitable for high-throughput studies

doi: 10.1101/2022.06.30.498115

Figure Lengend Snippet: ( A ) (i) Representative maximum intensity z-projection of syncytium microvilli stained for Ezrin (red) and DNA (blue). Scale bar: 25 μm. (ii) 3-dimensional reconstruction of the syncytium layer stained for Ezrin (red), Actin (yellow) and DNA (blue) shows a polarized syncytium. Scale bar: 50μm ( B ) mRNA expression of trophoblast differentiation markers Syncytin-2, hCG-α, hCG-ß and PlGF in undifferentiated or differentiated BeWo b30 (48h 25 μM Forskolin + 10 nM PMA). Each data point is derived from 5 pooled chips. Graphs show the mean of relative expression values normalized to GAPDH ± SEM and statistical analysis was performed using a one-way ANOVA (N=1, n=3). ( C ) Representative maximum intensity z-projection of undifferentiated and differentiated BeWo b30 stained for ZO-1 (red), Actin (yellow) and DNA (blue) shows the formation of multinucleated syncytium upon differentiation. Scale bar: 100μm ( D ) Representative and (i) maximum intensity z-projection of undifferentiated and differentiated BeWo b30 stained for ECAD (green), hCG-ß (red) and DNA (blue). Scale bar: 100μm. A zoom-in (ii) shows trophoblast hormone production within the formed syncytium. Scale bar: 20 μm. ( E) Fluorescence intensity analysis of SUM projections of hCG-ß immunostaining from undifferentiated or differentiated BeWo b30. The intensity is corrected for the isotype control. Data points represent individual chips. Data was presented as mean ± standard deviation (SD) and statistical analysis was performed using two-tailed unpaired t-test (N=3, n=4). (F) Apical secretion of hCG-ß from undifferentiated and differentiated epithelium at day 7 (before differentiation), day 8 (24h differentiation) and day 9 (48h differentiation). Data points represent individual chips. Data was presented as mean ± SD and statistical analysis was performed using ordinary two-way ANOVA (N=3, n=4). (G) Fold change of hCG-ß release in the lumen of differentiated over undifferentiated BeWo b30 at day 8 (24h differentiation) and day 9 (48h differentiation). Data points represent the average of hCG-β concentration of differentiated over undifferentiated epithelium of 4 chips. Data was acquired from 3 individual experiments. Data were presented as mean ± SD and statistical analysis was performed using two-tailed unpaired t-test (N=3, n=4). ns: non-significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: Human choriocarcinoma cell line BeWo b30 clone (#C0030002; AddexBio) was cultured in T75 flasks in Optimized-DMEM (#C0003-02; AddexBio) supplemented with 10% fetal bovine serum (FBS; F4135; Sigma) and 1% penicillin-streptomycin (#15070063; Thermo Fisher).

Techniques: Staining, Expressing, Derivative Assay, Fluorescence, Immunostaining, Standard Deviation, Two Tailed Test, Concentration Assay