Fig 2: Confocal microscopy for distinct trophoblast markers in STBin and STBout TOs.(A), Cross-sections of STBin (top) of STBout (bottom) TOs immunostained for SDC-1 (green), and cytokeratin-19 (red). DAPI-stained nuclei are in blue. At bottom and right are orthogonal views of three-dimensional stacked images. Movies demonstrating image reconstruction and sectioning are in Supplemental Movies 1 and 2. (B), Confocal micrographs of STBin (top) or STBout (bottom) TOs immunostained for ITGA6 (in green) or CGBs (in red). DAPI-stained nuclei are shown in grey. Right are individual channels, bottom is orthogonal views of three-dimensional stacked images. Movies demonstrating image reconstruction and sectioning are in Supplemental Movies 3 and 4. Scale bar, 30µm. (C), Levels of human chorionic gonadotropin (hCG) (shown as pg/mL normalized to total protein of wells from which CM was collected) in conditioned medium collected from STBin or STBout TOs wellsas determined by Luminex. (D), Confocal micrographs of STBin (top) or STBout (bottom) TOs immunostained for ITGA6 (in blue), SDC-1 (in green), and HLA-G (in red). DAPI-stained nuclei are shown in grey. At right are individual channels. Scale bar, 30µm (E), Quantification of the percentage of HLA-G+ positive cells in STBin versus STBout TOs. Individual points represent unique fields used for image analysis. A minimum of 15 organoids was used for analysis. Significance determined by a student’s t-test (** p<0.01).

Fig 3: Development of STBout trophoblast organoids.(A), Schematic of the workflow to generate STBout trophoblast organoids (TOs) from STBin TOs collected from Matrigel domes. (B), Brightfield images of STBin (top) or STBout (bottom) TOs at the end of their culture period. Scale, 25µm. (C), Left, schematic of STBin (top) or STBout (bottom) TOs representing the cellular orientation of cytotrophoblasts (CTBs, in green), extravillous trophoblasts (in blue), and the syncytiotrophoblast (in red). All schematics created using Biorender. Middle, hematoxylin and eosin (H&E) histological staining of cryosections of STBin and STBout TOs as noted at left. Right, immunostaining for ITGA6 (green) and CGBs (red) in cryosections of matched same TOs. DAPI-stained nuclei are shown in grey. Scale, 15µm (D), Confocal micrographs of TOs cultured as STBin (left panels) or in suspension to generate STBout for 21hrs (middle) or 48hrs (right) and immunostaining for SDC-1 (green) and cytokeratin-19 (red). DAPI-stained nuclei are in blue, Top panels were captured at the outermost surface of organoids (surface) and bottom panels were captured at the innermost layers (inner). Orthogonal views (Orth) are shown at bottom. (E), Image analysis of the extent of surface immunostaining for SDC-1 as assessed by image analysis of SDC-1 intensity on the surfaces of TOs three-dimensional whole organoid images (shown as a percent of STBin TOs) in STBout TOs cultures for 21hrs (light blue) or 48hrs (dark blue). Data are shown as mean ± standard deviation with significance determined by a student’s t-test (** p<0.01). Symbols represent unique fields of organoids from individual replicates.