snRNA-seq analysis of apical-out endometrial organoids including endometrial stromal cells and human umbilical vein endothelial cells

In this study, we developed an apical-out-endometrial organoids (AO-EMO + eSC/HUVEC), which exposes the apical surface of the epithelium and contains eSC and an endothelial network. To evaluate this model, we conducted a single-nucleus RNA-sequencing (snRNA-seq). The endometrial organoids (EMO) fragments were centrifuged at 500 × g for 3 min, after which the supernatant was meticulously aspirated. Subsequently, a blend of 70% type I collagen and 30% Matrigel was incorporated into a pellet of EMO fragments, and 20 µl of the resulting mixture was dispensed into a non-treated 48-well plate. The plates were then incubated at 37°C for 30 min. ECSY medium supplemented with 50 ng/ml FGF2 and 20 ng/ml VEGF were added and replaced every two or three days. The composition of the ECSY medium was as follows: DMEM/F-12 supplemented with 1% ITS-X supplement, 0.15% BSA, 1% Knockout Serum Replacement, 200 mM L-ascorbic acid, 50 ng/ml EGF, 2 μM CHIR99021, 2 μM SB202190, and 10 μM Y27632. We conducted snRNA-seq following nuclear extraction and DAPI+ sorting.