An in vitro model for equine placental research

A fundamental understanding of placental development and its associated pathologies is crucial to mitigating the losses associated with placental disorders. Organoids are widely used to investigate the developmental biology of organs and in disease modeling. In humans, placental organoids have been successfully developed using trophoblast cells. In this study, we aimed to 1) develop placental organoids using pre-term and term equine placentae, 2) evaluate the organoid structures, 3) compare the organoid transcriptome with their respective chorioallantois (CA), and 4) evaluate organoids based on their starting material (pre-term vs term). The CA was collected from healthy placentae following elective abortion (80-100d of gestation) and following spontaneous term parturition. CA samples were processed for culture, fixed for histology, and stored in RNAlater. For culture, each CA sample was enzymatically digested to a single cell suspension and was seeded into 50µL Matrigel Matrix droplets surrounded by medium. Passage-3 organoids (P3) were used for histology (H&E), immunohistochemistry (IHC), and transcriptomic analysis. Steroid production and conversion were measured by mass spectrometry (MS) of culture media. After optimization of culture conditions, we produced 3D-organoids (Pre-Term, n=6 and Term, n=3). Organoids were passaged up to 12 times over ±3 months and were successfully frozen and re-cultured post-thaw. Total extracted RNA from initial CA samples and organoids was sequenced (150PE; ∼43 million reads/sample). The genes expressed in the organoidsclosely resemble those of the tissue of origin, 19.4% of the expressed genes were differentially expressed (DEGs) between the initial CA and P3 (n=3,949 genes; FDR<0.001). H&E staining of both CA and the organoids indicates the epithelial origin of the cells within the organoids. The enriched expression of trophoblast-specific genes PLAC8B, GATA3, KRT7, VGLL1, and EGFR, and the presence of the trophoblast-specific genes TFAP2A and TFAP2C, and GATA2 in the organoids confirm their trophoblast identity. The presence of SOX2 and OCT4, as demonstrated by IHC, confirms the pluripotency of the organoid cells. The SRD5A1 and SRD5A3 genes were expressed in all organoids and our preliminary MS analysis demonstrated the conversion of progesterone to 5α-DHP, further suggesting functional integrity. Comparison between Pre-Term and Term organoids revealed 552 DEGs (FDR<0.001), with downregulation of immune/inflammatory/defense responses and blood vessel development, and upregulation of molecule transport, development, and adhesion in the Pre-Term group. To conclude, this is the first report of placental organoids in domestic animals. The organoids show structural, transcriptomic, and functional similarities to the equine placenta in vivo and consequently could be used as an in vitro model to study placental development and associated pathologies. Funded by The Foundation for The Horse, GJCRF, UGent BOF, BAEF, L'Oréal-UNESCO FWO, and the Center for Equine Health at UC Davis.