Establishment of functional trophoblast organoids from trophoblast cells of bovine placenta

The placenta is an organ that plays a vital role in successful pregnancies, and the failure of early placentation is a significant factor leading to abortion in ruminant species. However, the mechanisms involved in the development and differentiation of bovine placenta remain elusive due to the lack of suitable in vitro placental models. This study aimed to develop an effective method for generating the bovine functional trophoblast organoids by assembling bovine primary trophoblast cells (PBTCs) from the placenta or immortalized bovine placental trophoblast (BTCs) in a 3D culture system in vitro. PBTCs isolated from the 3-month-gestation placenta and BTCs rapidly proliferated and exhibited typical epithelioid morphology in the modified trophoblast organoid medium (TOM) for bovine. Furthermore, PBTCs and BTCs proliferating in the modified TOM were both CK7- and Ecadherin-positive. Both PBTCs or BTCs embedded into Matrigel droplets overlaid with modified TOM proliferated and formed trophoblast organoids after 15 days of culture. Moreover, the expression of syntrophoblast marker genes, including CD71, CD46, and chorionic somatomammotropin hormone 1 (CSH1), was detectable in both organoids derived from different types of trophoblast cells. Notably, the protein expression levels of various genes implicated in the establishment of early pregnancy in endometrial epithelium cells (EECs) was increased following coculture with bovine trophoblast organoids. Collectively, the bovine trophoblast organoids established in our study could serve as robust models for elucidating the essential physical functions of the placenta and the causes of pregnancy failures related to the placenta developmental disorders during early bovine pregnancy.