Establishment of a Trophoblast Stem Cell Model to Study Placenta Development in Cattle

Abdallah W. Abdelhady¹, Andrew M. Kelleher¹, M. Sofia Ortega Obando² and Thomas E. Spencer¹

¹Division of Animal Sciences and Department of Obstetrics, Gynecology, and Women’s Health, University of Missouri, Columbia, MO, USA.

 ² Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, USA.


Abstract Text:

Cattle have a synepitheliochorial placenta characterized as semi-invasive, with maternal caruncles and fetal cotyledons forming highly vascularized placentome structures to facilitate nutrient exchange and fetal development. As the conceptus begins implantation, trophoblast binucleate cells (BNCs) begin to differentiate from uninucleate cells (UNCs) within the chorion. The BNC are thought to be derived from UNC through endoreduplication, form the foundation of the placental cotyledons, and express unique placenta-specific genes including pregnancy-associated glycoproteins (PAGs) and placental lactogen (CSH2). Pregnancy loss in cattle can be caused by inadequate development and differentiation of the placenta. Importantly, defects in BNC differentiation and decreased PAG production are associated with pregnancy loss after day 30 in cattle. However, the cellular and molecular mechanisms regulating bovine placenta development and, particularly, trophoblast differentiation are not well understood. Therefore, the objective of this study was to establish a trophoblast stem cell model to better understand critical pathways regulating the development of BNC in the bovine placenta. Trophoblast stem cells (TSC) were derived from in vitro produced bovine blastocysts. The TSCs were cultured on collagen IV for 10 days in a differentiation media containing forskolin and a ROCK inhibitor, resulting in the generation of BNC based on the presence of two nuclei and cytoplasm filled with secretory granules. RNA-seq analysis determined that expression of BNC-specific marker genes (CSH2, HSD17B1, PAG7, PAG17, PRP3, PRP14) was present on day 10. Transcription factors involved in placenta differentiation in the human and mouse (CITED1, GCM1) or endoreduplication (DKK1, E2F1) were also up regulated on day 10. Single nuclei RNA sequencing (snRNA-seq) analysis revealed 4 distinct cell types on day 10. One UNC population (37 % cells) expressed trophoblast stem cell markers CDX2and ELF5, another population (41 %) of cells expressed cell cycle regulators MKI67, CDK1, BIRC5 and CCNB2 together with the UNC-specific PAG2, and another cell population (22 %) expressed cell differentiation marker CDKN1C together with TKDP4, PAG8 and PAG12. A low number of cells (1 %) expressed BNC-specific marker genes CSH2, PAG17, GCM1, PRP1, PRP2, PRP3 and PRP14. This in vitro model system will be useful to uncover the cellular and molecular mechanisms regulating trophoblast differentiation during the development of the bovine placenta. Supported by USDA NIFA Grant Award 2019-67015-28998.