Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.

Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.