Abstract
Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3⁄4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRβ, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.
Highlights
Mammalian blastocysts are composed of two distinct cell types: the inner cell mass and the trophectoderm
During trials for the generation of bovine-induced pluripotent stem cells from bovine amnion-derived cells (bADCs) using Dox-inducible PB vectors in culture medium used for human iPSCs, a small portion of the colonies appeared in a similar morphology as human iPSCs (Fig 2B and S1A–S1C Fig)
After introducing transcription factors by piggyBac vectors, morphologically different colonies from iPS cells appeared even in medium consisting of KSR and FGF2, which are usually used for the generation and maintenance of human iPSCs [38]
Summary
Mammalian blastocysts are composed of two distinct cell types: the inner cell mass and the trophectoderm. The trophectoderm is the first cell type that differentiates from pre-implantation embryos at the blastocyst stage. The trophectoderm cell lineage plays an important role in implantation and placental formation [1]. At the beginning of implantation and throughout pregnancy, trophoblastic binucleate cells are differentiated from mononuclear cells [4]. Binucleate cells are fused with uterine epithelial cells and formed trinucleate cells and these cells play similar roles to syncytiotrophoblasts [5]. During this peri-implantation period, trophoblastic cells produce a number of molecules such as interferon-tau (IFN-τ), placental lactogen (PL), prolactinrelated proteins (PRPs) and pregnancy-associated glycoproteins (PAGs). PL, PRPs, and PAGs are hormones that are secreted by binucleate cells and play a main role in the fetal-maternal interface [4]
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