Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors

• Published in: PloS one 2016-12, Vol.11 (12), p.e0167550-e0167550
• Main Authors: Kawaguchi, Takamasa, Cho, Dooseon, Hayashi, Masafumi, Tsukiyama, Tomoyuki, Kimura, Koji, Matsuyama, Shuichi, Minami, Naojiro, Yamada, Masayasu, Imai, Hiroshi
• Format: Article
• Language: English
• Subjects: Amnion; Amnion - cytology; Amnion - drug effects; Amnion - metabolism; Amniotic cells; Animals; Antibiotics; Beef cattle; Biology; Biology and Life Sciences; Biomarkers - metabolism; Biotechnology; c-Myc protein; Cattle; CDX2 protein; Cell culture; Cell Line; Cell lineage; Cell Lineage - drug effects; Cell lines; Colonies; Comparative analysis; Cytology; Differentiation; Dosage and administration; Doxycycline; Doxycycline - pharmacology; Ectoderm - cytology; Ectoderm - drug effects; Ectoderm - metabolism; Embryos; Female; Fibroblasts; Founder Effect; Gene Expression; Genes; Genetic aspects; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Grasslands; Growth factors; Implantation; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - metabolism; Interferon; KLF4 protein; Kruppel-Like Transcription Factors - genetics; Kruppel-Like Transcription Factors - metabolism; Laboratories; Life sciences; Livestock; Mammals; Morphology; Myc protein; Oct-4 protein; Octamer Transcription Factor-3 - genetics; Octamer Transcription Factor-3 - metabolism; Placenta; Pluripotency; Pregnancy; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Research and Analysis Methods; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - metabolism; Spheroids; Stem cells; Transcription factors; Trophectoderm; Trophoblasts - cytology; Trophoblasts - drug effects; Trophoblasts - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0167550

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.