title: Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.
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.
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Kawaguchi
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Dooseon
authors.identifier: http://orcid.org/0000-0002-0067-5804
authors.initials: D
authors.lastname: Cho
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: Research Center for Animal Life Science, Shiga University of Medical Science, Shiga, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Tsukiyama
authors.affiliation: Animal Feeding and Management Research Division, NARO Institute of Livestock and Grassland Science, Tochigi, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kimura
authors.affiliation: Animal Feeding and Management Research Division, NARO Institute of Livestock and Grassland Science, Tochigi, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Matsuyama
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Naojiro
authors.initials: N
authors.lastname: Minami
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Yamada
authors.affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imai
mesh_terms: D000650:Amnion / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D004475:Ectoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018703:Founder Effect; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D015415:Biomarkers; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; D004318:Doxycycline