title: Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.
abstract: Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.
authors.affiliation: Animal Development and Differentiation Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Ibaraki, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Furusawa
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Ohkoshi
authors.forename: Koji
authors.initials: K
authors.lastname: Kimura
authors.forename: Shuichi
authors.initials: S
authors.lastname: Matsuyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akagi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kaneda
authors.forename: Mitsumi
authors.initials: M
authors.lastname: Ikeda
authors.forename: Misa
authors.initials: M
authors.lastname: Hosoe
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Kizaki
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Tokunaga
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002678:Chimera / Q000378:metabolism*; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D016256:Lewis X Antigen; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D000469:Alkaline Phosphatase
keywords: blastocyst; bovine embryonic stem cell-like cell lines; camel); chimeric conceptuses; llama; nuclear transfer; ruminants (cows; sheep; stem cells