title: Establishment of bovine embryonic stem cell lines using a minimized feeder cell drop.
abstract: Bovine embryonic stem cells (ESCs) are a powerful tool for agricultural and biomedical applications. The purpose of this study was to introduce a new method for generating bovine ESCs. Mechanically isolated bovine inner cell masses (ICMs) from in vitro-produced blastocysts were cultured individually on a 10-μL mouse embryonic fibroblast (MEF) feeder cell drop covered with oil. From 126 blastocysts classified by their developmental stage and ICM size, 21 primary bovine ESC-like colonies were formed (16.7%) and established six JNU (Jeju National University)-ibES cell lines (28.6%, 6/21; hatched blastocyst×4, hatching blastocyst×1, and expanded blastocyst×1). These cells exhibited typical ESC morphology, and pluripotency markers were detected through immunocytochemistry, RT-PCR, and real-time RT-PCR, including Oct4, stage-specific embryonic antigen-1 (SSEA-1), Nanog, Tumor rejection antigen-1-81, Rex1, and alkaline phosphatase. Through RT-PCR analysis of spontaneous differentiation, gene expression of all three embryonic germ layers was detected: ectodermal (Pax6 and DBH), mesodermal (CMP and Enolase), and endodermal [alpha fetoprotein (α-FP) and albumin]. In addition, JNU-ibES cell lines were directed differentiated into neuronal (Map2 and Tuj1) and glial (GFAP) cells. Bovine ESC lines had a normal karyotype, with a chromosome count of 58+XY (JNU-ibES-05). This is the first trial investigating a minimized microdrop culture method for the generation of bovine ESCs. These results demonstrated that the minimized MEF feeder cell drop can support the establishment of bovine ESC lines.
authors.affiliation: Mirae Biotech, Seoul 143-854, Korea. jlokey@daum.net
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Noh
authors.forename: Hyo Young
authors.initials: HY
authors.lastname: Park
authors.forename: Min Jee
authors.initials: MJ
authors.lastname: Park
authors.forename: Eun Hyung
authors.initials: EH
authors.lastname: Noh
authors.forename: Jun Beom
authors.initials: JB
authors.lastname: Lee
authors.forename: Chang Jin
authors.initials: CJ
authors.lastname: Jeong
authors.forename: Dong Sun
authors.initials: DS
authors.lastname: Lee
authors.forename: Key Zung
authors.initials: KZ
authors.lastname: Riu
authors.forename: Se Pill
authors.initials: SP
authors.lastname: Park
mesh_terms: D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D033481:Chromosomes, Mammalian / Q000378:metabolism; D018920:Coculture Techniques / Q000379:methods; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology* / Q000378:metabolism; D005855:Germ Layers / Q000166:cytology* / Q000378:metabolism; D051379:Mice