Establishment of bovine embryonic stem cell lines using a minimized feeder cell drop

• Published in: Cellular reprogramming Vol. 14; no. 6; p. 520
• Main Authors: Kim, Eun Young, Noh, Eun Ji, Park, Hyo Young, Park, Min Jee, Noh, Eun Hyung, Lee, Jun Beom, Jeong, Chang Jin, Lee, Dong Sun, Riu, Key Zung, Park, Se Pill
• Format: Journal Article
• Language: English
• Published: United States 01-12-2012
• Subjects: Animals; Blastocyst Inner Cell Mass - cytology; Blastocyst Inner Cell Mass - metabolism; Cattle; Cell Line; Chromosomes, Mammalian - metabolism; Coculture Techniques - methods; Embryonic Development; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Feeder Cells - cytology; Feeder Cells - metabolism; Germ Layers - cytology; Germ Layers - metabolism; Mice
• ISSN: 2152-4998
• DOI: 10.1089/cell.2012.0038

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.