Establishment of Bovine Embryonic Stem Cell Lines Using a Minimized Feeder Cell Drop
| Autor: | Se Pill Park, Hyo Young Park, Key Zung Riu, Chang Jin Jeong, Eun Ji Noh, Dong-Sun Lee, Eun-Young Kim, Eun Hyung Noh, Min Jee Park, Jun Beom Lee |
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| Rok vydání: | 2012 |
| Předmět: |
Homeobox protein NANOG
Rex1 Cell Immunocytochemistry Embryonic Development Biology Cell Line Mice Gene expression medicine Animals Embryonic Stem Cells reproductive and urinary physiology urogenital system Feeder Cells Cell Biology Chromosomes Mammalian Molecular biology Embryonic stem cell Coculture Techniques medicine.anatomical_structure Cell culture Blastocyst Inner Cell Mass embryonic structures Alkaline phosphatase Cattle Germ Layers Developmental Biology Biotechnology |
| Zdroj: | Cellular Reprogramming. 14:520-529 |
| ISSN: | 2152-4998 2152-4971 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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