Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES‐like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11–16 of first estrus. A total of 80 normal embryos were obtained from 15...

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Bibliographic Details
Published in:Molecular reproduction and development 2006-03, Vol.73 (3), p.298-305
Main Authors: Hatoya, Shingo, Torii, Ryuzo, Kondo, Yasushi, Okuno, Tsuyoshi, Kobayashi, Kinji, Wijewardana, Viskam, Kawate, Noritoshi, Tamada, Hiromichi, Sawada, Tsutomu, Kumagai, Daijiro, Sugiura, Kikuya, Inaba, Toshio
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
blastocyst
Blastocyst - cytology
Blastocyst - physiology
canine
Cell Differentiation - physiology
Cell Separation - methods
Cells, Cultured
Cloning, Organism - methods
Coculture Techniques
Dogs
Embryo, Mammalian - cytology
Embryo, Mammalian - physiology
Embryology: invertebrates and vertebrates. Teratology
Embryonic Development - physiology
embryonic stem cell
Fibroblasts - cytology
Fundamental and applied biological sciences. Psychology
isolation
Mice
Molecular embryology
Morula - cytology
Morula - physiology
Stem Cells - cytology
Stem Cells - physiology
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Summary:Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES‐like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11–16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES‐like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell‐like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen‐1 and Oct‐4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron‐like, epithelium‐like, fibroblast‐like, melanocyte‐like, and myocardium‐like cells. These observations indicate that we successfully isolated and characterized canine ES‐like cells. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
ISSN:1040-452X
1098-2795
DOI:10.1002/mrd.20392