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

Authors

  • Shingo Hatoya,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Ryuzo Torii,

    1. Research Center for Animal Life Science, Shiga University of Medical Science, Shiga, Japan
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  • Yasushi Kondo,

    1. Advanced Medical Research Laboratories and Drug Safety Research Laboratories, Tanabe Seiyaku Co., Ltd., Osaka, Japan
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  • Tsuyoshi Okuno,

    1. Advanced Medical Research Laboratories and Drug Safety Research Laboratories, Tanabe Seiyaku Co., Ltd., Osaka, Japan
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  • Kinji Kobayashi,

    1. Advanced Medical Research Laboratories and Drug Safety Research Laboratories, Tanabe Seiyaku Co., Ltd., Osaka, Japan
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  • Viskam Wijewardana,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Noritoshi Kawate,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Hiromichi Tamada,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Tsutomu Sawada,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Daijiro Kumagai,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Kikuya Sugiura,

    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
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  • Toshio Inaba

    Corresponding author
    1. Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
    • Laboratory of Cell Pathobiology, Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
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Abstract

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.

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