title: Isolation and characterization of embryonic stem-like cells from canine blastocysts.
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.
authors.affiliation: Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Hatoya
authors.forename: Ryuzo
authors.initials: R
authors.lastname: Torii
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kondo
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Okuno
authors.forename: Kinji
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Viskam
authors.initials: V
authors.lastname: Wijewardana
authors.forename: Noritoshi
authors.initials: N
authors.lastname: Kawate
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Tamada
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Sawada
authors.forename: Daijiro
authors.initials: D
authors.lastname: Kumagai
authors.forename: Kikuya
authors.initials: K
authors.lastname: Sugiura
authors.forename: Toshio
authors.initials: T
authors.lastname: Inaba
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods; D018920:Coculture Techniques; D004285:Dogs; D004622:Embryo, Mammalian / Q000166:cytology* / Q000502:physiology; D047108:Embryonic Development / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology; D051379:Mice; D009028:Morula / Q000166:cytology / Q000502:physiology; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology