title: Generating porcine chimeras using inner cell mass cells and parthenogenetic preimplantation embryos.
abstract: BACKGROUND
  The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. METHODOLOGY/SIGNIFICANT PRINCIPAL FINDINGS: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.
  
  
  CONCLUSIONS
  Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.
authors.affiliation: Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakano
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Taisuke
authors.initials: T
authors.lastname: Matsuda
authors.forename: Kasumi
authors.initials: K
authors.lastname: Honda
authors.forename: Miki
authors.initials: M
authors.lastname: Maehara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kanai
authors.forename: Gota
authors.initials: G
authors.lastname: Hayashida
authors.forename: Mirina
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Momoko
authors.initials: M
authors.lastname: Kuramoto
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Arai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Umeyama
authors.forename: Shuh-Hei
authors.initials: SH
authors.lastname: Fujishiro
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Mizukami
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagaya
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hanazono
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D002678:Chimera / Q000196:embryology* / Q000254:growth & development; D005260:Female; D005307:Fertilization in Vitro; D005333:Fetus; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D008297:Male; D009028:Morula / Q000166:cytology / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D010312:Parthenogenesis / Q000235:genetics*; D027724:Reproductive Techniques, Assisted; D013552:Swine