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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P184 | DOI: 10.1530/repabs.1.P184

Derivation of naive-type induced pluripotent stem cells in cattle using piggyBac transposition of doxycycline-inducible transcription factors

Takamasa Kawaguchi1, Tomoyuki Tsukiyama2, Naojiro Minami1, Masayasu Yamada1, Shuichi Matsuyama3, Koji Kimura3 & Hiroshi Imai1

1Kyoto University, Kyoto, Japan; 2RIKEN Center for Developmental Biology (CDB), Kobe, Japan; 3National Institute of Livestock and Grassland Science, Nasushiobara, Japan.

Pluripotent stem cells (PSCs) in large domestic animals including cattle are expected to be used as means for genetic improvement and production of disease-model animals. There are two types of PSCs: naive and primed. Ontogenetically, naive PSCs correspond to an earlier developmental state compared to other types of PSCs and have the capacity for producing chimeric offspring. Previously reported PSCs in large domestic animals are classified as primed type. In this study, putative naive-type bovine induced pluripotent stem cells (biPSCs) were generated. Bovine amnion cells were transduced with piggyBac transposon (PB) vectors expressing the reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc and/or Nanog). Since the PB vectors shared with a doxycycline (Dox)-inducible system, the expression of reprogramming factors can control under the condition with or without Dox. Two weeks after the addition of Dox, two types of colonies were obtained in different culture conditions. Under a primed PSC culture condition, flattened colonies were obtained and were mechanically propagated over 60 passages. Under a naive PSC culture condition, colonies showed round morphology and could be passaged as a single cell over ten times. When the former were passaged and maintained under the latter condition, the naive-type colonies were appeared. Resultant naive-type biPSCs had alkaline phosphatase activity and expressed pluripotent-related genes (OCT3/4, NANOG, ESRRβ and REX1). They also showed a normal karyotype for over 60 passages, formed embryoid body and differentiated into all three germ layers in vitro. This study shows the generation of biPSCs having similar characteristics to those of naive PSCs.

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