title: A comprehensive system for generation and evaluation of induced pluripotent stem cells using piggyBac transposition.
abstract: The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell-specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis-specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse-rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC-derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan; Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), Kobe, Hyogo, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Tsukiyama
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan; JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Chiyoda-ku, Tokyo, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kato-Itoh
authors.affiliation: Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan; JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Chiyoda-ku, Tokyo, Japan.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan; Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), Kobe, Hyogo, Japan; Life Science Experimental Facility, Department of Biotechnology, Faculty of Life and Environmental Science, University of Yamanashi, Kofu, Yamanashi, Japan.
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Ohinata
mesh_terms: D000818:Animals; D065150:Cellular Reprogramming / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008297:Male; D051379:Mice; D010957:Plasmids / Q000235:genetics; D051381:Rats; D013087:Spermatids / Q000166:cytology / Q000378:metabolism; D014162:Transfection / Q000379:methods*