Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.
Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.
Laboratory of Reproductive Biology, Graduate School of Agricuture, Kyoto University, Kyoto, Japan.
Takamasa
T
Kawaguchi
Research Center for Animal Life Science, Shiga University of Medical Science, Shiga, Japan.
Tomoyuki
T
Tsukiyama
Animal Feeding and Management Research Division, NARO Institute of Livestock and Grassland Science, Tochigi, Japan.
Koji
K
Kimura
Animal Feeding and Management Research Division, NARO Institute of Livestock and Grassland Science, Tochigi, Japan.
Shuichi
S
Matsuyama
Laboratory of Reproductive Biology, Graduate School of Agricuture, Kyoto University, Kyoto, Japan.
Naojiro
N
Minami
Laboratory of Reproductive Biology, Graduate School of Agricuture, Kyoto University, Kyoto, Japan.
Masayasu
M
Yamada
Laboratory of Reproductive Biology, Graduate School of Agricuture, Kyoto University, Kyoto, Japan.
Hiroshi
H
Imai
D000650:Amnion / Q000166:cytology*; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D002678:Chimera / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000196:embryology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005819:Genetic Markers / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D005819:Genetic Markers; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D053605:Leukemia Inhibitory Factor; C489428:Myc protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; D004318:Doxycycline