Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Takamasa Kawaguchi,Naojiro Minami,Koji Kimura,Tomoyuki Tsukiyama,Hiroshi Imai,Masayasu Yamada,Shuichi Matsuyama,Yuin-Han Loh

doi:10.1371/journal.pone.0135403

Abstract

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.

Highlights

Somatic cells can be reprogrammed to a pluripotent state via ectopic expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc, thereby generating induced pluripotent stem cells [1, 2]
Naïve Pluripotent stem cells (PSCs) correspond to inner cell mass (ICM) of blastocysts, and are similar to mouse ES cells, whereas primed PSCs correspond to the epiblast at the postimplantation stage, as represented by mouse epiblast stem cells and human ES cells
After transfection with Dox-inducible piggyBac vectors containing reprogramming factors, the bADCS were cultured in piPSC medium, which was similar to medium generally used for maintenance of human ES cells

Summary

Introduction

Somatic cells can be reprogrammed to a pluripotent state via ectopic expression of the transcription factors Oct, Sox, Klf, and c-Myc, thereby generating induced pluripotent stem cells (iPSCs) [1, 2]. Naïve PSCs correspond to ICM of blastocysts, and are similar to mouse ES cells (mESCs), whereas primed PSCs correspond to the epiblast at the postimplantation stage, as represented by mouse epiblast stem cells (mEpiSCs) and human ES cells (hESCs). Naïve PSCs exhibit some distinctive characteristics, such as a compact and dome-shaped morphology, the ability to be passaged as a single cell, dependence of proliferation on the leukemia inhibitory factor (LIF)–Jak/Stat signaling pathway, two active X chromosomes (XaXa), and specific expression of REX1, ESRRβ, and STELLA [3, 4]. Naïve PSCs are a feasible potential source of material for production of PSC-derived offspring in domestic species

Methods

Results

Conclusion