Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins

Abstract

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…Speculatively, bovine iPSCs may need NANOG on top of OSKM, and FGF and knockout serum to maintain the pluripotency status (Han et al, 2011). The exogenous genes used for reprogramming in the current study were not silenced as reported in previous studies in which viral vectors had been used for reprogramming (Cao et al, 2012;Han et al, 2011;Sumer et al, 2011).…”

“…Previously, bovine iPSCs have been derived from bovine fetal and adult fibroblasts by retroviral or lentiviral transduction (Cao et al, 2012;Han et al, 2011;Sumer et al, 2011) or nonintegrating plasmids (Huang et al, 2011;Wang et al, 2013). Retroviral and lentiviral vectors are the most commonly used gene delivery systems because they have high transduction rates; lentiviral vectors even allow transduction of nonproliferating cells (Coroadinha et al, 2010;Segura et al, 2013).…”

“…Previously, bovine iPSCs were cultured in medium supplemented with growth factors including FGF and LIF (Cao et al, 2012;Sumer et al, 2011) and kinase inhibitors (PD0325901 and CHIR99021) or with N2/B27 medium with LIF (Huang et al, 2011). Here, we tested bFGF, hLIF, and kinase inhibitors alone or in combination ( Table 2).…”

“…Recent advances in the development of programmable DNA nucleases allow the targeted introduction of frameshift mutations of livestock genomes Gün and Kues, 2014;Hauschild-Quintern et al, 2013;Tan et al, 2013). Germ line-competent bovine iPSCs could be valuable for improving reproduction traits and biotechnological applications, including gene targeting and genetic modifications (Cao et al, 2012;Koh and Piedrahita, 2014). Bovine iPSCs may enhance the ability to develop transgenic cattle for the production of therapeutic proteins in milk and to introduce disease resistance and other valuable traits (Plews et al, 2012;Sumer et al, 2011).…”

“…iPSCs derived from somatic tissue are particularly promising in farm animals in which true, germline-competent embryonic stem cells (ESCs) could not yet be derived (Kumar et al, 2015). Previous approaches to derive bovine iPSCs employed retroviral (Han et al, 2011;Sumer et al, 2011), or lentiviral transduction (Cao et al, 2012) or plasmids (Huang et al, 2011;Wang et al 2013).…”