Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells

Abstract

Embryonic stem cell (ES cell) lines were first generated by culturing mouse inner cell mass (ICM) on feeder layers in 1981 [1]. However, in large domestic animals, attempts to establish ES cell lines from ICM of blastocysts or the later epiblast have not been successful. This has hindered the efficient production of genetically modified livestocks by using ES-based approaches. Recently, it was found that ectopic expression of various combinations of transcription factors is able to reprogram somatic cells to a pluripotent state [2][3][4][5]. These induced pluripotent stem (iPS) cells show similarities to embryo-derived ES cells and can be used to produce viable mice through tetraploid complementation [6,7]. So far, iPS cells of several mammalian species have been successfully generated [2,3,[8][9][10][11][12]. In this letter, we report the first establishment of bovine iPS cells using defined transcription factors and a modified culture medium.cDNAs coding for the bovine OCT4 (also named POU5F1), SOX2, KLF4, MYC, LIN28, and NANOG genes were cloned into pMXs retroviral vector. The pMXs plasmids containing human OCT4, SOX2, KLF4, and c-MYC genes were all purchased from Addgene. GP2-293 cells were used as the packaging cell line for retroviral production. Bovine fibroblasts used in this study were derived from an E55 Western Shandong Yellow cattle fetus. Three sets of factors, termed b4TF, b6TF, and h4TF, were used to transduce cells by overnight retroviral infection, respectively. Whereas the former two included only bovine factors (b4TF: bOCT4, bSOX2, bMYC, bKLF4; b6TF: bOCT4, bSOX2, bKLF4, bMYC, bLIN28, bNANOG), the latter employed only human factors (hOCT4, hSOX2, hc-MYC, hKLF4). On day 2, the infected cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeders at a density of 1 × 10 4 cells per 100-mm dish. The next day after being seeded onto feeders, growing cells were cultured in iPS media ( Figure 1A and Supplementary information, Table S1). Bovine iPS (hereinafter named biPS) cells with a mouse ES-like morphology were detected after ~3 Figure 1B). On day 21-35, colonies were isolated mechanically using a 200 µl pipette and transferred to feeder-coated tissue culture dishes. The biPS cells were split with trypsin at a ratio of 1:10 every 4-5 days afterwards (Supplementary information, Data S1). A total of 26 b6TF-derived colonies have been expanded into biPS cell lines. These lines maintained good ES-like morphology for more than 16 passages. However, none of the colonies generated by b4TF or h4TF could be passaged more than six times. Importantly, we showed that the combination of six transcription factors (b6TF set) significantly increased the efficiency of iPS cell generation (by three-fold) compared to the other two combinations ( Figure 1C).We tested eight different types of biPS culture media (Supplementary information, Table S1) by assessing the numbers of ES-like colonies obtained from b6TF-transduced BEFs on day 28. Three out of the eight media could efficiently generat...

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

“…The biPS-1 cell line was established in medium supplemented with bFGF (8 ng/mL) and hLIF (1000 U/mL), suggesting that the bovine iPSCs use different pluripotency signaling pathways and require different culture conditions than human or murine iPSCs. 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).…”

“…iPSCs were first derived from mouse fibroblasts by overexpression of four reprogramming factors-Oct4, Sox2, Klf4, and c-Myc-and subsequently from human fibroblasts by overexpression of OCT4, SOX2, LIN28, and NANOG (Takahashi and Yamanaka, 2006;Yu et al, 2007) after retroviral transfection. Retroviral and lentiviral delivery of core reprogramming factors allowed the derivation of iPSCs from several other mammals, including rat (Liao et al, 2009), dog (Shimada et al, 2010), pig (Esteban et al, 2009), rhesus monkey (Liu et al, 2008), sheep (Bao et al, 2011), goat , horse (Nagy et al, 2011), cattle (Han et al, 2011), buffalo (Deng et al, 2012), and of iPS-like cells from nonmammalian vertebrates (Rossello et al, 2013). 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).…”

“…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).…”

A special issue on cell signaling, disease, and stem cells

“…We chose to highlight these stem cell papers in this special issue as stem cell research is intrinsically connected to the study of cell signaling and disease. The collection of a large number of stem cell papers also reflects the fact that stem cell research has become an important part of the growing content of Cell Research, as highlighted by a large number of important discoveries published in the journal in the past year alone, such as the successful generation of iPS cells with a single factor Oct4 [3,4], efficient generation of human iPS cells from blood cells via a non-integrating method [5], generation of functional platelets from hESCs [6], directed differentiation of atrial and ventricular myocytes from hESCs [7], the successful generation of ovine, hircine and bovine iPS cells [8][9][10], somatic cell reprogramming using electrofused blastomeres [11], improved methods for iPS cell generation [12,13], gener-ating iPS cells using Bmi1 [14], comprehensive analyses of chromatin state dynamics in hESC differentiation [15], characterization of neuroblasts in the adult human brain [16], identification of lectin biomarkers for human pluripotent stem cell isolation [17], and efficient correction of disease-causing genetic mutations in patient iPS cells [18].…”