NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts1

Abstract

Since the first reports on isolation of pluripotent mouse embryonic stem (ES) cells 3 decades ago, there have been numerous attempts to derive ES cell lines from commercially important livestock species with limited success. The recent discovery that ectopic expression of a handful of stem cell-related genes was capable of inducing pluripotency in rodents and primates provided a novel approach to derivation of pluripotent stem cell lines. We used this approach in cattle and demonstrated that the ectopic expression of POU5F1 (also known as Oct4), SOX2, KLF4, and c-MYC alone was not sufficient for stable induction of pluripotency in bovine adult fibroblasts and that the additional expression of NANOG to the reprogramming cocktail was essential for the generation of stable bovine (b) induced pluripotent stem (iPS) cells. The resulting biPS cells were characterized by reverse-transcription PCR for a panel of ES marker genes. Immunocytochemical localization of POU5F1, SSEA-1, SSEA-4, and colorimetric alkaline phosphatase activity was measured in the iPS clones. The differentiation potential of the biPS cells was determined in vitro by expression of differentiation markers in embryoid bodies. Injection of biPS into immunocompromised mice resulted in teratomas containing cell types of the 3 germ lineages. This study reports the first generation of bovine induced pluripotent cell lines and paves the way for the use of biPS cells for biotechnological and agricultural purposes.

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

“…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 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).…”

“…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).…”

Insights on bovine genetic engineering and cloning

“…Whereas ES cells are not characterized/ maintained in vitro in farm animals, iPS cells (which are differentiated cells induced to pluripotency with the forced expression of some pluripotency transcription factors, most commonly Oct4, Klf4, Sox2 and c-Myc -OKSM), showed great similarity regarding self-renewing and ability to differentiate into tissues from the three germ layers. Hence, iPS cells are able to maintain the gene expression and typical morphology related to pluripotency even after a long period of in vitro culture (Esteban et al 2009, Nagy et al 2011, Sumer et al 2011.…”

“…Whereas ES cells are not characterized/ maintained in vitro in farm animals, iPS cells (which are differentiated cells induced to pluripotency with the forced expression of some pluripotency transcription factors, most commonly Oct4, Klf4, Sox2 and c-Myc -OKSM), showed great similarity regarding self-renewing and ability to differentiate into tissues from the three germ layers. Hence, iPS cells are able to maintain the gene expression and typical morphology related to pluripotency even after a long period of in vitro culture (Esteban et al 2009, Nagy et al 2011, Sumer et al 2011.Recent data from our research group have shown that cells from farm animals may be reprogrammed to pluripotency after lentiviral transduction of interspecific factors (OKSM), and reprogrammed cells presented several pluripotency markers, being characterized in a pluripotent status not yet seem in stem-cells like from farm animals (Fig.3). Bovine fetal fibroblasts, as well as mesenchymal prior to SCNT is now possible.…”