br Patients and methods br Introduction Embryonic stem

Patients and methods
Introduction Embryonic stem (ES) Sodium phosphate monobasic can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages (Reubinoff et al., 2000; Wobus and Boheler, 2005). This ability to form different cell types under appropriate conditions makes them a powerful tool in the study of biological mechanisms and treatment of disease. In vitro, mouse ES (mES) cells can be maintained in an undifferentiated state with the addition of the cytokine leukemia inhibitory factor (LIF) to culture media (Smith et al., 1988; Williams et al., 1988). LIF primarily acts through the JAK–STAT signaling pathway to maintain pluripotency (Niwa et al., 2009; Paling et al., 2004). Self-renewal also is enhanced by inhibition of the mitogen-activated protein kinase (MAPK) pathways. Members of the MAPK family, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAP kinases, have been extensively studied for their role in phosphorylation and activation of transcription factors that regulate genes that are instrumental in cell growth, adaptation, differentiation, and transformation. ERKs, JNKs, and p38 are also able to regulate ES commitment from early steps of the process to mature differentiated cells (Binetruy et al., 2007; Burdon et al., 1999; Lee et al., 2006; Van Hoof et al., 2009). The JNKs are encoded by three genes (jnk1, jnk2, and jnk3) and participate in cellular decisions following diverse forms of stress as well as responses to growth factors, oncogenes, cytokines, and morphogens (Bode and Dong, 2007). Moreover, JNKs are involved in both ectoderm and primitive endoderm differentiation (Aubert et al., 2002; Haegele et al., 2003). Studies in model organisms in which the jnk genes have been disrupted support a role for JNKs in embryonic development and morphogenesis. Mouse somatic cells can also acquire a pluripotent state in vitro after the introduction of a defined combination of transcription factors that are highly enriched in embryonic stem (ES) cells (Aasen et al., 2008; Lowry et al., 2008; Nakagawa et al., 2008; Park et al., 2008; Takahashi and Yamanaka, 2006; Wernig et al., 2008). Mouse iPS cells (iPSCs) are similar to ES cells in most aspects and can generate entire individuals after tetraploid complementation. iPSCs have raised the possibility of clinical application of personalized stem cell-based therapies without immune rejection or ethical concerns. Human iPSCs also provide a unique platform for studying genetic diseases in vitro. However, the low efficiency of iPSC generation is a significant impediment for mechanistic studies and high throughput screening, and also makes bona fide colony isolation time consuming and costly (Maekawa et al., 2011). Therefore, understanding the molecular mechanisms governing the commitment of ES cells is an essential challenge in this field. Krüppel-like factor 4 (Klf4/GKLF/EZF), also known as gut-enriched Krüppel-like factor (GKLF), belongs to the KLF family of evolutionarily conserved zinc finger transcription factors that regulate numerous biological processes, including proliferation, differentiation, development, and apoptosis. As a key factor in reprogramming, Klf4 functions as both a transcriptional activator and repressor to regulate proliferation and differentiation of different cell types (Rowland and Peeper, 2006). RNA interference experiments confirm that Klf4 is redundant with two other family members, Klf2 and Klf5, in regulating expression of pluripotency related genes. In ES cells, Klf4 has been shown to be important to activate Lefty1 together with Oct4 and Sox2 (Chan et al., 2009; Nakatake et al., 2006; Wei et al., 2009). Genome-wide chromatin immunoprecipitation with microarray analysis (ChIP-Chip) demonstrates that the DNA binding profile of Klf4 overlaps with that of Oct4 and Sox2 on promoters of genes specifically underlying establishment of iPSCs (Nakatake et al., 2006), suggesting transcriptional synergy among these factors. Furthermore, studies also indicate that Klf4 might function in establishing a pluripotent state in various pluripotent cell types. Recent studies show that post-translational modifications of iPS factors regulate their activity. For example, phosphorylation of human Sox2 inhibits Sox2 DNA binding activity (Tsuruzoe et al., 2006; Van Hoof et al., 2009). Acetylation of mouse Sox2 enhances nuclear export and degradation of Sox2 through a ubiquitin-mediated degradation pathway (Baltus et al., 2009). Furthermore, phosphorylation of human Oct4 might partially regulate Oct4 transactivation (Saxe et al., 2009). Klf4, as a critical transcriptional regulator, controls the switch from somatic cell to stem cell. Klf4 is regulated by various post-translational modifications, including phosphorylation, acetylation and sumoylation (Du et al., 2010; Evans et al., 2007; Hu and Wan, 2010). These findings indicate that post-translational modifications of iPS factors are likely involved in the regulation of their activity, which could result in modulation of ES cell self-renewal activity and efficiency of iPSC generation. However, the underlying mechanism remains largely unknown.