**5. Overview of the JAK/STAT pathway**

tion of pluripotent stem cells. p65 binds to the regulatory regions of similar differentiation genes as RelB with opposing effects on their activation or silencing. It regulates cell prolifera‐ tion by direct binding to the CYCLIN D1 promoter [89]. There are different levels of inhibiting canonical NFκB signaling: first, p65 translational repression by the microRNA cluster miR-290 to maintain low p65 protein amounts and second, the inhibition of translated p65 by physical interaction with NANOG. Similarly, *OCT4* expression is reversely correlated with canonical NFκB signaling [105]. In contrast to most observations in mouse ESC, NFκB probably plays a more important role in the maintenance of human ESC pluripotency [106]. Finally, active TLRs are expressed on embryonic, hematopoietic and mesenchymal stem cells (MSC), thus impli‐

**4. Role of NFκB signaling during reprogramming to pluripotency**

of pluripotency, transdifferentiation and even malignant transformation [112-116].

Undifferentiated human iPSC have elevated NFκB activities, which play important roles in maintaining *OCT4* and *NANOG* expression in pluripotent hiPSC [111]. Innate immune TLR signaling was recently shown to enhance nuclear reprogramming probably through the induction of an open chromatin state, and global changes of epigenetic modifiers [39]. This normally increases cell plasticity in response to a pathogen, but may also enhance the induction

The EBNA (Epstein-Barr virus nuclear antigen) is a virus-derived protein that is not only a critical component of episomal reprogramming vectors, where it mediates extra-chromosomal self-replication, but it is also known to activate several TLRs [117-119]. These include TLR3, which is known to augment reprogramming efficiencies through the activation of inflamma‐ tory pathways [39, 120]. TLR3 recognizes double-stranded RNA from retroviruses and signals through TRAF6 and NFκB [121-123]. The TLR3 agonist poly I:C was shown to have the same effect as retroviral particles in enhancing Yamanaka factor-induced iPSC production. TLR3 causes widespread changes in the expression of epigenetic modifiers and facilitates nuclear reprogramming by inducing an open chromatin state through down-regulation of histone deacetylases (HDACs) and H3K4 (histone H3 at lysine 4) trimethylations [38, 39, 124]. These epigenetic modifications mark transcriptionally active genes, whereas the H3K9me3 (Histone H3 at lysine 9) modification marks transcriptionally silenced genes [125, 126]. Histone deacetylation is generally associated with a closed chromatin state and HDAC inhibitors were shown to enhance nuclear reprogramming [127, 128]. Histone acetylation favors an open chromatin state, and is maintained by proteins containing histone acetyltransferase (HAT) domains, such as p300 and CBP [129, 130]. Interestingly, p300/CBP is able to interact with NFκB [131, 132]. RelB directly interacts with the methyltransferase G9a to mediate gene silencing of differentiation genes [133]. Epigenetic changes that allow an open chromatin state are crucial for giving the Yamanaka factors access to promoter regions necessary for the induction of pluripotency. Epigenetic chromatin modifications by TLRs are normally involved in the expression of host defense genes during infections [134-136]. This capability can be deployed to enable nuclear reprogramming as TLR3 was shown to change the methylation status of the *Oct4* and *Sox2* promoters. Interestingly, changes in these methylation marks were

cating their roles in a variety of stem cell types [107-110].

8 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

The JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription) integrates a complex network of exterior signals into the cell, and can be activated by a variety of ligands and their receptors [139]. These receptors are associated with a JAK tyrosine kinase at their cytoplasmic domain. The JAK family consists of the four members JAK1, JAK2, JAK3 and TYK2 [140, 141]. Many cytokines and growth factors signal through this pathway to regulate immune responses, cell proliferation, differentiation and apoptosis [142-146]. Ligand binding induces the multimerization of gp130 receptor subunits, which brings two JAKs close to each other inducing trans-phosphorylation. Such activated JAKs phosphorylate their receptor at the C-terminus and the transcription factor STAT at tyrosine residues. This allows STAT dimerization and their nuclear translocation to induce target gene transcription. [147, 148] STAT3 acetylation is critical for stable dimer formation and DNA binding [149]. From the 7 mammalian STATs, STAT3 and STAT5 are expressed in many cell types, are activated by a plethora of cytokines and growth factors, and integrate complex biological signals [150, 151]. The other STAT proteins mainly play specific roles in the immune response to bacterial and viral infections. STAT3 is an acute phase protein with important functions during immediate immune reactions [152-154]. STAT3 can be recruited by receptor tyrosine kinases that harbor a common STAT3 binding motif in their cytoplasmic domain (e.g., GCSF (granulocyte colonystimulating factor), LIF (leukemia inhibitory factor), EGF (epidermal growth factor), PDGF (platelet-derived growth factor), interferons (IFNγ) and interleukins (IL-6, IL-10)) [155-158]. Many cytokines signal through IL-10/STAT3 to achieve an immunosuppressive function or anti-apoptotic effect [159, 160]. IL-10 is also required during terminal differentiation of immunoglobulins [161]. STAT3 can be phosphorylated at tyrosine or serine residues. The phosphorylation site can play distinct roles in the regulation of downstream gene transcription [162]. Stat3-deficient mice die during early embryogenesis due to Stat3 requirement for the self-renewal of ESC [163].

Negative feedback regulation of the JAK/STAT circuitry is mediated by the SOCS family of target genes (suppressors of cytokine signaling) in a way that activated STAT induces SOCS transcription [164, 165]. SOCS proteins can bind to phosphorylated JAKs as a pseudo-substrate to inhibit JAK kinase activity and turn off the pathway [166, 167]. SOCS are negative regulators of the immune response [168, 169]. A small peptide antagonist of SOCS1 was shown to bind to the activation loop of JAK2 leading to constitutive STAT activation and TLR3 induction. This boosts the immune system to exert broad antiviral activities [170]. The JAK/STAT pathway also interacts with many other signaling pathways in a complex manner to regulate cell homeostasis and immune reactions [149, 171].
