**7. The role of STAT3 signaling during reprogramming**

### **7.1. STAT3 is a master reprogramming factor**

**6. Functional role of the JAK/STAT pathway in stem cells**

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

**6.1. Stat3 maintains naïve pluripotency in mouse embryonic stem cells**

cy is conserved among species and includes OCT4, SOX2 and NANOG [174].

maintenance and their inhibition during cell differentiation [193].

**6.3. STAT3 signaling in immune cells**

**6.2. The LIF-IL6-STAT3 circuitry**

ESC pluripotency is regulated by transcriptional networks that maintain self-renewal and inhibit differentiation [172-174]. Stat3 and Myc are necessary to maintain mouse ESC (mESC) self-renewal andbindtomanyESC-enrichedgenes [175].Theirtargetgenes includepluripoten‐ cy-related transcription factors, polycomb group repressive proteins, and histone modifiers [176, 177]. The transcription factor Stat3 is a key pluripotency factor required for ESC selfrenewal [178, 179]. Mouse ESC require LIF-Stat3 (leukemia inhibitory factor) and Bmp4 (bone morphogenic protein 4) to remain pluripotent in *in vitro* cultures, whereas human ESC require FGF2/MAPK (fibroblast growth factor / mitogen-activated protein kinase) and TGFβ/Activin/ Nodal (transforming growth factor β) [180-183]. Nevertheless, the core circuitry of pluripoten‐

LIF belongs to the IL-6 family of cytokines and acts in parallel through the Jak/Stat3 and PI3K/ Akt (Phosphatidylinositide 3-kinase) pathways to maintain *Oct4, Sox2* and *Nanog* expression viaKruppel-like factor 4 (Klf4) andT-box factor 3 [184, 185].Lif andIL-6 arenecessaryfor STAT3 phosphorylation mediated by Jak1 [186]. Stat3 phosphorylation positively regulates *Klf4* and *Nanog* transcripts andfacilitatesLif-dependent maintenance ofpluripotency in a signaling loop [106].Stat3directlybinds togenomic sitesof*Oct4*and*Nanog*,regulates theOct4-Nanogcircuitry and is necessary to maintain the self-renewal and pluripotency of mESC [187-189]. Overexpres‐ sion of *Stat3* maintains mESC self-renewal even in the absence of Lif [190]. Withdrawal of LIF up-regulates the NFκB pathway and results in ESC differentiation as well as STAT3 disrup‐ tion [191-193]. The interleukin 6 (IL-6)response element (IRE) is activated by STAT3, vice versa IL-6 stimulation leads to STAT3 phosphorylation and transactivation of IRE- containing promoters providing a positively regulated STAT3-IL6 loop. STAT3 directly associates with c-Jun and c-Fos in response to IL-6 [194]. c-Jun and c-Fos are DNA binding proteins and compo‐ nentsoftheAP-1 (activationprotein-1)transcriptionfactor complex [195].AP-1 canbe activated by TLR2/4, IL-10 or STAT3 to regulate inflammatory responses or drive keratinocyte differen‐ tiation in interplay with STAT3 and c-MYC [196]. Tlr2 also plays an important role in the maintenance of mESC [107]. STAT3 is important to tune appropriate amounts of AP-1 pro‐ teins requiredforproperdifferentiation.DNAbinding sites for both*AP-1* and*STAT3*have been found in many gene promoters [194, 197]. It is important to note that c-Jun is able to capture or release the NuRD (nucleosome remodeling and deacetylation) repressor complex, an impor‐ tant epigenetic modulator of gene silencing [198, 199]. STAT3 is able to bind to bivalent histone modifications enabling a quick switchbetweenthe activationofpluripotency genesduringESC

STAT3 also has complex functions during hematopoietic development, immune regulation, cell growth, and leukemic transformation [200-202]. It is critically important for the survival and differentiation of lymphocytes and myeloid progenitors [171]. STAT3 signaling can be Activation of Stat3 is a limiting factor for the induction of pluripotency, and its over-expression eliminates the requirement for additional factors to establish pluripotency [233]. These key properties have positioned Stat3 signaling as one of the master reprogramming factors that dominantly instructs naïve pluripotency [175]. Elevated Stat3 activity overcomes the pre-iPSC reprogramming block and enhances the establishment of pluripotency induced by SOKM [234]. Stat3 and Klf4 co-occupy genomic sites of *Oct4, Sox2* and *Nanog*. Klf4 and c-Myc are downstream targets of Stat3 signaling and part of the transcriptional network governing pluripotency. The Stat3 effect is combinatorial with other reprogramming factors, which implies that additional targets of Stat3 play a pivotal role [235].

#### **7.2. STAT3 is an epigenetic regulator**

Stat3 activation regulates major epigenetic events that induce an open-chromatin state during late-stage reprogramming to establish pluripotency [236-238]. For example, Stat3 signaling stimulates DNA methylations to silence lineage commitment genes and facilitates DNA demethylations to activate pluripotency-related genes [106, 239, 240]. Other chromatin modifications include histone acetylation and deacetylation, which are catalyzed by enzymes with histone acetyltransferase (HAT) or histone deacetylase (HDAC) activities. Histone acetylation is associated with an open chromatin state that allows active gene transcription. HDAC inhibitors are known to significantly improve the efficiency of iPSC generation by allowing promoter accessibility [128, 241, 242]. STAT3 suppresses HDAC expression and repressive chromatin regulators to establish an open-chromatin structure giving full access to transcriptional machineries. The key pluripotency factor Nanog cooperates with Stat3 to maintain ESC pluripotency [173]. Interestingly, HDAC inhibitors but not *NANOG* overexpression rescues complete reprogramming in the presence of STAT3 inhibition.

NFκB signaling and inflammation during infections, whereas extensive ITAM activation inhibits JAK/STAT signaling to limit the immune reaction [256, 257]. Pleiotropic cytokines like interferons and IL-6 regulate the balance of pro-and anti-inflammatory functions by activating

The Role of an NFκB-STAT3 Signaling Axis in Regulating the Induction and Maintenance of the Pluripotent State

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NFκB and STAT3 are also part of an important stem cell pathway axis [259, 260]. A functional link between NANOG, NFκB and LIF/STAT3 signaling was shown in the maintenance of pluripotency [228]. Non-canonical NFκB signaling is activated by STAT3 through activation of IKKα and p100 processing [58]. Conversely, STAT3 inhibits TLR-induced canonical NFκB activity probably through up-regulated SOCS3. C-terminal binding of NANOG inhibits the pro-differentiation activities of canonical NFκB signaling and directly cooperates with STAT3 to maintain ESC pluripotency. NANOG and STAT3 bind to each other and synergistically

The STAT3 pathway also interacts with many signaling pathways that are critically involved in the reprogramming process. For example, STAT3 signaling activates the MYC transcriptome and signals in loop with LIN28 [229]. LIN28 is expressed in undifferentiated hESC and is able to enhance the reprogramming efficiency of fibroblasts. It is down-regulated upon ESC differentiation [262-265]. Proto-oncogene tyrosine-protein kinase Src activation triggers an inflammatory response mediated by NFκB that directly activates *IL6* and *Lin28B* expression through a binding site in the first intron. IL6-mediated activation of STAT3 transcription is necessary for monocyte activation and tumorigenesis. IL6 itself further activates NFκB, thereby completing a positive NFκB-STAT3-IL6 feedback loop that links inflammation to cell trans‐ formation [229]. Constitutive STAT3 signaling maintains constitutive NFκB activity in tumors by inhibiting its nuclear export through p65 acetylation, although STAT3 signaling inhibits

**9. The role of epigenetic regulators during the induction of pluripotency**

A panoply of chromatin remodelers play active, regulatory roles during the reprogramming process [266, 267]. For example, the Mbd3/NuRD complex is an important epigenetic regulator that restricts the expression of key pluripotency genes [268]. MBD3 (Methyl-CpG-binding domain protein 3) is part of the NuRD (nucleosome remodeling and deacetylation) repressor complex, which mediates chromatin remodeling through histone deacetylation via HDAC1/2 and ATPase activities [269-271]. The NuRD complex interacts with methylated DNA to mediate heterochromatin formation and transcriptional silencing of ESC-specific genes. Whereas MBD2 recruits NuRD to methylated DNA, MBD3 fails to bind methylated DNA as it evolved from a methyl-CpG-binding domain to a protein–protein interaction module [272]. Mbd3 antagonizes the establishment of pluripotency and facilitates differentiation [273].

variable levels of STAT1 and STAT3 [258].

**8.2. NFκB and STAT3 synergies in stem cells**

activate STAT3-dependent promoters [106, 261].

NFκB activation during normal immune responses [52].

**9.1. The NuRD complex**

Finally, DNA demethylation is regulated in mammalian cells by Tet proteins (tet methylcyto‐ sine dioxygenase), which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet1 suppresses ESC differentiation and Tet1 knockdown leads to defects in ESC selfrenewal. Tet1 up-regulation is positively regulated by Stat3 during the late-reprogramming stage [243-246].
