**1. Introduction**

Induced pluripotent stem cells (iPSC) are generated by reprogramming differentiated somatic cells to a pluripotent cell state that highly resembles embryonic stem cells (ESC) [1]. Fully reprogrammed iPSC can differentiate into any adult cell type [2-6]. Takahashi and Yamanaka generated the first iPSC in 2006 by transfecting fibroblasts with four defined factors: SOX2, OCT4, KLF4, c-MYC (SOKM; also referred to as Yamanaka factors) [7]. The clinical use of iPSC offers great potential for regenerative medicine as any cell type can be generated from true pluripotent cells [8-10]. However, human clinical iPSC applications are currently limited by inefficient methods of reprogramming that often generate incompletely reprogrammed pluripotent states that harbor potentially cancerous epigenetic signatures, and possess limited or skewed differentiation capacities [11-13]. Many standard iPSC lines do not fully resemble pluripotent ESC, and often retain an epigenetic memory of their cell of origin [14, 15]. Such incompletely reprogrammed iPSC also display limited differentiation potential to all three germ layers (e.g., endoderm, ectoderm, mesoderm) [16, 17].

To avoid integrating retroviral constructs that may carry mutagenic risks, many non-viral methods have been described for hiPSC derivation [18, 19]. For example, one successful approach is to transiently express reprogramming factors with EBNA1-based episomal vectors [20-22]. It was initially intuitive to reprogram skin fibroblasts due to their easy accessibility. However, standard episomal reprogramming in fibroblasts occurs at even lower efficiencies (< 0.001-0.1%) than reprogramming with retroviral vectors (0.1%–1%) [23-25]. Subsequent studies revealed that various cell types possess differential receptive‐ ness for being reprogrammed to pluripotency [26-30]. One highly accessible human donor source is blood, which has been demonstrated to reprogram with significantly greater efficiency than fibroblasts [4, 20, 31-33].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The innate immune system possesses highly flexible cell types that are able to adapt quickly to various pathogens by eliciting defense responses that protect the host [34-36]. Innate immune cells derived from the myeloid lineage (eg, monocyte-macrophage, dendritic cells, neutrophils) are able to reactivate some unique features of pluripotent stem cells that may give them greater flexibility for being reprogrammed to a pluripotent cell state than other differ‐ entiated cells [37]. Additionally, the differentiation state of the cell seems to be of critical importance for its reprogramming efficiency [38].

A proteomics and bioinformatics analysis of this reprogramming system implicated significant activation of MSC-induced inflammatory TLR-NFκB and STAT3 signaling [20]. A combination of cell contact-dependent and soluble factors mediate these effects. A recent study similarly implicated inflammatory TLR3 signaling as a novel trigger for enhanced fibroblast reprog‐ ramming, albeit at much lesser efficiencies than observed in our myeloid reprogramming system. TLR3 signaling leads to epigenetic modifications that favor an open chromatin state, which increases cell plasticity and the induction of pluripotency [39]. Lee *et al.* termed this novel link between inflammatory pathways and cell reprogramming 'Transflammation' [40].

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

http://dx.doi.org/10.5772/57602

5

In this chapter we will discuss hypotheses why inflammation-activated myeloid cells may be highly receptive to factor-mediated reprogramming. Specifically, we will explore the role of the NFκB-STAT3 signaling axis in mediating the unique susceptibility of myeloid cells to high-

Multipotent myeloid progenitors are derived from hematopoietic stem cells and differentiate to monocytes macrophages, dendritic cells, and granulocytes, which elicit the initial innate immune response toward pathogens [41]. NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a central transcription factor that regulates these innate immune responses during microbial infections [42-44]. The NFκB system belongs to a group of early-acting transcription factors that are present in the cytoplasm in an inactive state but can be quickly

The NFκB family consists of 5 members; p65 (RelA), p50 and c-Rel are involved in canonical signaling, and p52 and RelB are involved in non-canonical signaling. Canonical NFκB signaling is characterized by activation of the IκB kinase complex (IKK), which contains two kinases, IKK1/α and IKK2/β along with a non-catalytic subunit called IKKγ (NEMO) [47, 48]. Unstimulated NFκB is sequestered in the cytoplasm by IκBα protein. In contrast, activation of the IKK complex (e.g., by TLRs) leads to IKKβ-mediated serine phosphorylation of IκBα triggering its proteasome-mediated degradation and its dissociation from NFκB [49, 50]. This activates the p65:p50 dimer through p65 phosphorylation and leads to NFκB translocation into the nucleus where it induces target gene expression. Subsequent acetylation keeps p65 in the nucleus [51]. This can be reverted by HDAC3 (histone deacetylase 3)-induced deacetylation of p65, which increases the affinity of NFκB proteins for IκBα and nuclear export [52, 53]. Canonical NFκB signaling is a fast and transient process that regulates complex inflammatory processes that includes the initial pro-inflammatory phase, the induction of apoptosis, and even tumorigenesis [54]. It can be activated by toll-like receptors (TLR), which recognize

characteristic pathogenic molecules to activate innate immune responses [55-57].

**2. Overview of the canonical and non-canonical NFκB pathway**

quality human iPSC derivation.

activated by multiple inflammatory stimuli [45, 46].

**2.1. The canonical NFκB signaling pathway**

Our group established a reprogramming method that solves many of the technical caveats cited above (Figure 1). We have generated high-fidelity human iPSC (hiPSC) from stromalprimed (sp) myeloid progenitors [20]. This system can reprogram >50% of episome-expressing myeloid cells to high-quality hiPSC characterized by minimal retention of hematopoieticspecific epigenetic memory and a molecular signature that is indistinguishable from bona fide human ESC (hESC). The use of bone marrow-, peripheral-or cord blood (CB)-derived myeloid progenitor cells instead of fibroblasts, and a brief priming step on human bone marrow stromal cells / mesenchymal stem cells (MSC) appeared to be critical for this augmented reprogram‐ ming efficiency. In this system, CD34+ - enriched cord blood cells (CB) are expanded with the growth factors (GF) FLT3L (FMS-like tyrosine kinase 3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) for 3 days, subsequently nucleofected with non-integrating episomes expressing the Yamanaka factors (4F, SOX2, OCT4, KLF4, c-MYC), and then co-cultured on irradiated MSC for an additional 3 days. Cells are then harvested, and passaged onto MEF (mouse embryonic fibroblasts), and hiPSC are generated via standard methods and culture medium. The initial population of enriched CD34+ CB progenitors quickly differentiates to myeloid and monocytic cells in this system, and reprogrammed cells arise from CD34- myeloid cells. The first iPSC colonies appear around day 10, and stable mature iPSC colonies can be established after ~21-25 days. The episomal constructs are partitioned after relatively few cell divisions (e.g., 2-9 passages) to generate high quality non-integrated hiPSC.

**Figure 1.** Schema of the stromal-primed myeloid reprogramming protocol for the generation of high quality human iPSC. 4F: four Yamanaka factors, GF: hematopoietic growth factors.

A proteomics and bioinformatics analysis of this reprogramming system implicated significant activation of MSC-induced inflammatory TLR-NFκB and STAT3 signaling [20]. A combination of cell contact-dependent and soluble factors mediate these effects. A recent study similarly implicated inflammatory TLR3 signaling as a novel trigger for enhanced fibroblast reprog‐ ramming, albeit at much lesser efficiencies than observed in our myeloid reprogramming system. TLR3 signaling leads to epigenetic modifications that favor an open chromatin state, which increases cell plasticity and the induction of pluripotency [39]. Lee *et al.* termed this novel link between inflammatory pathways and cell reprogramming 'Transflammation' [40].

In this chapter we will discuss hypotheses why inflammation-activated myeloid cells may be highly receptive to factor-mediated reprogramming. Specifically, we will explore the role of the NFκB-STAT3 signaling axis in mediating the unique susceptibility of myeloid cells to highquality human iPSC derivation.
