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

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 activated by multiple inflammatory stimuli [45, 46].

#### **2.1. The canonical NFκB signaling pathway**

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

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

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

**Figure 1.** Schema of the stromal-primed myeloid reprogramming protocol for the generation of high quality human

divisions (e.g., 2-9 passages) to generate high quality non-integrated hiPSC.

CB progenitors quickly differentiates to

importance for its reprogramming efficiency [38].

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

medium. The initial population of enriched CD34+

iPSC. 4F: four Yamanaka factors, GF: hematopoietic growth factors.

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].

#### **2.2. The non-canonical NFκB signaling pathway**

Non-canonical NFκB signaling is stimulated via the NFκB-inducing kinase (NIK), which leads to phosphorylation of the p100 precursor protein and generation of the p52:RelB dimer that translocates to the nucleus to activate gene transcription. This pathway is uniquely dependent on steady state levels of *NIK* expression, which are controlled under normal conditions through TRAF3-directed ubiquitination and proteasomal degradation. Non-canonical NFκB signaling is slow but persistent and requires de novo NIK protein synthesis and NIK stabilization [58]. It is activated by receptors that belong to the TNFR (tumor necrosis factor receptor) super‐ family like BAFF (B-cell-activating factor), CD40 or lymphotoxin β-receptor (LTβR) [59-62].

**3. Functional role of NFκB signaling in stem cells**

and *GATA6* (endodermal marker) [89].

FGF4 are highly expressed in CD34+

CD34+

**3.2. Canonical NFκB signaling in hematopoietic stem cells**

to direct early HSC commitment to the myeloid lineage [73, 91].

Nuclear p65 can be detected in 90% CB-derived CD34+

**3.3. Canonical NFκB signaling during ESC differentiation**

in granulocytosis, splenomegaly and impaired immune responses [73, 103].

**cells**

**3.1. Differential roles of canonical and non-canonical NFκB signaling in embryonic stem**

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

TLR activation is not only important for mediating innate immune responses, but also for stem cell differentiation. For example, hESC are characterized by the expression of pluripotency genes and markers such as OCT4, NANOG, alkaline phosphatase (AP) and telomerase [82-86]. NFκB signaling has been demonstrated to be crucial for maintaining ESC pluripotency and viability, and drives lineage-specific differentiation [87, 88]. A balance of canonical and noncanonical NFκB signaling regulates these opposing functions; non-canonical pathway signaling maintains hESC pluripotency, and canonical pathway signaling regulates hESC viability and differentiation [89, 90]. For example, non-canonical NFκB signaling has to be silenced during cell differentiation, which allows this pathway to act like a switch between hESC self-renewal and differentiation. RelB positively regulates several key pluripotency markers and represses lineage markers by direct binding to their regulatory units. RelB downregulation reduces the expression of pluripotency genes like *SOX2* and induces differentiationassociated genes like *BRACHYURY* (mesodermal marker), *CDX2* (trophoectodermal marker)

RelB/p52 signaling also positively regulates hematopoietic stem-progenitor cell (HSPC) selfrenewal in response to cytokines (e.g., TPO and SCF) and maintains osteoblast niches and the bone marrow stromal cell population. It negatively regulates HSPC lineage commitment through cytokine down-regulation in the bone marrow microenvironment, although it is able

Canonical p65 signaling also regulates hematopoietic stem cell functions and lineage commit‐ ment by controlling key factors involved in hematopoietic cell fate [92-94]. Canonical NFκB signaling is positively regulated by Notch1, which facilitates nuclear retention of NFκB proteins and promotes self-renewal [95-98]. FGF2 (fibroblast growth factor 2) is important for hESC self-renewal and preserves the long-term repopulating ability of HSPC through NFκB activation [99-102]. Deletion of p65, p52 and RelB dramatically decreases HSC differentiation, function and leads to extramedullary hematopoiesis [103]. NFκB pathway components and

 cells [104]. The important role of NFκB in regulating myeloid cell lineage development has been most potently revealed via genetic deletion of IKKβ, IκBα, and RelB, which resulted

Canonical NFκB signaling is very low in the undifferentiated pluripotent state, where it maintains hESC viability. However, it strongly increases during lineage-specific differentia‐

HSPC from cord blood, where they regulate clonogenicity.

cells but only in 50% BM-derived

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

7

The common feature of these receptors is the possession of a TRAF-binding motif, which recruits TRAF members (e.g., TRAF2 and TRAF3) during ligand ligation [63, 64]. Receptor recruitment of TRAF members triggers their degradation, and leads to NIK activation and p100 processing [65]. Additionally, BAFF is an important component of pluripotency-supporting growth media for the culture of ESC and a regulator of B-cell maturation [66]. It predominantly activates non-canonical NFκB signaling due to its possession of an atypical TRAF-binding sequence, which interacts only with TRAF3 but not with TRAF2 [67]. TRAF3 degradation is sufficient to trigger non-canonical NFκB signaling, whereby activation of the canonical NFκB pathway requires TRAF2 recruitment [68].

#### **2.3. CD40 stimulates both NFκB pathway components**

Another receptor associated with NFκB signaling is CD40, which is expressed on various cell types including B cells and monocytes. The CD40 receptor interacts with its ligand CD40L, which is primarily expressed on activated T cells. This signaling is majorly involved in B-cell activation, dendritic cell maturation, antigen presentation and acts as a co-stimulatory pathway of T-cells [69]. Upon ligation by CD40L, CD40 targets both the canonical and noncanonical NFκB pathways via proteolysis of TRAF2 and TRAF3 [70-72]. Non-canonical NFκB signaling regulates hematopoietic stem cell self-renewal via regulating their interactions with the microenvironment [73]. The deregulation of non-canonical hematopoietic NFκB signaling is associated with auto-immunity, inflammation and lymphoid malignancies [58, 74].

#### **2.4. NFκB subunit functions**

A third NFκB signaling pathway is activated following response to DNA damage that results in IκB degradation independent of IKK. This results in dimerization of free NFκB subunits that are mobilized similarly to canonical NFκB signaling [47]. Unlike RelA, RelB, and c-Rel, the p50 and p52 NFκB subunits do not contain transactivation domains in their C-terminus. Never‐ theless, the p50 and p52 NFκB members play critical roles in modulating the specificity of NFκB functions and form heterodimers with RelA, RelB, or c-Rel [75]. Cell contact-dependent signals are crucial during immune responses and can be mediated through NFκB signaling [76]. This can be augmented by co-stimulatory signals like CD40 or CD28 that directly bind to NFκB proteins like p65 [77-81].
