*2.1.3. Ligand-activated TFs*

that can subsequently recruit signaling molecules. Cytokine receptors are often expressed on a limited set of cell types/differentiation stages. IL-3 and IL-6 are cytokines that promote survival of hematopoietic stem/progenitor cells (HSPCs), but they do not act on EBL-specific stages. IL-9 synergizes with IL-3 to enhance both erythropoiesis and megakaryopoiesis [31, 32]. IL-1β is an inflammatory cytokine that signals via MyD88 to IRAK and NF-κB to inhibit cell growth, to induce expression of growth factors and the extracellular matrix, and to cause apoptosis in most tissues. In megakaryopoiesis, it enhances commitment and differentiation [33, 34].

*Erythropoietin* (*EPO*) is mainly produced by the kidney (80%) and partially in the liver (10–15%; 30% upon stress erythropoiesis) [35, 36]. EPO binds and activates EPO receptor (EPOR) and functions as a survival factor during erythropoiesis [37, 38]. Deficiency of Epo and/or Epor causes embryonic lethality due to the failure of definitive fetal liver erythropoiesis leading to the lack of mature RBCs [24, 39–41]. Fetal livers of EPO-deficient embryos contain normal numbers of erythroid progenitors that can form colony-forming unit-erythroid (CFU-E) *in vitro* in the presence of exogenous Epo [39]. EPO shows cross-reactivity in human and mouse.

*Thrombopoietin* (*TPO*) is the ligand for the MPL receptor. MPL is well conserved between species and TPO showing cross-reactivity between mouse and human. TPO has a direct effect on self-renewal and expansion of HSCs in the BM, but controls megakaryopoiesis as well [42]. MKs and Plts bind TPO and sequester it from the circulation. Upon activation, Plts release TPO into the plasma, thereby stimulating megakaryopoiesis. Thus, the number of Plts and their activation controls TPO levels [43]. TPO is mainly produced by hepatocytes in the human liver. TPO production is increased when hepatocytes bind damaged and aged desialylated Plts via the Ashwell-Morell receptor. In a JAK2/STAT3-dependent manner, this activates TPO transcription and thereby regulates Plt production, HSC renewal, and expansion [42, 44, 45].

The tyrosine kinase receptors directly cross-phosphorylate tyrosine residues in their own cytoplasmic tail, and they phosphorylate downstream effector molecules. They are more

*Stem cell factor* (*SCF*; first described as the *Steel* locus) signals via the mast/stem cell growth factor receptor KIT (CD117; *White locus*). SCF cooperates with other cytokines in order to maintain the viability of HSPCs, and their proliferation/differentiation ability [46–48]. SCF is produced by the stromal cells in the BM as a secreted soluble factor but also as a membranebound factor. A specific mutation in SCF (*Steel-Dickie*) disrupting the membrane form of SCF leads to severe anemia, indicating that the membrane-bound form is crucial at least for erythropoiesis [49]. In addition, Kit-deficient mice (*W/W*) suffer from neonatal lethality due to severe anemia [50, 51]. Besides its function on HSPC, SCF is particularly important upon blood loss, to enhance proliferation and delay differentiation of the erythroid and megakaryocytic progenitors [52–54]. In contrast to TPO and EPO, SCF is not interchangeably reactive

*FLT-3-ligand* (*FL*) binds to Fms-like tyrosine kinase 3 (FLT-3 (CD135)) as its receptor. FL is an

HSPC by controlling their proliferation. Underscoring its

between mouse and human—mouse SCF activating both, while human does not.

*2.1.2. Growth factor-binding tyrosine kinase receptors*

248 Cell Culture

widely expressed compared to cytokine receptors.

important growth factor for CD34<sup>+</sup>

Steroid hormone receptors are the best known nuclear hormone receptors. In addition to these ligand-dependent TFs that bind DNA through a Zinc-finger domain, other ligand-activated TFs exist such as the aryl hydrocarbon receptor (AhR) that binds DNA through a helix-loophelix domain.

*Glucocorticoids* bind the glucocorticoid receptor (GR), a nuclear hormone receptor, which translocates to the nucleus upon association with its ligand, where it functions as a transcriptional repressor and activator of gene expression. The GR homodimer binds to glucocorticoid response elements that consists of two inverted repeats. As a heterodimer with for instance STAT5, it only needs a "halfmer" GRE, i.e., a single repeat combined with the STAT5 binding site [63, 64]. The ligand of the GR is glucocorticoid (*in vivo*) produced by the adrenal gland. Several synthetic ligands have been designed to be used as immunosuppressive agents (e.g., dexamethasone—DEX). The GR exerts its immunosuppressive function at pharmaceutical levels of glucocorticoids, and as a monomer that binds and inhibits other TFs such as NF-κB or FOS/Jun dimers [65]. In contrast, stress erythropoiesis *in vivo*, and expansion of EBL cultures *in vitro*, is induced at physiological levels of glucocorticoids and depends on dimerization of the GR and on the ligand-dependent transcription activation domain [66, 67]. Polymorphisms of the GR may alter the expansion potential of EBLs [68].

*3,5,3 -triiodothyronine* (*T3*) binds the α and β thyroid hormone receptors (TRα, TRβ). T3 deficiency is associated with anemia, although it is not clear whether this is caused by a direct effect on erythropoiesis [69, 70]. The effect of T3 appears to be highly species and developmental stage specific. T3 has a potent differentiation promoting effect on avian erythropoiesis, but mouse EBLs are only sensitive to T3 during neonatal spleen erythropoiesis [71, 72]. In cultures of human erythroid cells, T3 enhances synchronous differentiation to enucleated reticulocytes [73].

*StemRegenin 1* (*SR-1*) binds and inhibits the AhR, which mediates toxicity of environmental pollutants, by binding to specific DNA enhancer sequences. This receptor can be activated by endogenous or exogenous ligands and contributes to several physiological processes, among which cell migration, apoptosis, and cell growth [74–77]. Importantly, AhR-deficient mice have increased numbers of BM HSCs [78]. The AhR functions in complex with the AhR Nuclear Translocator to induce differentiation by regulating genes directly such as *c-MYC* and *C/EBP*. During hematopoietic differentiation, its targets also include *PU.1, β-CATENIN, CXCR4,* and *STAT5* [79]. Currently, the antagonist of AhR (SR-1) is used in clinical trials to expand CB-CD34<sup>+</sup> cells prior to transplantation, thereby reducing the number of CB units needed per transplant from 5 to 1 [80]. Importantly, SR-1 also enhances *in vitro* proplatelet formation [81]. To achieve proper Plts production, MKs have to produce an extensive membrane system; the demarcation membrane system. This requirs extensive lipid biosynthesis which can be inhibited by AhR [82]. Thus, blocking the inhibition of AhR with SR-1 may increase the proplatelet formation, through increasing the lipid membrane biosynthesis.

BM transplantation. Exposure of IL-11 on HSPCs directly leads to an inhibition of NF-κB signaling, which suppresses miR-204-5p, that targets and represses the expression of TPO [97]. Because of this effect on TPO, it has a dual role in MK differentiation, first, in the expansion of HSCs, second, in the terminal differentiation of MKs. In a similar way, IL-9 synergizes with IL-3 as well as with IL-4 and SCF to increase the yield of MKBL [33, 98]. Furthermore, the addition of IL-1β promotes selective megakaryocytic differentiation. IL-1β can increase the production of Plts by enhancing the effects of SDF1 and FGF4 that are produced by BM niche cells. IL-1α was shown to induce MK rupture and is considered as a stress megakaryopoiesis

Inhibition of AhR by SR-1 slows the differentiation program in HSCs, leading to increased expansion, and has a direct effect on MK differentiation, by slowing and/or conditioning cells to have a more synchronized maturation [74, 79, 81]. Through its effect on PU.1, it can influence RUNX1 which is one of the TF-regulating megakaryopoiesis. The introduction of SR-1 into *in vitro* MK cultures results in increased cell size, higher polyploidization, and proplatelet production. In these cultures, a specific MK precursor population is identified that has an

The most widely used immortalized mouse erythroid cell lines are MEL cells, which are EBLs transformed by Friend Leukemia virus. The viral gp55 protein activates the Epor to sustain cell growth, whereas integration of the virus upstream of *PU.1* or Friend leukemia integration 1 (*Fli-1*) induces constitutive high expression and inhibits differentiation. Leukemogenesis and the establishment of cell lines require additional inactivation of tumor suppressor protein p53 [100]. Under conditions that enable transient expansion of primary mouse cultures (serum-free medium supplemented with Epo, SCF, and glucocorticoids), lack of p53 is sufficient to establish immortalized cell lines with full differentiation potential in the absence of

Deletion of p53 is not sufficient in human to establish immortalized erythroid cell lines. However, expression of the human papilloma virus E6/E7 proteins in EBLs differentiated from iPSC gave rise to the HiDEP cell line [101]. The E6/E7 proteins inactivate both p53 and retinoblastoma tumor suppressor proteins [102]. They are expressed from a doxycycline (dox) inducible vector allowing for unlimited growth in the presence of EPO, SCF, and dox, and differentiation in the presence of EPO but without dox and SCF. Lack of p53 does not affect differentiation, but retinoblastoma is required for terminal erythroid differentiation [103–105]. Dox-inducible expression was also used to establish immortalized erythroid cells lines from

HUDEP, and BEL-A express embryonic, fetal, and adult Hbs, respectively [101, 107].

HSPC (BEL-A) [106, 107]. The HiDEP,

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 251

regulator [99].

**2.2. Cell culture models**

*2.2.1.1. Mouse cell lines*

SCF and glucocorticoids [13].

CB (HUDEP) and from adult EBLs cultured from CD34<sup>+</sup>

*2.2.1.2. Human cell lines*

*2.2.1. Erythroid immortalized cell lines*

increased potential to produce proplatelets [81].

#### *2.1.4. Erythroid-specific regulation*

EPO is sufficient for steady-state erythropoiesis, when proliferative signals are mediated through the EPOR-associated RON receptor and EPO-induced differentiation is dependent on STAT5 [83, 84]. Increased erythropoiesis during development and upon blood loss requires the cooperative action of the EPOR and KIT [85]. Activation of KIT prevents differentiation and propagates the long-term proliferation of erythroid progenitors through inhibition of *FOXO3a* and activation of mRNA translation via the PI3K/mTOR pathway [86, 87]. Activation of the GR is required for stress erythropoiesis and to inhibit *in vitro* differentiation. Interestingly, glucocorticoids activate largely the same growth inhibitory genes in EBLs and in immune cells, but growth inhibition is counteracted by EPOR/KIT activation [88]. Even in the presence of serum, glucocorticoids promote selective proliferation of erythroid progenitors by both supporting erythroid proliferation and inhibiting proliferation of other myeloid and lymphoid cells [57].

Erythropoiesis is also regulated by the availability of iron, which is imported into the cell as holotransferrin via the transferrin receptor (TfR; CD71), and by selenium through selenoproteins [89, 90]. *In vivo*, erythropoiesis is dependent on the formation of erythropoietic islands that form around a central macrophage [91, 92]. Whereas CD169 macrophages are essential for erythropoiesis *in vivo*, high level of enucleation can be achieved *in vitro* in the absence of macrophages (van den Akker and von Lindern, manuscript in preparation) [93]. The complete understanding of which macrophage signals control enucleation *in vivo* may further optimize the *in vitro* production of erythrocytes for transfusion purposes.

#### *2.1.5. MK-specific regulation*

TPO is the main regulator of megakaryopoiesis, and multiple other factors can work synergistic with it. IL-6 can, in conjunction with TPO, increase hepatic TPO synthesis [94]. This effect is through the shared usage of gp130 and amplification of the same downstream JAK pathways [95]. The direct admission of IL-6, besides its effect on TPO, results in increased polyploidization and subsequently leads to an enhanced Plts production in patients [96]. IL-11 is not constitutively expressed but was shown to be induced in thrombocytopenia patients undergoing

BM transplantation. Exposure of IL-11 on HSPCs directly leads to an inhibition of NF-κB signaling, which suppresses miR-204-5p, that targets and represses the expression of TPO [97]. Because of this effect on TPO, it has a dual role in MK differentiation, first, in the expansion of HSCs, second, in the terminal differentiation of MKs. In a similar way, IL-9 synergizes with IL-3 as well as with IL-4 and SCF to increase the yield of MKBL [33, 98]. Furthermore, the addition of IL-1β promotes selective megakaryocytic differentiation. IL-1β can increase the production of Plts by enhancing the effects of SDF1 and FGF4 that are produced by BM niche cells. IL-1α was shown to induce MK rupture and is considered as a stress megakaryopoiesis regulator [99].

Inhibition of AhR by SR-1 slows the differentiation program in HSCs, leading to increased expansion, and has a direct effect on MK differentiation, by slowing and/or conditioning cells to have a more synchronized maturation [74, 79, 81]. Through its effect on PU.1, it can influence RUNX1 which is one of the TF-regulating megakaryopoiesis. The introduction of SR-1 into *in vitro* MK cultures results in increased cell size, higher polyploidization, and proplatelet production. In these cultures, a specific MK precursor population is identified that has an increased potential to produce proplatelets [81].
