*2.1.1. Lineage- and stage-specific cytokines*

RBCs express Hbe consisting of ζ and ε isoforms (Portland 1: ζ<sup>2</sup>

leading to donor-independent blood cell production (**Figure 3**).

Gower 2: α<sup>2</sup>

246 Cell Culture

ε2

γ2

); fetal RBCs are characterized by HbF consisting of α and γ isoforms; adult RBCs

express HbA consisting of α and β isoforms (HbA1) plus a small amount of HbA2 consisting of α and δ isoforms. Hbs can be used to distinguish RBCs originated from different developmental

**Figure 2.** Human erythropoiesis/megakaryopoiesis during development. (A) Schematic depiction of site-specific (yolk sac/fetal liver-AGM/bone marrow) blood production during ontogeny focusing on erythroid and megakaryocytic lineages. (B) Representative HPLC tracks, showing the Hb content of *in vitro*-cultured erythroid cells derived from primary sources originating from distinct anatomical sites. Fetal liver-erythroid cells express fetal Hbs (HbF). Cord blood is obtained at the time of birth when the fetal to adult Hb switch takes place, resulting a mix of HbF and HbA. PBMC/ MPB-derived erythroid cells produced by the bone marrow mainly express adult hemoglobins (HbA1, HbA2).

Biochemical and molecular analysis of erythroid/megakaryocytic cells requires large cell numbers. The *in vitro* expansion and differentiation of erythroid and megakaryocytic progenitors from human fetal liver (HFL), cord blood (CB), BM, or peripheral blood enable the production of large cell numbers from distinct ontology and at defined stages of differentiation for basic research, drug testing, disease modeling, or translational purposes (**Figure 3**). Differentiation of pluripotent stem cell types such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) toward hematopoietic lineages allows to study early blood ontogeny, which is difficult to study *in vivo* as of ethical issues and availability of human material. The knowledge gained by using the abovementioned culture systems is subsequently of great value to control the expansion and differentiation of erythroid/megakaryocytic cells from ESCs/iPSCs

stages; however, in the megakaryocytic lineages, there is a lack of such markers.

; Portland 2: ζ<sup>2</sup>

β2

; Gower 1: ζ<sup>2</sup>

ε2 ;

> *Interleukins* (*ILs*) activate cytokine receptors, which do not have enzymatic activity and recruit Janus Kinases (JAK1, JAK2, JAK3, TYK) to phosphorylate tyrosines in their intracellular tail

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].

function, activating mutations in the FLT-3 receptor are prominent in acute myeloid leukemia [55]. FL, however, has a limited role in terminal MKBL/EBL differentiation because FLT-3 is

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 249

*Insulin* (*Ins*) *and Insulin-like growth factor-I* (*IGF-I*) bind the Ins receptor (InsR) and IGF-I receptor (IGF1R). Both receptors are very well conserved between species to the extent that Ins even cross-reacts between mammals and birds. The homology between the receptors suggested that Ins may also act through the IGF1R, which is not true. IGF-I has a general cell survival function mediated by PI3K activation [56]. Human erythroid progenitors (pro-EBLs, early basophilic EBLs) express the IGF1R, and not the InsR, which changes during differentiation when the InsR becomes the prominent receptor [57]. InsR signaling is particularly important to control trafficking of GLUT4 glucose transporters to the cell membrane [58]. Late EBLs and mature RBCs depend on glycolysis and EBLs express high levels of glucose transporters [59]. Upregulation of GLUT4, however, is also required for import of glutamine, required for nucleotide synthesis [60]. Because Ins and IGF-I act in physiological concentrations, their effect on *in vitro* cultures is

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-loop-

*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

*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

not expressed on committed cells.

*2.1.3. Ligand-activated TFs*

helix domain.

reticulocytes [73].

not noticed in the presence of serum or plasma [61, 62].

of the GR may alter the expansion potential of EBLs [68].

*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].

#### *2.1.2. Growth factor-binding tyrosine kinase receptors*

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

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

*FLT-3-ligand* (*FL*) binds to Fms-like tyrosine kinase 3 (FLT-3 (CD135)) as its receptor. FL is an important growth factor for CD34<sup>+</sup> HSPC by controlling their proliferation. Underscoring its function, activating mutations in the FLT-3 receptor are prominent in acute myeloid leukemia [55]. FL, however, has a limited role in terminal MKBL/EBL differentiation because FLT-3 is not expressed on committed cells.

*Insulin* (*Ins*) *and Insulin-like growth factor-I* (*IGF-I*) bind the Ins receptor (InsR) and IGF-I receptor (IGF1R). Both receptors are very well conserved between species to the extent that Ins even cross-reacts between mammals and birds. The homology between the receptors suggested that Ins may also act through the IGF1R, which is not true. IGF-I has a general cell survival function mediated by PI3K activation [56]. Human erythroid progenitors (pro-EBLs, early basophilic EBLs) express the IGF1R, and not the InsR, which changes during differentiation when the InsR becomes the prominent receptor [57]. InsR signaling is particularly important to control trafficking of GLUT4 glucose transporters to the cell membrane [58]. Late EBLs and mature RBCs depend on glycolysis and EBLs express high levels of glucose transporters [59]. Upregulation of GLUT4, however, is also required for import of glutamine, required for nucleotide synthesis [60]. Because Ins and IGF-I act in physiological concentrations, their effect on *in vitro* cultures is not noticed in the presence of serum or plasma [61, 62].
