*2.2.1.2. Human cell lines*

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 CB (HUDEP) and from adult EBLs cultured from CD34<sup>+</sup> HSPC (BEL-A) [106, 107]. The HiDEP, HUDEP, and BEL-A express embryonic, fetal, and adult Hbs, respectively [101, 107].

#### *2.2.2. MK-cell lines*

Multiple cell lines have been generated to study megakaryopoiesis with among them; MEG-01 (suspension cells) and DAMI (adherent/suspension cells) both megakaryoblastic leukemia cell lines [108, 109]. These cell lines are mostly positive for MK-specific markers (see in Section 2.3.3) but are not a homogenous population. Although they proliferate in a MKBL-like state with some spontaneous differentiation and limited terminal differentiation, they can be induced to differentiate by the addition of phorbol myristate acetate. Under these conditions, the cells can become polyploid, increase their expression of MK-associated proteins, like von Willebrand factor, and are able to produce proplatelets although with low efficiency. Mechanistic insights were uncovered with these lines; for example, the formation of long, beaded cytoplasmic extensions of MKs that yield platelets upon shear stress. This process was also observed in normal healthy MKs *in vivo*, showing the usefulness of this artificial system to study fundamental processes [110]. Despite the usefulness of these lines, their genetic background (patients with genetic abnormalities) can influence megakaryopoiesis, resulting in incomplete differentiation and potential abnormalities that could possibly be linked to their immortalization process. As such, they are suboptimal models to study megakaryopoiesis and specifically MK polyploidization, synthesis of granules, and proplatelet formation.

caused by the downregulation of adhesion and chemokine processes and by the loss of BM macrophages [15, 112–115]. The mobilization can cause side effects for the donor, including headaches, fatigue, vomiting, muscle pain, bone pain, thrombocytopenia, citrate toxicity, etc., in which females experience the most adverse events [116]. These MPBs are ideal for RBC/Plt

The MKs can be cultured in Cellgro (Corning), and both cell types can be cultured in StemSpan (Stem Cell Technologies) or other Iscove's modified Dulbecco's medium (IMDM)-based media. We generated a completely defined GMP-grade medium called Cell-Quin (Migliaccio et al. with minor modifications) that is highly efficient in expanding and differentiating EBLs/RBCs and MKs/Plts, with the ability to culture other hematopoietic progenitors and blood cell types [117].

Several parameters characterize the differentiation stage of erythroid cells. Expansion of EBL cultures is only possible when they maintain cell size control during their cell cycle, which is achieved by the cooperative action of SCF and glucocorticoids [12, 13, 118]. Terminal differentiation in the presence of EPO involves 3–4 cell divisions during which cells' surface marker expression changes and gets smaller due to loss of cell size control until cell cycle arrest and extrusion of their nuclei, concurrently, accumulating Hb [119]. Thus, surface marker expression pattern, cell size and morphology (enucleation), Hb content, and cumulative cell numbers are a measure of differentiation (**Figure 4A–C**). Morphological features of the cells (nuclei-cytoplasm ratio, hemoglobinization, nuclei condensation, and polarization) are commonly assessed by cytospins coupled with Giemsa/benzidine stainings (**Figure 4A**). The purity of the erythroid population and its distribution over different maturation stages can be assessed by monitoring the progression of various cell surface markers. Commonly used markers are CD36, CD71 (transferrin receptor), CD117, and the erythroid-specific markers band 3 (SLC4A1) and CD235 (glycophorin A). The generally accepted dynamics of these markers during erythroid differentiation: pro-EBLs (immature EBL stage) are characterized

/CD71<sup>+</sup>

/CD235<sup>+</sup>

remain positive for CD235 and lose their expression of CD117 followed by the gradual loss of

The first human erythroid culture systems utilized the knowledge obtained from genetics, e.g., discoveries in the field of cytokines, growth factors, and their receptors. In these first protocols, HSPCs were expanded in the presence of IL-3, SCF plus or minus IL-6. It is followed by a step in which the resulting erythroid progenitors were further expanded and differentiated in the presence of EPO [121]. This protocol was modified, using low EPO concentrations in step 1 (0.5 U/ml) and high concentrations (>3 U/ml) in step 2 [122]. Others used high concentrations of EPO throughout step 1 and step 2 [123, 124]. These two-step protocols are based on the original protocol of Fibach and coworkers who employ IMDM supplemented with serum or plasma [121]. Serum and plasma contain factors that support erythropoiesis in these cultures. The major factor in the serum is transforming growth factor β (TGFβ), which is a potent differentiation factor for erythropoiesis [57, 125]. These cultures

/CD71high/CD235low/−, while during expansion phase, EBLs gain

. In terminal differentiation phase, EBLs

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 253

production because of their HSPC richness.

*2.3.2. Erythroid-specific culture system*

by CD34−/CD36<sup>+</sup>

/CD117<sup>+</sup>

CD71, which is associated with reticulocyte formation [120].

CD235 expression and become CD117<sup>+</sup>

### **2.3. Primary cell culture**

### *2.3.1. Cell source and media*

RBCs and MKs can be cultured for research and clinical applications from multiple primary tissue sources including HFL, CB, BM, mobilized peripheral blood (MPB), and peripheral blood mononuclear cells (PBMC). HFL is obtained from abortions on medical indication. HFL-derived erythroid cells express HbF and can be expanded to large numbers and differentiated to hemoglobinized enucleated RBCs. For MK culture, this source is less ideal, mainly because of the harshness of the isolation method. Ethical concerns rule out HFL as a general source for transfusion, but with proper consent allows research into fetal hematopoiesis development. A widely available and ethically accepted source is CB which is commonly used for production of both erythroid cells and MKs. CB is obtained at birth when Hb-switch occurs from HbF to HbA1 (γ to β switch) and both Hb types are expressed in CB-derived cultures. The presence of HSPCs with a fetal hematopoietic program in CB has notable effects in the MK cultures. MKBL expansion is high; MK polyploidization and proplatelet formation are decreased compared to cultures of adult cells. As adult hematopoietic source, either BM or PBMC can be applied. BM is a limited source that can be more difficult to obtain, but does yield large quantities of HSPCs that can differentiate to erythroid and MK lineages. HSPCs can also be isolated from PBMCs, which is less invasive therefore a less limited source. This makes PBMC an ideal source to scale-up RBC production. The HSPC percentage in PBMC is significantly lower compared to BM, which can be enhanced by leukophoresis and by mobilizing BM HSPCs using G-CSF (10 μg/kg) alone or in combination with CXCR4/CXCL12 inhibition [19, 111]. G-CSF alone leads to 5–30% mobilization [15]. HSPC mobilization is caused by the downregulation of adhesion and chemokine processes and by the loss of BM macrophages [15, 112–115]. The mobilization can cause side effects for the donor, including headaches, fatigue, vomiting, muscle pain, bone pain, thrombocytopenia, citrate toxicity, etc., in which females experience the most adverse events [116]. These MPBs are ideal for RBC/Plt production because of their HSPC richness.

The MKs can be cultured in Cellgro (Corning), and both cell types can be cultured in StemSpan (Stem Cell Technologies) or other Iscove's modified Dulbecco's medium (IMDM)-based media. We generated a completely defined GMP-grade medium called Cell-Quin (Migliaccio et al. with minor modifications) that is highly efficient in expanding and differentiating EBLs/RBCs and MKs/Plts, with the ability to culture other hematopoietic progenitors and blood cell types [117].
