*2.3.2. Erythroid-specific culture system*

*2.2.2. MK-cell lines*

252 Cell Culture

proplatelet formation.

**2.3. Primary cell culture**

*2.3.1. Cell source and media*

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

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 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 by CD34−/CD36<sup>+</sup> /CD117<sup>+</sup> /CD71high/CD235low/−, while during expansion phase, EBLs gain CD235 expression and become CD117<sup>+</sup> /CD71<sup>+</sup> /CD235<sup>+</sup> . In terminal differentiation phase, EBLs remain positive for CD235 and lose their expression of CD117 followed by the gradual loss of CD71, which is associated with reticulocyte formation [120].

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

expansion [73, 119]. Using Cell-Quin, we can obtain 2 × 10<sup>10</sup> EBLs within 16 days, starting from

Commitment of MKBL and differentiation of MKs can be monitored by the expression of cell surface markers and by the morphological features of the cells (**Figure 4D** and **E**). MKBLs are

and late MKs are CD34−/CD41a<sup>+</sup>

To obtain large numbers of MKBLs, SCF/FL and TPO are used during the first 4–7 days of

tion to proplatelet-forming cells. To increase the expansion potential, IL-3 can be included only in the initial phase as its prolonged exposure directs the HSPC toward the monocyte/ granulocyte lineage. With the addition of IL-6, the MK specification and TPO signaling can be enhanced. With the addition of either IL-1β, IL-9, or IL-11 during the first phase of CD34<sup>+</sup>

ferentiation, MK commitment is enhanced instead of progenitor proliferation. It is important to determine the main goal of an experiment before starting the culture: does the experiment require large numbers of MKBLs, or should MK enrichment be maximal, because a good expansion of MKBL tends to compromise terminal differentiation and vice versa (Hansen and van den Akker, unpublished results). Factors such as IL-1β and IL-9 increase polyploidization, formation of proplatelets, and Plts shedding. There is some concern about using IL-1β, because of its proinflammatory nature. Particularly, as it is closely related to IL-1α, and the increased Plt shedding may cause rupture of MKs [33, 99]. To introduce proplatelet formation, IL-6 can be used in high concentrations (>100 ng/ml), by itself or in combination with TPO. SR-1 influences megakaryopoiesis on an early and late stage of the culture, as described above, having a positive effect on the expansion of HSC and terminal differentiation of MK [80, 81]. During the terminal stages of MK cultures (during proplatelet formation), it becomes increasingly essential to prevent the activation of the MKs and produced proplatelets. The addition of heparin prevents the coagulation of plasma added to the media but cells are still able to clump together, thereby having a negative impact on the differentiation and

expression and blast-like morphology. In terminal differen-


/CD42<sup>+</sup> .

, leading to a subdivision of stages: early

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 255

dif-

 PBMCs (**Figure 4B**). Expansion of EBLs in the presence of serum and in the absence of glucocorticoids irrevocably results in differentiation and transfer of the cells to differentiation conditions. Of note, addition of glucocorticoid agonists in a serum-based culture will still induce spontaneous differentiation due to the presence of TGFβ [57]. At any moment during expansion phase, cells can be transferred to differentiation conditions in which the medium is supplemented with EPO, Ins, and low level of plasma/serum [119, 120]. Although expansion of EBL cultures is achieved in serum-free medium, terminal differentiation to enucleated cells requires at least 2% serum or plasma [119, 120]. Using Cell-Quin medium, we currently obtain >90% enucleation, a deformability that corresponds to values between freshly isolated reticulocytes and erythrocytes, and normal oxygen association and dissociation values (van den Akker and von Lindern, manuscript in preparation). We use DRAQ5 staining coupled with flow cytometry analyses to quantify reticulocyte/nuclei/nucleated cell ratio (**Figure 4C**). Flexibility is measured on a ARCA, and oxygen binding by the Hemox analyzer [127, 128].

5 × 10<sup>7</sup>

*2.3.3. MK-specific culture system*

/CD41a<sup>+</sup>

/CD41a<sup>+</sup>

tiation, MKs gradually lose their expression of CD34<sup>+</sup>

/CD42<sup>+</sup>

characterized by CD34<sup>+</sup>

cultures started from CD34<sup>+</sup>

MKs are CD34<sup>+</sup>

**Figure 4.** Characteristics of erythroid and megakaryocytic cultures. (A) Erythroid-specific morphology by cytospin with Giemsa/benzidine staining. Left: pro-EBL, right reticulocytes. (B) Erythroid expansion growth curve from PBMCs (n = 4). (C) Flow cytometry of terminal erythroid differentiation, DNA staining by DRAQ5 resulting in three distinct populations: DRAQ5<sup>+</sup> big cells: nucleated EBLs (red); DRAQ5<sup>+</sup> small cells: nuclei (blue); DRAQ5− cells: enucleated reticulocytes (purple). (D) MK-specific morphology by cytospin with MGG-staining. Left: MKBL; right: polyploid MK (arrows). (E) Proplatelet-forming MK (arrows (beads on a string)).

show a high degree of spontaneous differentiation, which is often used to study expansion and differentiation of EBLs carrying a genetic defect. The quality differences between serum batches and the use of different cytokines make these culture protocols difficult to compare. Two major changes increase the yield of these erythroid cultures and enable synchronous differentiation. First, glucocorticoids cooperate with SCF to retain pro-EBLs and early basophilic EBLs in their undifferentiated state [57, 126]. Second, serum-free medium avoids the differentiation promoting effect of TGFβ. However, the available serum-free media are suboptimal and require complementation with lipids [73]. Even better expansion is achieved with a serum-free medium optimized for expansion of EBLs [117]. The differentiation arrest in the presence of glucocorticoids and the absence of serum enables the expansion of a homogeneous early EBLs culture that can undergo up to 20 cell divisions to achieve a million-fold expansion [73, 119]. Using Cell-Quin, we can obtain 2 × 10<sup>10</sup> EBLs within 16 days, starting from 5 × 10<sup>7</sup> PBMCs (**Figure 4B**). Expansion of EBLs in the presence of serum and in the absence of glucocorticoids irrevocably results in differentiation and transfer of the cells to differentiation conditions. Of note, addition of glucocorticoid agonists in a serum-based culture will still induce spontaneous differentiation due to the presence of TGFβ [57]. At any moment during expansion phase, cells can be transferred to differentiation conditions in which the medium is supplemented with EPO, Ins, and low level of plasma/serum [119, 120]. Although expansion of EBL cultures is achieved in serum-free medium, terminal differentiation to enucleated cells requires at least 2% serum or plasma [119, 120]. Using Cell-Quin medium, we currently obtain >90% enucleation, a deformability that corresponds to values between freshly isolated reticulocytes and erythrocytes, and normal oxygen association and dissociation values (van den Akker and von Lindern, manuscript in preparation). We use DRAQ5 staining coupled with flow cytometry analyses to quantify reticulocyte/nuclei/nucleated cell ratio (**Figure 4C**). Flexibility is measured on a ARCA, and oxygen binding by the Hemox analyzer [127, 128].
