**1. Introduction**

Blood cells are by far the most abundant cells of which our body is comprised. Red blood cells (RBCs, or erythrocytes) and platelets (Plts, or thrombocytes) circulate in the vascular system, whereas the white blood cells that form our immune system locate both in the vascular system

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and in the tissues. RBCs are best known for their function as oxygen transporters and for the clearance of CO<sup>2</sup> . Plts exert a crucial function in homeostasis upon vascular damage but they also function during angiogenesis, innate immunity, inflammation, wound healing, cancer, and hemostasis [1, 2]. This chapter focuses on erythropoiesis and megakaryopoiesis. RBCs in the periphery have an average life span of 120 days, constituting approximately 45% of the blood volume. To maintain the population of RBCs, humans generate daily ~2 × 1011 reticulocytes [3]. Plts are shed by megakaryocytes (MK) and live approximately 8–9 days in humans, which require a production of ~8.5 × 10<sup>10</sup> Plts/day [4, 5]. The generation of RBCs and Plts occurs mainly in the bone marrow (BM) in adults, although the lung has also been found to host megakaryocytic progenitors as well as Plts-shedding MKs [6]. A small population of hematopoietic stem cells (HSCs) ensures the life-long generation of blood cells, although the HSCs themselves divide rarely. Mostly, HSCs that divide give rise to one new HSC and a daughter cell that develops to an actively dividing multipotent progenitor (MPP) (**Figure 1**) [7]. These MPPs undergo specification through reciprocal actions of transcription factors (TF) that enhance or repress expression of lineage-specific TFs and direct the cells to a lineage-specific gene expression program [7]. Erythropoiesis and megakaryopoiesis were long thought to arise from a common progenitor, the megakaryocytic-erythroid progenitor, but recent lineage tracing indicates that MKs can also differentiate directly from HSCs [8–10]. Not only MPPs, also erythroid progenitors (erythroblasts:

EBLs) and megakaryocytic progenitors (megakaryoblasts: MKBLs) have extensive potential to undergo cell divisions before they commit to the final differentiation program to generate RBCs/ MKs. The final differentiation stages of both lineages have unique features. Erythroid progenitors undergo 3–4 additional cell divisions with a short G1 cell cycle phase and without regaining the cell volume (i.e., loss of cell size control) [11–13]. MKBLs, instead, undergo 4–5 cell division cycles without cytokinesis, which results in a single cell with 64–128 genome copies (N = 64–128) [14]. Erythropoiesis and megakaryopoiesis also show spatiotemporal regulation. All blood cell progenitors including erythroid progenitors and MKBLs propagate in close contact with stromal cells that produce membrane-bound factors such as stem cell factor (SCF). Upon terminal differentiation, erythroid progenitors bind to central macrophages that express receptors such as CD163, VCAM1, ICAM4, and CD163 to associate with EBLs [15–19]. Each macrophage binds several progenitors that undergo synchronous differentiation, which ends with phagocytosis of the extruded erythroid nucleus by the macrophage and release of reticulocytes into the circulation. The mature MKs have to interact with the endothelial cells of the vasculature and protrude proplatelets into the capillaries, where shear stress contributes to the shedding of Plts [20].

Whereas steady state erythropoiesis and megakaryopoiesis of adult mammals take place in the BM and lung (MK), distinct anatomic sites of hematopoiesis are employed during development (**Figure 2A**). After gastrulation, in humans, mesodermal precursor cells arise in the primitive streak, migrate to the yolk sac, and develop into blood islands (hemangioblasts), which produce primitive RBCs, primitive MKs, and macrophages [21]. During this process, basic fibroblast growth factor (bFGF) influences the proliferation of the hemangioblast and thereby the production of hematopoietic cells [22]. bFGF is synergistic with vascular endothelial growth factor (VEGF) signaling in this process [23]. The primitive RBCs express embryonic type of hemoglobins (Hbs), retain the size of the early EBLs, and lose their nucleus only after prolonged circulation. Their erythropoietin (EPO)-dependence is unclear at this early stage of development [24, 25]. The primitive MKs are thrombopoietin (TPO) independent, have low ploidy compared to adult MKs, and produce fewer Plts, but contrary to primitive erythroid cells, these cells migrate to the fetal liver, where their polyploidization is TPO dependent [26, 27]. Erythroid-myeloid progenitors (EMPs) arise in the yolk sac from hemogenic endothelium (HE) through endothelial to hematopoietic transition (EHT) and give rise to the first intermediate definitive wave, producing RBCs with fetal type of Hbs, MKs, and other myeloid cells [21, 28]. The EMPs migrate and colonize the developing fetal liver where they transiently produce definitive fetal RBCs and MKs. Permanent definitive hematopoiesis in the fetal liver depends on the "birth" of HSC in the aorta main arteries, and more specifically in the aorta-gonad-

are dependent on bone morphogenetic protein 4 (BMP4), VEGF, and bFGF secreted by "feeder cells," which are located near the endothelial cells undergoing EHT, thereby promoting this transition [23, 29]. These HSCs home to the fetal liver to produce definitive fetal blood cells. From the fetal liver, the HSCs migrate to the final site of hematopoiesis; the BM, where they give rise to adult definitive blood cells. Perinatally, hematopoiesis also occurs in the spleen [30]. RBCs and Plts generated at distinct anatomic sites have distinct characteristics; for example, RBCs express different Hb molecules arising from different sites (**Figure 2B**). Hb consists of two α and two β subunits each bound to an iron-containing heme molecule. The α locus expresses ζ and α protein isoforms, the β locus expresses ε, γ (γ1 and 2), and β (β and δ) isoforms. Primitive

HSC arises through EHT. These early HSCs

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 245

mesonephros (AGM) region, where the first CD34<sup>+</sup>

**Figure 1.** HSC commitment to the erythroid/megakaryocytic lineages with lineage-specific marker expression pattern.

EBLs) and megakaryocytic progenitors (megakaryoblasts: MKBLs) have extensive potential to undergo cell divisions before they commit to the final differentiation program to generate RBCs/ MKs. The final differentiation stages of both lineages have unique features. Erythroid progenitors undergo 3–4 additional cell divisions with a short G1 cell cycle phase and without regaining the cell volume (i.e., loss of cell size control) [11–13]. MKBLs, instead, undergo 4–5 cell division cycles without cytokinesis, which results in a single cell with 64–128 genome copies (N = 64–128) [14]. Erythropoiesis and megakaryopoiesis also show spatiotemporal regulation. All blood cell progenitors including erythroid progenitors and MKBLs propagate in close contact with stromal cells that produce membrane-bound factors such as stem cell factor (SCF). Upon terminal differentiation, erythroid progenitors bind to central macrophages that express receptors such as CD163, VCAM1, ICAM4, and CD163 to associate with EBLs [15–19]. Each macrophage binds several progenitors that undergo synchronous differentiation, which ends with phagocytosis of the extruded erythroid nucleus by the macrophage and release of reticulocytes into the circulation. The mature MKs have to interact with the endothelial cells of the vasculature and protrude proplatelets into the capillaries, where shear stress contributes to the shedding of Plts [20].

and in the tissues. RBCs are best known for their function as oxygen transporters and for the

also function during angiogenesis, innate immunity, inflammation, wound healing, cancer, and hemostasis [1, 2]. This chapter focuses on erythropoiesis and megakaryopoiesis. RBCs in the periphery have an average life span of 120 days, constituting approximately 45% of the blood volume. To maintain the population of RBCs, humans generate daily ~2 × 1011 reticulocytes [3]. Plts are shed by megakaryocytes (MK) and live approximately 8–9 days in humans, which require a production of ~8.5 × 10<sup>10</sup> Plts/day [4, 5]. The generation of RBCs and Plts occurs mainly in the bone marrow (BM) in adults, although the lung has also been found to host megakaryocytic progenitors as well as Plts-shedding MKs [6]. A small population of hematopoietic stem cells (HSCs) ensures the life-long generation of blood cells, although the HSCs themselves divide rarely. Mostly, HSCs that divide give rise to one new HSC and a daughter cell that develops to an actively dividing multipotent progenitor (MPP) (**Figure 1**) [7]. These MPPs undergo specification through reciprocal actions of transcription factors (TF) that enhance or repress expression of lineage-specific TFs and direct the cells to a lineage-specific gene expression program [7]. Erythropoiesis and megakaryopoiesis were long thought to arise from a common progenitor, the megakaryocytic-erythroid progenitor, but recent lineage tracing indicates that MKs can also differentiate directly from HSCs [8–10]. Not only MPPs, also erythroid progenitors (erythroblasts:

**Figure 1.** HSC commitment to the erythroid/megakaryocytic lineages with lineage-specific marker expression pattern.

. Plts exert a crucial function in homeostasis upon vascular damage but they

clearance of CO<sup>2</sup>

244 Cell Culture

Whereas steady state erythropoiesis and megakaryopoiesis of adult mammals take place in the BM and lung (MK), distinct anatomic sites of hematopoiesis are employed during development (**Figure 2A**). After gastrulation, in humans, mesodermal precursor cells arise in the primitive streak, migrate to the yolk sac, and develop into blood islands (hemangioblasts), which produce primitive RBCs, primitive MKs, and macrophages [21]. During this process, basic fibroblast growth factor (bFGF) influences the proliferation of the hemangioblast and thereby the production of hematopoietic cells [22]. bFGF is synergistic with vascular endothelial growth factor (VEGF) signaling in this process [23]. The primitive RBCs express embryonic type of hemoglobins (Hbs), retain the size of the early EBLs, and lose their nucleus only after prolonged circulation. Their erythropoietin (EPO)-dependence is unclear at this early stage of development [24, 25]. The primitive MKs are thrombopoietin (TPO) independent, have low ploidy compared to adult MKs, and produce fewer Plts, but contrary to primitive erythroid cells, these cells migrate to the fetal liver, where their polyploidization is TPO dependent [26, 27]. Erythroid-myeloid progenitors (EMPs) arise in the yolk sac from hemogenic endothelium (HE) through endothelial to hematopoietic transition (EHT) and give rise to the first intermediate definitive wave, producing RBCs with fetal type of Hbs, MKs, and other myeloid cells [21, 28]. The EMPs migrate and colonize the developing fetal liver where they transiently produce definitive fetal RBCs and MKs. Permanent definitive hematopoiesis in the fetal liver depends on the "birth" of HSC in the aorta main arteries, and more specifically in the aorta-gonadmesonephros (AGM) region, where the first CD34<sup>+</sup> HSC arises through EHT. These early HSCs are dependent on bone morphogenetic protein 4 (BMP4), VEGF, and bFGF secreted by "feeder cells," which are located near the endothelial cells undergoing EHT, thereby promoting this transition [23, 29]. These HSCs home to the fetal liver to produce definitive fetal blood cells. From the fetal liver, the HSCs migrate to the final site of hematopoiesis; the BM, where they give rise to adult definitive blood cells. Perinatally, hematopoiesis also occurs in the spleen [30]. RBCs and Plts generated at distinct anatomic sites have distinct characteristics; for example, RBCs express different Hb molecules arising from different sites (**Figure 2B**). Hb consists of two α and two β subunits each bound to an iron-containing heme molecule. The α locus expresses ζ and α protein isoforms, the β locus expresses ε, γ (γ1 and 2), and β (β and δ) isoforms. Primitive

**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).

RBCs express Hbe consisting of ζ and ε isoforms (Portland 1: ζ<sup>2</sup> γ2 ; Portland 2: ζ<sup>2</sup> β2 ; Gower 1: ζ<sup>2</sup> ε2 ; Gower 2: α<sup>2</sup> ε2 ); 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 stages; however, in the megakaryocytic lineages, there is a lack of such markers.

The aim of this chapter is twofold. First, we provide background information of the basic processes of erythropoiesis and megakaryopoiesis that underly the various cell culture models. Second, we provide details, interpret and compare results on current protocols to expand and

**Figure 3.** Application of *in vitro*-derived blood products from different primary sources in basic research, drug discovery,

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 247

*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

differentiate erythroid and megakaryocytic progenitors.

**2. Erythropoiesis/megakaryopoiesis**

**2.1. Growth factors and major regulators**

*2.1.1. Lineage- and stage-specific cytokines*

and transfusion.

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

**Figure 3.** Application of *in vitro*-derived blood products from different primary sources in basic research, drug discovery, and transfusion.

The aim of this chapter is twofold. First, we provide background information of the basic processes of erythropoiesis and megakaryopoiesis that underly the various cell culture models. Second, we provide details, interpret and compare results on current protocols to expand and differentiate erythroid and megakaryocytic progenitors.
