*2.3.3. MK-specific culture system*

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

reticulocytes (purple). (D) MK-specific morphology by cytospin with MGG-staining. Left: MKBL; right: polyploid MK

**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

small cells: nuclei (blue); DRAQ5− cells: enucleated

big cells: nucleated EBLs (red); DRAQ5<sup>+</sup>

(arrows). (E) Proplatelet-forming MK (arrows (beads on a string)).

populations: DRAQ5<sup>+</sup>

254 Cell Culture

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 characterized by CD34<sup>+</sup> /CD41a<sup>+</sup> expression and blast-like morphology. In terminal differentiation, MKs gradually lose their expression of CD34<sup>+</sup> , leading to a subdivision of stages: early MKs are CD34<sup>+</sup> /CD41a<sup>+</sup> /CD42<sup>+</sup> and late MKs are CD34−/CD41a<sup>+</sup> /CD42<sup>+</sup> .

To obtain large numbers of MKBLs, SCF/FL and TPO are used during the first 4–7 days of cultures started from CD34<sup>+</sup> -HSPCs. TPO without SCF and FL allows terminal differentiation 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> differentiation, 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 proplatelet production. To prevent activation, signaling via the GPIIb/IIIa (ITGA2B) receptor can be blocked with tirofiban hydrochloride monohydrate. Whereas an MK sheds thousands of Plts *in vivo*, shedding large amounts *in vitro* from a single MK has not yet been achieved. *In vivo* of this process requires that proplatelets extrude between the endothelial cells of the blood vessel wall into the capillaries. This increases level of SP1 among others, combined with shear stress of the blood flow is required for the Plts to be released. To mimic this *in vitro*, several specialized bioreactors are being tested (see Section 2.5).

VEGF that drive the cells toward mesoderm, followed by the addition of IL-3, IL-6, SCF, and TPO stimulating hematopoietic specification. Lineage-committed progenitors can be further differentiated toward mature cell types, which is achieved by the combination of medium, growth factors, and hormones. Although most of the differentiation protocols are following the abovementioned scheme, including the listed growth factors, there are multiple technical variations during iPSCs differentiation toward hematopoietic lineages. Two main technical details underlie the major differences in the applied protocols: (i) the induction of differentiation as a 2D monolayer versus 3D embryoid body formation and (ii) the use of coculture with feeder versus feeder-free systems [141–144]. The choice of the differentiation system depends on the application need. 3D systems more closely resemble the *in vivo* process in comparison to 2D systems, offering a tool to study embryogenesis [141, 144–147]. 2D systems, however, are relatively simple, more reproducible, and therefore suitable to scale up production, enabling clinical application [77, 148–150]. The choice of feeder-based or feederfree differentiation similar to 2D/3D systems also depends on the purpose of the specific research question. Feeder-based coculture systems more resemble the niche, including secretome and cell-cell contact; however, for future clinical application, feeder-free systems are imperative [142, 143, 151, 152]. Protocols can also differ in timing, in the applied media and cytokine cocktails used, which makes comparisons between research groups and methods difficult. Stemline II (SIGMA) is a widely applied base medium during the first and, in some cases, the second phase in feeder-free settings [138, 149, 153]. From the second/third phase onward, the same basic medium are applied that are generally used for other definitive blood cell types such as IMDM with serum/holotransferrin/lipid/Ins supplementation or StemSpan (Stem Cell Technologies) [137, 138, 150, 153]. From the second-step onward, we apply Cell-Quin which in comparison to StemSpan was more efficient in their iPSC-erythroid expansion potential (**Figure 6A**). Some methods still include BMP4, VEGF, and bFGF (or either of them) at this second stage, typically in embryoid body-based system because the 3D structures are less homogenous. Therefore, the transition between phases is not entirely uniform and clear [140, 149, 154, 155]. The few feeder-free 2D systems that have been published mostly do not rely on these three additional factors during commitment phase [148, 149]. FL is also used in erythroid/MK commitment cytokine mixes to improve progenitor expansion [140, 154]. MK commitment/expansion is always based on TPO with or without the addition of multiple

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 257

other cytokines (e.g., IL-1β, IL-9, IL-11) [99, 136, 156–158].

As pointed out before, early erythropoiesis/megakaryopoiesis (yolk sac) in humans is not well studied, resulting in a lack of knowledge on the regulatory program at these developmental stages. Therefore, the generally applied cytokines might not ideally mimick the *in vivo* situation or the iPSC-driven hematopoietic program. This could underlie inefficient iPSC to RBC/MK differentiation. For example, EPO is applied in all systems to induce erythropoiesis; however, the role of EPO during primitive wave is not entirely clear. Disruption of *EPO* and/ or *EPOR* causes embryonic lethality in mice due to the failure of the definitive fetal liver erythropoiesis with reduced primitive erythropoiesis, suggesting that *EPO* and *EPOR* are already functional in early yolk sac [24, 25, 39–41]. However, others showed that additional *EPO* did not affect heme synthesis in early mouse embryos [159]. Furthermore, Malik et al. found that *EPOR*-null embryos have normal number of primitive, early stage progenitors but subsequently develop anemia with loss of primitive EBL [24]. In line with these findings, the

#### **2.4. Erythropoiesis/megakaryopoiesis from iPSC**

Pluripotent stem cells offer a novel approach for developmental studies, drug screening/ discovery, disease modeling, and regenerative medicine. ESCs originate from the inner cell mass of a blastocyst stage embryo, while iPSCs are somatic cells that are reprogrammed back to this embryonic stage [129–132]. Hematopoietic differentiation of ESC/iPSC cells follows the various stages of blood development from early embryonic stages (**Figure 2A**). This offers a valuable tool to study early human hematopoiesis which is difficult because of ethical issues and tissue availability. Besides, differentiation of iPSCs opens opportunities for large-scale manufacture of blood products with the expectancy of clinical application [133]. Several groups showed the potential of ESCs in blood cell production, the source which was later replaced by iPSCs with similar outcome including our group (**Figure 5**) [134–140]. The published protocols generally include four culture phases: (1) mesoderm induction, (2) hematopoietic/erythroid/megakaryocytic commitment, (3) expansion of the specific cell pool, and (4) terminal maturation. The hematopoietic differentiation phases *in vitro* are directed by stepwise addition of cytokines. This is commonly achieved by BMP4, bFGF, and

**Figure 5.** Erythroid/MK differentiation of iPSC according to Hansen et al. showing the different phases of differentiation, with their corresponding growth factor combination and morphological changes [136].

VEGF that drive the cells toward mesoderm, followed by the addition of IL-3, IL-6, SCF, and TPO stimulating hematopoietic specification. Lineage-committed progenitors can be further differentiated toward mature cell types, which is achieved by the combination of medium, growth factors, and hormones. Although most of the differentiation protocols are following the abovementioned scheme, including the listed growth factors, there are multiple technical variations during iPSCs differentiation toward hematopoietic lineages. Two main technical details underlie the major differences in the applied protocols: (i) the induction of differentiation as a 2D monolayer versus 3D embryoid body formation and (ii) the use of coculture with feeder versus feeder-free systems [141–144]. The choice of the differentiation system depends on the application need. 3D systems more closely resemble the *in vivo* process in comparison to 2D systems, offering a tool to study embryogenesis [141, 144–147]. 2D systems, however, are relatively simple, more reproducible, and therefore suitable to scale up production, enabling clinical application [77, 148–150]. The choice of feeder-based or feederfree differentiation similar to 2D/3D systems also depends on the purpose of the specific research question. Feeder-based coculture systems more resemble the niche, including secretome and cell-cell contact; however, for future clinical application, feeder-free systems are imperative [142, 143, 151, 152]. Protocols can also differ in timing, in the applied media and cytokine cocktails used, which makes comparisons between research groups and methods difficult. Stemline II (SIGMA) is a widely applied base medium during the first and, in some cases, the second phase in feeder-free settings [138, 149, 153]. From the second/third phase onward, the same basic medium are applied that are generally used for other definitive blood cell types such as IMDM with serum/holotransferrin/lipid/Ins supplementation or StemSpan (Stem Cell Technologies) [137, 138, 150, 153]. From the second-step onward, we apply Cell-Quin which in comparison to StemSpan was more efficient in their iPSC-erythroid expansion potential (**Figure 6A**). Some methods still include BMP4, VEGF, and bFGF (or either of them) at this second stage, typically in embryoid body-based system because the 3D structures are less homogenous. Therefore, the transition between phases is not entirely uniform and clear [140, 149, 154, 155]. The few feeder-free 2D systems that have been published mostly do not rely on these three additional factors during commitment phase [148, 149]. FL is also used in erythroid/MK commitment cytokine mixes to improve progenitor expansion [140, 154]. MK commitment/expansion is always based on TPO with or without the addition of multiple other cytokines (e.g., IL-1β, IL-9, IL-11) [99, 136, 156–158].

As pointed out before, early erythropoiesis/megakaryopoiesis (yolk sac) in humans is not well studied, resulting in a lack of knowledge on the regulatory program at these developmental stages. Therefore, the generally applied cytokines might not ideally mimick the *in vivo* situation or the iPSC-driven hematopoietic program. This could underlie inefficient iPSC to RBC/MK differentiation. For example, EPO is applied in all systems to induce erythropoiesis; however, the role of EPO during primitive wave is not entirely clear. Disruption of *EPO* and/ or *EPOR* causes embryonic lethality in mice due to the failure of the definitive fetal liver erythropoiesis with reduced primitive erythropoiesis, suggesting that *EPO* and *EPOR* are already functional in early yolk sac [24, 25, 39–41]. However, others showed that additional *EPO* did not affect heme synthesis in early mouse embryos [159]. Furthermore, Malik et al. found that *EPOR*-null embryos have normal number of primitive, early stage progenitors but subsequently develop anemia with loss of primitive EBL [24]. In line with these findings, the

**Figure 5.** Erythroid/MK differentiation of iPSC according to Hansen et al. showing the different phases of differentiation,

proplatelet production. To prevent activation, signaling via the GPIIb/IIIa (ITGA2B) receptor can be blocked with tirofiban hydrochloride monohydrate. Whereas an MK sheds thousands of Plts *in vivo*, shedding large amounts *in vitro* from a single MK has not yet been achieved. *In vivo* of this process requires that proplatelets extrude between the endothelial cells of the blood vessel wall into the capillaries. This increases level of SP1 among others, combined with shear stress of the blood flow is required for the Plts to be released. To mimic this *in vitro*,

Pluripotent stem cells offer a novel approach for developmental studies, drug screening/ discovery, disease modeling, and regenerative medicine. ESCs originate from the inner cell mass of a blastocyst stage embryo, while iPSCs are somatic cells that are reprogrammed back to this embryonic stage [129–132]. Hematopoietic differentiation of ESC/iPSC cells follows the various stages of blood development from early embryonic stages (**Figure 2A**). This offers a valuable tool to study early human hematopoiesis which is difficult because of ethical issues and tissue availability. Besides, differentiation of iPSCs opens opportunities for large-scale manufacture of blood products with the expectancy of clinical application [133]. Several groups showed the potential of ESCs in blood cell production, the source which was later replaced by iPSCs with similar outcome including our group (**Figure 5**) [134–140]. The published protocols generally include four culture phases: (1) mesoderm induction, (2) hematopoietic/erythroid/megakaryocytic commitment, (3) expansion of the specific cell pool, and (4) terminal maturation. The hematopoietic differentiation phases *in vitro* are directed by stepwise addition of cytokines. This is commonly achieved by BMP4, bFGF, and

several specialized bioreactors are being tested (see Section 2.5).

**2.4. Erythropoiesis/megakaryopoiesis from iPSC**

256 Cell Culture

with their corresponding growth factor combination and morphological changes [136].

more mature erythroid cell production from iPSCs if certain TFs are included; however, the *in vitro* feasibility is not provided presently. To direct the cells more lineage-specific, other lineage instructive TFs may be used. GATA1, FLI-1, and TAL1 (MK-specific TFs) can be overexpressed to direct iPSC to MKs, thereby achieving ~100% MK yield within 15 days [158]. Besides this set of genes, there are other combinations that are used to achieve a similar aim

The technical differences between published differentiation methods are leading to slight discrepancies in marker expression pattern, purity, yield, and stage of development. However, currently all published methods are limited by technical pitfalls, including the production of developmentally immature (nonadult) cell types which may be the cause of low yield and difficulty to terminally differentiate toward functional end stage blood cell types (e.g., low enucleation potential of iPSC-erythroid cells and low efficiency of iPSC-Plt

The purity of the iPSC-derived erythroid population, and its distribution over different maturation stages can be assessed by the erythroid-specific markers used for definitive erythroid culture systems (Section 2.3.2); however, their progression differs in some aspects. Based on our differentiation scheme (**Figure 5**), we recognize three maturation stages: (i) an early erythroid population (harvest at day 10–14) is CD71high/CD235high/CD36med/high, which is not yet hemoglobinized and displays big nuclei [136]. Furthermore, the cells are negative for CD18 (myeloid lineage marker) confirming specification toward the erythroid lineage; (ii) a 100% pure erythroid population (day 7–9 expansion) is CD71/CD235/CD36med with some spontaneous differentiation, which is recognized by hemoglobinization and condensation of the nuclei; (iii) a mature erythroid population (D7-14 terminal differentiation) gives rise to CD235high/ CD71high/med/CD36low cells. However, there is a slight CD71 decrease associated with reticulocyte formation, and iPSC-derived erythroid cells do not become CD71 negative. Morphologically, these cells were somewhat different from their definitive counterparts. Despite hemoglobinization, nuclear condensation, and polarization, we do not observe a decrease of cytoplasm size and the enucleation potential is poor. Technical variations in the published methods (timing, added growth factors) cause notable differences in the erythroid marker expression pattern; therefore, it is hard to compare and/or draw general conclusions. The emergence of CD71<sup>+</sup>

population is generally reported with purity discrepancies. For example, Yang et al.

Salvagiotto et al. [148] by a feeder-free monolayer system reached 40% pure population, while Kobari et al. [135] with EB-based induction reached 98–99% comparable to our findings. The pattern of CD36 expression is not entirely clear. Mao et al., for example, used a four-step differentiation scheme, including an AGM coculture induction step, and defined the following

sequence [164]. Others including us found high CD36 expression during the early erythroid

/CD43<sup>+</sup>

/CD34−/CD36−/low, CD235<sup>+</sup>

/

preselection and OP9 coculture),

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 259

/CD34low/CD36−, and they

/CD34−/CD36− cells in

[156, 162].

formation).

CD235<sup>+</sup>

develop to CD235<sup>+</sup>

stage [136].

*2.4.1. Marker expression pattern of differentiating iPSC cells*

[163] reported 80% CD71/CD235 purity (with CD34<sup>+</sup>

gene expression profile: early definitive EBLs derived from CD235<sup>+</sup>

/CD34−/CD36−, CD235<sup>+</sup>

**Figure 6.** Optimization of iPSC-erythroid differentiation cultures. (A) iPSC-derived erythroid cells arose (D12+0) and expanded in Cell-Quin or StemSpan media. (B) Representative FACS-plots of iPSC-derived erythroid cell (D12 harvest), with or without EPO and SCF from day 6 onward.

same group concluded that EPO signaling is not critical for the survival of human primitive erythroid progenitors, but have a less understood role to promote proliferation and maturation of these cells. Since the role of EPO is controversial in yolk sac erythropoiesis, we tested whether iPSC-erythroid commitment is EPO-dependent. We have tested the requirement of the two most important growth factors for definitive erythropoiesis in various combinations; EPO and SCF (Control), without EPO (-EPO), without SCF (-SCF), and without both cytokine (-EPO and -SCF) [136]. Without EPO, we noticed lower harvest rate/colony number and the loss of CD71/CD235 population. The erythroid commitment was not affected by the deprivation of SCF; however, the addition of SCF together with EPO resulted in a more pure CD71/CD235 population (**Figure 6B**). These data suggest that EPO is required to allow early erythroid commitment while the role of SCF is not entirely clear.

Introduction of erythroid/MK-specific TFs into iPSC-derived hematopoietic cells, often named "forward reprogramming", is being pursued as an approach to improve differentiation outcome. HOXA9, ERG, RORA, SOX4, and MYB have been introduced into human pluripotent stem cells. Engraftment into NSG mice resulted in erythroid cells, which were more skewed to definitive erythropoiesis (lack of embryonic Hbs, mainly HbF and some HbA, some enucleation) compared to TF-free counterparts [160, 161]. These results suggest the possibility of more mature erythroid cell production from iPSCs if certain TFs are included; however, the *in vitro* feasibility is not provided presently. To direct the cells more lineage-specific, other lineage instructive TFs may be used. GATA1, FLI-1, and TAL1 (MK-specific TFs) can be overexpressed to direct iPSC to MKs, thereby achieving ~100% MK yield within 15 days [158]. Besides this set of genes, there are other combinations that are used to achieve a similar aim [156, 162].

The technical differences between published differentiation methods are leading to slight discrepancies in marker expression pattern, purity, yield, and stage of development. However, currently all published methods are limited by technical pitfalls, including the production of developmentally immature (nonadult) cell types which may be the cause of low yield and difficulty to terminally differentiate toward functional end stage blood cell types (e.g., low enucleation potential of iPSC-erythroid cells and low efficiency of iPSC-Plt formation).
