*2.4.2. Developmental stage of iPSC-derived erythroid/MK cells*

Human ESC/iPSC-derived erythropoiesis/megakaryopoiesis, with the current knowledge, do not reach the adult definitive stage, but give rise to a mixture of primitive and definitive fetal/ adult cells. Very little is known about human erythropoiesis/megakaryopoiesis in the early stages, between days 17 and 23 of embryogenesis (yolk-sac, AGM region) due to the fact that abortions are primarily performed at later fetal stages and in addition have serious ethical concerns. Hbs are commonly used to distinguish between developmental waves; however, HbF expressing RBCs both arises from yolk sac and later from fetal liver, and momentarily, there is a lack of markers, which can clearly distinguish these two waves (**Figure 2A** and **B**). The iPSCderived erythroid cells predominantly express HbF, in addition to embryonic types of Hb. We and others also showed the presence of a small portion of adult Hb [135, 136, 153–155]. From the embryonic type of globins, both the presence of Gower 1 and Gower 2 Hb has been reported, but the ratio greatly differs between methods [135]. The presence of Gower 2 Hb indicates that the cells are capable of the first globin switch (ζ to α) to provide more mature primitive-state RBCs. Interestingly, the presence of adult types of Hb also differs between protocols as some group were able to show HbA1 or the presence of β chain, whereas others, including us, observed mainly HbA2 [135, 155]. It is unknown whether primitive erythropoiesis *in vivo* goes through a stage that corresponds to iPSC-derived erythroid cells (Hbemid, HbFhigh, HbAlow/−) or whether globin synthesis is impaired in these cells. The globins, however, may not be the most accurate markers to define the waves. Altogether, these findings make it difficult to define the state of iPSC-RBCs with respect to their developmental stage. From a technical point of view, these cells are able to produce definitive RBCs; therefore, they potentially can give rise to the required therapeutic product. Better understanding of the underlying regulatory mechanisms such as the site where erythropoiesis/megakaryopoiesis takes place, and Hb switches during development might pave the way for the necessary improvement of the current methods.

Unlike in the erythroid lineages where the expression of stage-specific Hbs can be used to determine the ontogeny phase, this type of readout is not available in the MK lineage. There are, however, intrinsic differences between megakaryocytic cells derived at different sites during ontogeny. For instance, polyploidy is a measure of ontogeny *in vivo* as yolk sack MK exhibits low ploidy (4–8N), whereas adult MK reach >64N [14, 169, 170]. Besides the lower polyploidization, the number of Plts that are produced follows the same trend from a low number per MK in embryonic/fetal tissues too high numbers of in adult MK, linking polyploidization and MK size to Plts production [170]. However, ploidy levels and Plt production *in vitro* are generally lower compared to their *in vivo* counterparts; therefore, it is not a good marker to access iPSC-MK development stages. The current best approach would be to use data from erythroid cultures and their ontogeny stage/wave and extrapolated this to the MK development because of their close relationship. However, investigation of purified megakaryocytic cultures of defined ontogeny stages through, e.g., RNAseq or mass spectrometry could yield specific makers.

#### *2.4.3. Expansion potential/yield of iPSC-erythroid/MK cells*

The kinetics during differentiation/maturation of MK from iPSC follow the same steps as from

proplatelets but also in low numbers [157, 165–168]. These Plts can be activated and contribute to clot formation and wound healing when transfused to injured mice, showing that iPSC megakaryopoiesis, although still inefficient, leads to functional Plts. The MK-specific cell surface markers can be also used for iPSC-MKs with the exception of CD41a. This marker is also an early endothelial/hematopoietic marker. Therefore, it is essential to always use it in a combination, for example, with CD42 to confirm the specific MK commitment. Despite this, most groups report MK percentage only based on CD41a expression (~30–80% MK induction) [136, 150, 168]. With our method, we are able to achieve an average purity of 78% (CD41a) that

Human ESC/iPSC-derived erythropoiesis/megakaryopoiesis, with the current knowledge, do not reach the adult definitive stage, but give rise to a mixture of primitive and definitive fetal/ adult cells. Very little is known about human erythropoiesis/megakaryopoiesis in the early stages, between days 17 and 23 of embryogenesis (yolk-sac, AGM region) due to the fact that abortions are primarily performed at later fetal stages and in addition have serious ethical concerns. Hbs are commonly used to distinguish between developmental waves; however, HbF expressing RBCs both arises from yolk sac and later from fetal liver, and momentarily, there is a lack of markers, which can clearly distinguish these two waves (**Figure 2A** and **B**). The iPSCderived erythroid cells predominantly express HbF, in addition to embryonic types of Hb. We and others also showed the presence of a small portion of adult Hb [135, 136, 153–155]. From the embryonic type of globins, both the presence of Gower 1 and Gower 2 Hb has been reported, but the ratio greatly differs between methods [135]. The presence of Gower 2 Hb indicates that the cells are capable of the first globin switch (ζ to α) to provide more mature primitive-state RBCs. Interestingly, the presence of adult types of Hb also differs between protocols as some group were able to show HbA1 or the presence of β chain, whereas others, including us, observed mainly HbA2 [135, 155]. It is unknown whether primitive erythropoiesis *in vivo* goes through a stage that corresponds to iPSC-derived erythroid cells (Hbemid, HbFhigh, HbAlow/−) or whether globin synthesis is impaired in these cells. The globins, however, may not be the most accurate markers to define the waves. Altogether, these findings make it difficult to define the state of iPSC-RBCs with respect to their developmental stage. From a technical point of view, these cells are able to produce definitive RBCs; therefore, they potentially can give rise to the required therapeutic product. Better understanding of the underlying regulatory mechanisms such as the site where erythropoiesis/megakaryopoiesis takes place, and Hb switches during development might pave the way for the necessary improvement of

Unlike in the erythroid lineages where the expression of stage-specific Hbs can be used to determine the ontogeny phase, this type of readout is not available in the MK lineage. There are, however, intrinsic differences between megakaryocytic cells derived at different sites

/CD41a<sup>+</sup>

), early MK (CD34<sup>+</sup>

). The MKs can undergo some polyploidization albeit not in

cultures. MKs derived from mouse iPSC can form

/CD41a<sup>+</sup>

/CD42<sup>+</sup>

), and

cells, namely MKBL (CD34<sup>+</sup>

/CD42<sup>+</sup>

similar level as *in vivo* or primary CD34<sup>+</sup>

can be easily used in scaleup production [136].

*2.4.2. Developmental stage of iPSC-derived erythroid/MK cells*

definitive CD34<sup>+</sup>

260 Cell Culture

late MK (CD34−/CD41a<sup>+</sup>

the current methods.

The final yield of our method is relatively high; however, the comparison with other methods is difficult due to technical discrepancies. There are different ways to calculate the final yield, which also depends on the iPSC maintenance system (single cell vs. clumps) and on the induction system (2D or 3D), resulting various ways to report the final yield. Single cell-seeded iPSC cultures can be normalized both to the initial number of seeded cells or to colony number. Furthermore, the comparison of absolute cell number produced (harvested) between 2D and 3D systems is not entirely realistic because of the different nature of the two cultures. We use single cell-seeding, which allows to calculate the yield/iPSC and yield/colony number to represent differentiation efficiency. In our hands, one iPSC colony can give rise on average 5.6 × 106 erythroid cells after 9 days of expansion. While a single iPSC gives rise to ~8 × 10<sup>3</sup> erythroid cells (harvest) and subsequent 9 days expansion results in ~2 × 105 erythroid cells/iPSC on average [136]. The expansion potential (from harvest day) compared to definitive cell types remains relatively low, in line with other methods irrespective of culture condition (**Figure 4B**). The terminal maturation of iPSC-erythroid cells toward enucleating reticulocytes is inefficient with the existing methods and is currently one of the major hurdles to overcome. In our hands, matured iPSC-erythroid cultures had 30–40% enucleation rate based on their nuclei count; however, the resulting reticulocytes appeared to be instable. Altogether, iPSC-derived erythroid cells are able to expand but for limited time and length, with suboptimal enucleation capacity. Probably, this is also coming from the fact that iPSC-derived erythroid cells, based on their globin expression, do not entirely correspond to a fetal/adult definitive wave.

MK yield from iPSCs is higher than CD34<sup>+</sup> differentiation (6.9 × 10<sup>3</sup> cells/iPSC). Unlike the erythroid system in which differentiation can be inhibited for several days by the addition of glucocorticoid analogues, the megakaryocytic system currently lacks such a specific expansion advantage. As a result, iPSC-MK yield and purity is currently low compared to the iPSC-erythroid yield. Even though the yield of MK is low, a small number of MK still could produce significantly large amount of Plts. The *in vivo* production of 2000–8000 Plts/MK is not achieved by far (1–50 Plts/MK in static condition) [4, 5, 81, 168]. Initiatives to increase this yield are needed but will require increasing our knowledge on the later stages of proplatelet formation, which is a current hurdle to overcome in culture conditions and further discussed in the next paragraph devoted to bioreactors (Section 2.5).

#### **2.5. Bioreactors**

The production of cultured red blood cells for transfusion purposes has been the holy grail for transfusion medicine. However, a main challenge is the well-described limitation in cell density during the expansion phase [171]. A single transfusion unit contains 2 × 10<sup>12</sup> RBCs. Conventional cultivation systems using dishes or flasks can reach up to 10 × 10<sup>6</sup> cells/mL, meaning that more than 1000 L of culture would be required for the manufacture of a single transfusion unit [172]. Handling of such large volumes is impractical if static culture conditions are maintained. Thus, multiple bioreactor designs have been proposed to improve the volumetric productivity of the process (produced cells/volume of medium). A static culturing system mimicking BM tissue has been proposed, in which cells are grown in a porous scaffold and nutrients are continuously fed through hollow fibers, while used media containing waste from metabolism are removed [173]. This system allows to have continuous replenishment of spent components in the media, while it is possible to re-use some of the most expensive components such as growth factors and transferrin. Also, it separates cells from large shear stress sources. Although an optimal design of this system could lead to the production of transfusion units at competitive costs compared to the price of rare blood units, it would require significant improvements in transfer of nutrients and matured RBCs between the scaffold and the inflow/outflow streams [174]. Mass transfer limitations in diffusion-governed systems can be tackled with agitation. It is relevant to note that conflicting reports have been made on the effect of shear due to agitation in *in vitro* erythropoiesis [172, 175]. Nevertheless, important advances have been made toward culture of EBLs in stirred reactors. Enucleated RBCs were produced in microbioreactors (<20 mL) in which the effect of shear stress on expansion and enucleation of EBLs was evaluated. Gas sparging caused cell death, whereas stirring enhanced enucleation [172]. Expansion can also be performed in shake flasks (40 mL) and stirred tank bioreactors of larger volumes (500 mL) with similar growth kinetics [176]. Hybrid systems combining mechanical agitation and growth of cells in porous materials have also been proposed. This type of systems could improve mass transfer while protecting cells from shear, but harvesting of mature cells from these carriers is still a challenge that must be addressed.

The produced Plts in these systems can be harvested and used for functional test. The aforementioned approaches can also be combined in a way that the culturing and differentiation of iPSC toward MK is performed on coated beads in a stirred spinner flask bioreactor, where subsequently also the Plt production is induced and enhanced, although with a relatively low Plt yield (1 Plt/MK) [168]. Recently, Ito et al. showed that in the vasculature, MKs are not only exposed to shear stress but also to turbulence during Plt sheading, where the process was implemented in their bioreactor [178]. By using their iPSC lines with inducible c-MYC-BMI-1 and BCL-XL expression, they were able to produce a thrombocyte transfusion unit in an 8 L system. This approach proves the possibility of large-scale Plt production but still with a discrepancy between *in vitro* (80–100 Plts) and *in vivo* (2000–8000 Plts) Plt production capacity.

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 263

The ability to produce large numbers of enucleated, hemoglobinized RBCs opens the perspective of producing cultured red blood cells (cRBC) for transfusion purposes. The feasibility to do so has been demonstrated by the team of Luc Douay who cultured 1 mL of packed cRBC

after injection [133, 179]. Donor-derived RBC transfusion is a cornerstone of modern medicine in the treatment of trauma, chronic anemia, and in surgery. The existence of 30 blood group systems, such as the ABO and Rhesus system, generates at least 300 distinct blood group antigens [180]. Recurrent transfusions carry an inherent risk on alloimmunization to nonidentical blood group antigens, which complicate further transfusions. Besides, this cellular therapy is dependent on donor availability with a potential risk of blood-borne diseases. *In vitro* derivation of cRBCs allows their thorough characterization, therefore providing access to better matched product. Improved cell culture protocols may eventually enable us to generate cRBCs for transfusion purposes from iPSCs that were selected and/or modified to lack most blood group antigens with the advance of donor-independency offered by iPSCs source.

In a recent publication, Ito et al. [178] were able to generate Plts in transfusion quantities, where the functionality of these Plts was shown in *in vivo* mouse experiments. This work gives a feasible prospect of clinical trials, which still requires the system to be converted to GMP

• day 0 expansion: purify PBMCs by Ficoll and seed in StemSpan or Cell-Quin supplemented

• Optional: to remove remaining RBCs after Ficoll purification, the use of RBC lysis buffer

• Optional: to remove remaining lymphocytes, purify the culture by density centrifuga-

• day 2 and 4: replace half of the medium; add all factors to the medium except for IL-3.

cells/ml, 37°C,

with 1 ng/ml human IL-3, 2 U/ml EPO, 10 ng/ml hSCF, 10−<sup>6</sup> M DEX at 10 × 10<sup>6</sup>

HSCs and transfused it to a healthy volunteer, with a cRBCs half-life of 26 days

**2.6. Clinical trial**

from CD34<sup>+</sup>

grade [178].

and 5% CO<sup>2</sup>

is suggested.

.

**Our culture protocol from human PBMC to RBC**

• day 5: EBLs appear as large (nongranulated) blasts.

tion on a 1.075 g/ml Percoll gradient.

The high sensitivity of MK to shear stress renders culturing and flow cytometry assays challenging. However, it can also be exploited to generate *in vitro* Plts. Multiple techniques can be used to induce the culturing and shedding of Plts like pipetting or the use of flow chambers [157, 165–168]. These approaches mimic more the *in vivo* situation of Plts formation where they are shed in the vasculature, in contrast to static cultures. Plt shedding can be induced by repetitive pipetting of the cultured cells with a hand pipette (p1000) [81]. There is a risk that too much pressure is enforced on the cells, causing them to lyse instead of shed. A more sophisticated method of *in vitro* Plts production is the use of flow chambers, which come in different versions [167, 177]. The most basic one has a linear flow with a surface that is coated to which MKs can attach [165, 167]. These versions were further developed to chambers that include small gaps where MKs are trapped and can extend their cytoplasmic extensions through the gap where they start forming Plts [165, 177]. Another flow chamber setup is the use of pillars in the chamber instead of gaps where the MKs can attach. Coating with a various matrices like fibronectin, von Willebrand factor, or TPO can enhance the efficiency of the Plts generation. In addition, the flow rates that are produced in these chambers can be regulated. The produced Plts in these systems can be harvested and used for functional test. The aforementioned approaches can also be combined in a way that the culturing and differentiation of iPSC toward MK is performed on coated beads in a stirred spinner flask bioreactor, where subsequently also the Plt production is induced and enhanced, although with a relatively low Plt yield (1 Plt/MK) [168]. Recently, Ito et al. showed that in the vasculature, MKs are not only exposed to shear stress but also to turbulence during Plt sheading, where the process was implemented in their bioreactor [178]. By using their iPSC lines with inducible c-MYC-BMI-1 and BCL-XL expression, they were able to produce a thrombocyte transfusion unit in an 8 L system. This approach proves the possibility of large-scale Plt production but still with a discrepancy between *in vitro* (80–100 Plts) and *in vivo* (2000–8000 Plts) Plt production capacity.
