*2.4.1. Marker expression pattern of differentiating iPSC cells*

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

**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),

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

erythroid commitment while the role of SCF is not entirely clear.

with or without EPO and SCF from day 6 onward.

258 Cell Culture

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> / CD235<sup>+</sup> population is generally reported with purity discrepancies. For example, Yang et al. [163] reported 80% CD71/CD235 purity (with CD34<sup>+</sup> /CD43<sup>+</sup> preselection and OP9 coculture), 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 gene expression profile: early definitive EBLs derived from CD235<sup>+</sup> /CD34low/CD36−, and they develop to CD235<sup>+</sup> /CD34−/CD36−, CD235<sup>+</sup> /CD34−/CD36−/low, CD235<sup>+</sup> /CD34−/CD36− cells in sequence [164]. Others including us found high CD36 expression during the early erythroid stage [136].

The kinetics during differentiation/maturation of MK from iPSC follow the same steps as from definitive CD34<sup>+</sup> cells, namely MKBL (CD34<sup>+</sup> /CD41a<sup>+</sup> ), early MK (CD34<sup>+</sup> /CD41a<sup>+</sup> /CD42<sup>+</sup> ), and late MK (CD34−/CD41a<sup>+</sup> /CD42<sup>+</sup> ). The MKs can undergo some polyploidization albeit not in similar level as *in vivo* or primary CD34<sup>+</sup> cultures. MKs derived from mouse iPSC can form 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 can be easily used in scaleup production [136].

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 spec-

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 ×

erythroid cells after 9 days of expansion. While a single iPSC gives rise to ~8 × 10<sup>3</sup>

their globin expression, do not entirely correspond to a fetal/adult definitive wave.

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

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

differentiation (6.9 × 10<sup>3</sup>

cells (harvest) and subsequent 9 days expansion results in ~2 × 105

erythroid

erythroid cells/iPSC on

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 261

cells/iPSC). Unlike the

trometry could yield specific makers.

MK yield from iPSCs is higher than CD34<sup>+</sup>

in the next paragraph devoted to bioreactors (Section 2.5).

106

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