**2.2. Cell culture models**

*StemRegenin 1* (*SR-1*) binds and inhibits the AhR, which mediates toxicity of environmental pollutants, by binding to specific DNA enhancer sequences. This receptor can be activated by endogenous or exogenous ligands and contributes to several physiological processes, among which cell migration, apoptosis, and cell growth [74–77]. Importantly, AhR-deficient mice have increased numbers of BM HSCs [78]. The AhR functions in complex with the AhR Nuclear Translocator to induce differentiation by regulating genes directly such as *c-MYC* and *C/EBP*. During hematopoietic differentiation, its targets also include *PU.1, β-CATENIN, CXCR4,* and *STAT5* [79]. Currently, the antagonist of AhR (SR-1) is used in clinical trials to

needed per transplant from 5 to 1 [80]. Importantly, SR-1 also enhances *in vitro* proplatelet formation [81]. To achieve proper Plts production, MKs have to produce an extensive membrane system; the demarcation membrane system. This requirs extensive lipid biosynthesis which can be inhibited by AhR [82]. Thus, blocking the inhibition of AhR with SR-1 may increase the

EPO is sufficient for steady-state erythropoiesis, when proliferative signals are mediated through the EPOR-associated RON receptor and EPO-induced differentiation is dependent on STAT5 [83, 84]. Increased erythropoiesis during development and upon blood loss requires the cooperative action of the EPOR and KIT [85]. Activation of KIT prevents differentiation and propagates the long-term proliferation of erythroid progenitors through inhibition of *FOXO3a* and activation of mRNA translation via the PI3K/mTOR pathway [86, 87]. Activation of the GR is required for stress erythropoiesis and to inhibit *in vitro* differentiation. Interestingly, glucocorticoids activate largely the same growth inhibitory genes in EBLs and in immune cells, but growth inhibition is counteracted by EPOR/KIT activation [88]. Even in the presence of serum, glucocorticoids promote selective proliferation of erythroid progenitors by both supporting erythroid proliferation and inhibiting proliferation of other myeloid and lymphoid cells [57]. Erythropoiesis is also regulated by the availability of iron, which is imported into the cell as holotransferrin via the transferrin receptor (TfR; CD71), and by selenium through selenoproteins [89, 90]. *In vivo*, erythropoiesis is dependent on the formation of erythropoietic islands that form around a central macrophage [91, 92]. Whereas CD169 macrophages are essential for erythropoiesis *in vivo*, high level of enucleation can be achieved *in vitro* in the absence of macrophages (van den Akker and von Lindern, manuscript in preparation) [93]. The complete understanding of which macrophage signals control enucleation *in vivo* may further

proplatelet formation, through increasing the lipid membrane biosynthesis.

optimize the *in vitro* production of erythrocytes for transfusion purposes.

TPO is the main regulator of megakaryopoiesis, and multiple other factors can work synergistic with it. IL-6 can, in conjunction with TPO, increase hepatic TPO synthesis [94]. This effect is through the shared usage of gp130 and amplification of the same downstream JAK pathways [95]. The direct admission of IL-6, besides its effect on TPO, results in increased polyploidization and subsequently leads to an enhanced Plts production in patients [96]. IL-11 is not constitutively expressed but was shown to be induced in thrombocytopenia patients undergoing

cells prior to transplantation, thereby reducing the number of CB units

expand CB-CD34<sup>+</sup>

250 Cell Culture

*2.1.4. Erythroid-specific regulation*

*2.1.5. MK-specific regulation*
