| Summary: | Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the <i>HBB</i> gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. Methods: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of <i>HBB</i> and <i>HBG2</i>. Results: Both 2D and 3D differentiation protocols led to the induction of CD34<sup>+</sup>/CD43<sup>+</sup> HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71<sup>+</sup>/CD235a<sup>+</sup> cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a<sup>+</sup>/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal <i>HBG2</i> compared to adult <i>HBB</i>. Conclusion: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.
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