Scalable expansion and erythrocyte production of human induced pluripotent stem cells
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In vitro production of erythrocytes in physiologic numbers from human induced pluripotent stem cells (hiPSCs) holds great promise for improved transfusion medicine and novel cell therapies. However, large-scale production of hiPSCs and their progeny by robust and economic methods has been one of the major challenges for translational realization of hiPSC technology. First, this thesis demonstrates a scalable culture system for hiPSC expansion using the E8 chemically defined and xeno-free medium under either adherent or suspension conditions. To optimize suspension conditions guided by a computational simulation, we developed a method to efficiently expand hiPSCs as undifferentiated aggregates in spinner flasks. Serial passaging of two different hiPSC lines in the spinner flasks using the E8 medium for more than 10 passages preserved their normal karyotype and pluripotency. Second, an optimized differentiation medium was developed for robust and consistent production of hematopoietic stem and progenitor cells (HSPCs) and progeny erythrocytes from hiPSCs, minimizing risk and variation due to the animal-derived products in cell cultures. Several crucial reagents were evaluated and replaced with regulatory-approved pharmacological reagents, such as Romiplostium, to establish a feeder-free and xeno-free condition, in which all the animal-derived products were eliminated. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies, showing enucleation and hemoglobin expression. Finally, a strategy for scalable generation of HSPCs from hiPSCs and subsequent erythrocytes specification was established, by using stepwise cell culture conditions and by integrating spinner flasks and rocker platform. This system supported robust and reproducible definitive hematopoietic differentiation of multiple hiPSC lines. An ultra-high yield of up to 4×10^9 CD235a+ erythrocytes at >98% purity was achieved when using a 1-liter spinner flask for suspension culture. Erythrocytes generated from this system can reach a mature stage with red blood cell (RBC) characteristics of enucleation, β-globin protein expression and oxygen-binding ability. In conclusion, this thesis demonstrates a xeno-free and cGMP-compliant system that provides the opportunity to produce clinical-grade erythrocytes from hiPSCs in a large-scale bioprocess. Therefore, this study is a significant step towards the goal of large-scale generation of RBCs for therapeutic purposes.