Development of a scalable agitation suspension culture differentiation platform for generating erythroid cells from O-negative human induced pluripotent stem cells

Abstract

Background & Aim Universal red blood cells (RBCs) derived from differentiation of O-negative (neg) human induced pluripotent stem cells (hiPSCs) can potentially supplement the emergency transfusion needs of the healthcare industry. Given that each unit of blood requires 1 trillion RBCs, there is a need to develop efficient bioprocesses that could allow for large-scale differentiation and generation of very high numbers of RBCs. While many groups have described means to differentiate hiPSCs into erythroid cells, most of these have not yet been demonstrated to be amenable for scale-up. Methods, Results & Conclusion Using multifactorial process optimization, we have developed a continuous suspension agitation culture platform for differentiating hiPSCs-microcarrier aggregates towards erythroid cells. We have efficiently scaled-up the process starting from 5 ml ultra-low attachment 6 well plates to 125 ml spinner culture flasks. The best performing hiPSC line was efficiently differentiated, generating close to 1.5 billion erythroblasts in 100 ml culture volumes with peak cell densities of erythroblasts approaching 1.5 × 107 cells/ml. By flow cytometry, as much as 60% of hiPSC derived erythroblasts were shown to be enucleated following an optimized scalable co-culture system that we developed. Functional (oxygen binding, hemoglobin characterization and membrane deformability) and transcriptomics evaluation show minimal differences between hiPSC derived RBCs as compared to cord-blood and adult derived RBCs, making them potentially useful for future transfusion applications. The agitation suspension culture differentiation bioprocess developed by our group could serve as a key platform for future scale-up of hiPSCs differentiation towards RBCs and other hematopoietic lineages in controlled bioreactors.