Session: 101. Red Cells and Erythropoiesis, Structure and Function, Metabolism, and Survival, Excluding Iron: Mechanisms, Diagnosis and Treatment of Inherited
Hematology Disease Topics & Pathways:
HSCs, cell division, iPSCs, cell regulation, blood banking, red blood cells, Biological Processes, Technology and Procedures, Cell Lineage, erythropoiesis, hematopoiesis
Methods: Human erythroblasts, derived from peripheral blood of human donors, were transduced with a lentiviral vector expressing Bmi-1 to produce cultures of E3 cells. Following the transfer of proliferating E3 cells to differentiation culture conditions, we characterized changes in cell morphology and expression of erythroid cell surface markers CD49, CD71 and CD235a at various time points. Additionally, we tracked cells numbers, cell viability, and enucleation. At the end of the differentiation process we also analyzed cells for hemoglobinization and blood group expression and compared our results to the original donor cells. Culture conditions were optimized to increase the yield of viable and enucleated cells at the end of the differentiation process.
Findings: In vitro maturation and differentiation of Bmi-1-E3 cells is characterized by progressive enucleation (up to 50% of total erythroid cells in some experiments), as well as down-regulation of transferrin receptor (CD71) and α4 integrin (CD49d). As maturation progressed, cells also underwent additional changes including decrease in size, nuclear condensation, and accumulation of hemoglobin in the cytoplasm. We were able to optimize conditions to demonstrate that cells were terminally maturing over time in the culture system.
Conclusion(s): The data shows that Bmi-1-E3 cells can be cultured in vitro to produce mature erythroid cells with characteristics desirable for transfusion for those patients who may not otherwise have a suitable source for transfusion. Further work is required in order to increase enucleation and evaluate the survival and functionality of these cells in vivo, as once completely enucleated, cultured red cells do not pose concerns over genotoxicity and should be of adequate quality to be used for transfusion purposes. Simultaneously, our group is developing a mouse model to test the in-vivo performance of these cultured red blood cells.
Disclosures: Jajosky: Biconcavity Inc.: Other: CEO and partial owner; BioMarin Pharmaceuticals: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company.
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