Characteristics of in Vitro Differentiated Erythrocytes Derived from Human Bmi-1 Extensively Expanded Erythroblasts (E3)
Authors: Lancelot Moira M. , Saakadze Natia , Deng Jiusheng , Stowell Sean R. , Jajosky Ryan , Cheng Linzhao , Liu Senquan , Roback John D 
Published: February 2021

Background and significance: Blood transfusion is an important and sometimes life-saving therapeutic intervention that may not always be available due to donor shortages, alloimmunization, and rare blood groups. Red blood cells (RBCs) produced in vitro may provide an inexhaustible alternative or complimentary source of blood products for those patients without compatible donors, while eliminating the risk of infection and transfusion reactions. Bmi-1 overexpression has been shown to increase the self-renewal potential of mouse erythroblasts. We describe studies employing Bmi-1 overexpression in human erythroblasts to produce cultures of extensively expanded erythroblasts (E3) that can be differentiated into mature erythrocytes, and used for the study and optimization of ex-vivo methods of erythropoiesis with the ultimate goal of mass production of engineered blood red blood cells for transfusion purposes.
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.