Effective Erythropoiesis from Human iPSC-Derived RBC in Immunodeficient Mice

Transfusion of red blood cells (RBCs) was the earliest developed form of cell therapy and is still a highly effective life-saving treatment for many patients. Induced pluripotent stem cells (iPSC) can differentiate into RBCs (iPSC-RBCs) and may provide a novel source for blood transfusion and a cellular model for erythroid differentiation. Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. Human iPSC were generated from peripheral blood mononuclear cells of healthy donors by transfection of plasmids containing OCT4, SOX2, MYC, KLF4 and BCL-XL genes. iPSC lines expressed TRA-1-60, SSEA4 and Nanog markers, and showed a normal karyotype. iPSCs were induced to differentiate along the erythroid lineage using a 3-stage culture system requiring 33 days. At the end of the culture period, iPSC-RBCs were CD34-CD235a+CD41+CD43+CD71low; about 10% of cells were enucleated (CD235a+DRAQ5-). iPSC-RBCs were harvested and transfused into immunodeficient NSG mice which had been pretreated with clodronate liposomes and cobra venom factor (CL/CVF). CL/CVF treatment of NSG mice markedly promoted the survival of transfused human iPSC-RBC in vivo, which could be detected with anti-human CD235a antibodies for at least 7 days, although the numbers progressively decreased with time. Interestingly, a large number of transfused iPSC-derived cells homed to bone marrow of NSG mice. In NSG mice that were repetitively treated with CL/CVF every 3 days, nucleated iPSC-derived cells were still detectable in the bone marrow 4 weeks after transfusion. Furthermore, at 3 weeks after transfusion, human iPSC-RBCs reappeared in the peripheral circulation. These circulating iPSC-RBCs were > 90% enucleated and were present at levels more than 4-fold higher than at 1 hour after transfusion. These results suggest that iPSC-RBCs which homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into the peripheral blood. The results offer a new model using human peripheral blood iPSC and CL/CVF-treated NSG mice to investigate the development of human erythroid cells in vivo. 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.