Production of Cultured Red Blood Cells

Albert Einstein College Of Medicine, Bronx NY

Investigators

Linked publications & trials

Abstract

Summary: We have developed an innovative and highly efficient method for producing enucleated cultured Red Blood Cells (cRBCs) from induced Pluripotent Stem cells (iPSCs). This method involves introducing specific genetic mutations, the D816V mutation in the kit gene (SCF receptor) and the V617F mutation in the Jak2 gene, resulting in the constitutive activation of the SCF receptor and Jak2 adaptor, responsible for transducing Epo signaling. This allows us to generate KitJak2 self- renewing erythroblasts (SREs) that can continuously proliferate without the need for cytokines in a cost-effective manner. Importantly, these KitJak2 SREs retain the ability to differentiate into cRBCs and enucleate at a remarkably high rate. Our primary objectives in this proposal are to leverage this approach to produce and thoroughly characterize cRBCs from a panel of donors. These cRBCs can serve as a valuable source for creating reagent RBCs, which, in turn, can assist in resolving complex allo-antibody identification challenges in patients with Sickle Cell Disease (SCD). Furthermore, these cRBCs have the potential for use in transfusion support, representing a significant advancement in this field. In Aim 1.1, we propose to develop in collaboration with FiberCells Systems a method for the large-scale production of SRE-derived cRBCs in hollow-fiber bioreactors. In Aim 1.2 we will undertake a thorough characterization of the resultant cells using a variety of methods to assess their morphology and functionality in vitro and in vivo in animal models. In Aim 2, we will produce and assess cultured reagent RBCs. We have established a panel of four rare blood donors to simplify the identification of allo-antibodies in patients with SCD. These donors' RBCs express combinations of antigens that enable the identification of allo-antibodies in SCD patients, especially in cases that pose significant challenges. We have generated lines of iPSCs and stored primary cells from all four donors. Our proposal involves producing KitJak2 SREs and cRBCs from these four donors and thoroughly characterizing their antigen profiles. These cRBCs will undergo extensive testing with sera from allo-immunized patients to assess their suitability for use as reagent RBCs. In Aim 3, we propose to generate adult KitJak2 SREs capable of sustained proliferation without the need for cytokines. A limitation of iPSC-derived cRBCs is their embryonic nature, resulting in larger cell size and predominantly Hb F expression. To address this limitation, we propose to generate KitJak2 SREs that express mostly adult hemoglobin and that have the capacity for sustained proliferation. This will be achieved by introducing both the kit and Jak2 mutations into hematopoietic stem and progenitor CD34+ cells obtained from cord or peripheral blood, with or without the over- expression of hTERT or HoxA9. In summary, our innovative approach holds great promise for advancing the field of transfusion medicine by providing a reliable source of cRBCs with known antigen profiles, addressing critical challenges in SCD patients, and potentially enhancing transfusion support by providing a novel source of rare red blood cells.