3007 – POLYVINYL ALCOHOL AND THE EMERGING TRANSLATABILITY OF IPSC-DERIVED RED BLOOD CELLS FOR DISEASE MODELING AND CLINICAL USE

Abstract

Human induced pluripotent stem cells (iPSC) are an invaluable resource in tissue and blood cell engineering due to their multi-lineage potential in culture systems. iPSC-derived hematopoietic progenitors that undergo successful erythropoiesis in vitro would allow for the modeling of inheritable red blood cell (RBC) diseases, such as Sickle Cell Disease (SCD), for novel therapeutic design and pre-clinical testing. Here, we describe an optimized method of generating induced RBCs (iRBCs) in vitro from healthy and sickle cell homozygous iPSCs, using a novel differentiation media formula containing Polyvinyl Alcohol (PVA). iPSC-derived iRBCs mature into GlyA+Band3+CD71lo cells which successfully enucleate at a high rate (65-80%). mRNA profiles demonstrate iRBCs undergo three distinct waves of globin activation and suppression throughout in vitro erythroid development, which closely mimics the in vivo globin-switching profile modeled from humans. Ultimately, iRBCs become robust adult β-globin expressing cells, which is confirmed at the protein level by HPLC. Sickling is easily observable in SCD iRBCs, regardless of oxygen environment, along with other morphological abnormalities characteristic of the human disease (microcytosis, hypochromasia, cell-cell adhesion, fragmentation etc). The use of PVA in this novel erythroid differentiation protocol successfully replaces BSA as a non-toxic, xeno-free media supplement that significantly boosts early erythroblast proliferation in culture, and ultimately primes the blood products for safe use in live human trials. Future blood products engineered using this method have the capacity to supplement donor-derived blood products for clinical use and can be used as a modeling platform to design and test novel drugs against human globinopathies.