A xeno-free red blood cell differentiation formula models sickle cell disease from somatically sourced patient iPSCs.

Human-derived induced pluripotent stem cells (iPSCs) are an invaluable resource in both two-dimensional (2D) and three-dimensional (3D) tissue engineering due to their multilineage potential in culture systems. To date, modeling red blood cell (RBC) disorders such as sickle cell disease (SCD) from iPSCs has been challenging due to the tendency for differentiation protocols to produce immature erythrocytes that lack robust β-globin expression and enucleate poorly. Here, we demonstrated an optimized three-stage erythroid differentiation protocol that generates enucleated, β-globin-expressing RBCs from somatically sourced iPSCs, derived from both healthy donors and patients with homozygous SCD. Induced RBCs (iRBCs) present phenotypically as GlyA⁺Band3⁺CD71^lo and express adult hemoglobin tetramers. SCD iRBCs displayed sickling phenotypes in vitro when exposed to hypoxia. RNA-sequencing analysis of iPSC-derived SCD reticulocytes revealed dysregulated disease-relevant molecular pathways, suggesting future therapeutic avenues of investigation can be identified in this model. We further refined the protocol into a xeno-free formulation by replacing albumin sources with polyvinyl alcohol (PVA), significantly enhancing iRBC production without loss of terminal maturation. The ability to generate patient-specific iRBCs from somatic cell sources provides a valuable in vitro tool for the study of SCD and the development of novel treatments.