Large‐Scale Production of Transfusion‐Ready Red Blood Cells From Induced Pluripotent Stem Cells

Abstract There is a constant worldwide need for blood products, traditionally obtained from donations. In vitro red blood cell (RBC) production could supplement this demand and offer benefits such as thorough screening for improved safety, the possibility of genetic manipulation to restore genetic deficiencies, and therapeutic loading. Induced pluripotent stem cells (iPSCs) are a promising cell source for transfusable RBCs due to their immortality and independence from donors. However, current iPSC differentiation protocols—including both monolayer and embryoid body‐based systems—have failed to produce sufficient erythroid cells (10 ^11–12 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (5–25%), and suboptimal, static culture conditions lacking physiological relevance. This study describes the optimization of an iPSC to RBC differentiation platform and its step‐by‐step translation process to dynamic culture conditions, allowing scalability and eventual bioreactor application. The optimized dynamic culture yields ≈4.6 × 10 ³ RBC/iPSC, requiring an estimated ≈4.9 × 10 ⁷ iPSCs to produce a minitransfusion unit, achieving a consistent 40–70% enucleation rate and bona fide function, demonstrated by both in vitro and in vivo assays. Our feeder‐free, GMP‐compatible system accomplishes an enucleated RBC production rate sufficient for large‐scale application and serves as a bridge to large‐scale bioreactor RBC production, facilitating clinical application.