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 and blood products 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 (1,012 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (<20%), and suboptimal, static culture conditions lacking physiologic relevance. Here, we developed a hematopoietic organoid production protocol through embryoid body formation starting from iPSCs using low growth factor culture conditions. Optimization of this—iPSC to RBC differentiation platform—and its step-by-step translation process to complete dynamic culture conditions allows scalability and bioreactor application. The optimized dynamic culture yields ∼5 × 103 RBC/iPSC, requiring an estimated ∼5 × 107 iPSCs to produce a minitransfusion unit, achieving a consistent ∼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.
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