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 (1011-12 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 × 103 RBC/iPSC, requiring an estimated ≈4.9 × 107 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.

Understanding how mechanical stress affects erythropoiesis is crucial to produce transfusable erythrocytes in fluid-turbulent bioreactors. We investigated the effects of shear-stress on differentiating CD49d+CD235a+ primary human erythroblasts (EBL) at molecular, cellular, and functional level. Shear-stress, at differentiation onset, enhanced EBL maturation and induced upregulation of genes regulating cholesterol/lipids biosynthesis, causing changes in cell lipid composition. Of note, the osmotic resistance, and the expression of 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthesis pathway, were higher in dynamic cultures. Inhibition of the S1P-induced proteolytic cleavage, activating SREBPs, led to abrogation of HMCGR expression, and loss of EBL in dynamic cultures, similar to lovastatin administration. This data reveals a role for the S1P-SREBP-HMGCR-axis in the regulation of shear-stress induced adaptation during erythropoiesis, shedding light into mechanisms that will assist the upscaling of erythroid differentiation into bioreactors. Moreover, as shear-stress on hematopoietic cells occurs within the bone-marrow, these results introduce a novel signalling axis in the transduction pathways controlling erythropoiesis.