Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43⁺ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34⁺/CD45⁺ progenitors with high erythroid colony-forming potential and CD36⁺ erythroid precursors. A total of 1.5 × 10⁷ cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA⁺ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

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