Advances in the characterization of in vitro-generated red blood cells: from biophysical properties to functional applications.

BACKGROUND: Red blood cells (RBCs), essential for oxygen transport and carbon dioxide removal, are pivotal for maintaining systemic metabolic homeostasis. However, global blood shortages and limitations in current transfusion practices underscore the urgent need for alternative sources, such as in vitro-generated RBCs. Among these, induced pluripotent stem cell (iPSC)-derived RBCs have gained attention for their potential patient-specific, pathogen-free, and immunologically compatible solutions. Yet, conventional assays of RBC function provide only a partial view of the complex molecular programs that govern erythropoiesis and maturation. These gaps motivate the integration of multi-omics platforms to comprehensively profile the developmental and functional states of in vitro-generated RBCs.

MAIN BODY: iPSC-derived RBCs hold broad translational promise, yet recapitulating erythropoiesis in vitro remains difficult given the niche's hypoxia, extracellular cues, and multilayered regulation. We review erythroid development from primitive to definitive programs, the roles of HSCs and erythroblastic islands, and key pathways (e.g. EPO, GATA1, KLF1, TGF-β), with emphasis on transcriptional networks and chromatin remodeling that drive maturation. Integrating multi-omics provides a quantitative view of erythropoietic state transitions, explicitly linking gene-regulatory programs with metabolic and proteomic remodeling. By clarifying the mechanisms behind incomplete enucleation and hemoglobin switching, this systems-level perspective guides optimization toward scalable iPSC-derived RBC manufacture. We outline how transcriptomic, epigenomic, proteomic, and metabolomic readouts together map the regulatory landscape, and we discuss translational applications-transfusion medicine, rare blood type management, disease modeling, and drug delivery-alongside safety, ethical, and regulatory considerations.

CONCLUSION: This review underscores both current advances and persistent challenges in characterizing in vitro-generated RBCs, and it proposes a translational framework that integrates multi-omics data into their development and evaluation. By explicitly linking molecular regulation with process control and quality metrics, this approach provides a roadmap to optimize functional performance and ensure clinical readiness of in vitro-generated RBCs.