BCL-XL overexpression enables efficient in vitro erythropoiesis in a human erythroid progenitor cell line

Ex vivo manufacturing of cultured red blood cells (cRBCs) from immortalized erythroid progenitor lines represents a promising cell therapy platform, yet its clinical translation remains hindered by two critical bottlenecks: heavy reliance on expensive recombinant cytokines (EPO and SCF) for expansion, and massive apoptotic loss during terminal erythroid differentiation. To address these challenges, we developed a genetically engineered, BCL-XL–overexpressing human erythroid progenitor cell line (HuBoEP) via lentiviral transduction of the parental human erythroid progenitor cell (HuEPC) line and systematically evaluated its cellular kinetics, viability, and enucleation efficiency. Lentiviral-mediated BCL-XL overexpression did not alter baseline erythroid surface marker expression, confirming phenotypic preservation of the progenitor state. Under cytokine-reduced conditions, HuBoEP cells maintained higher cell numbers and viability than parental HuEPC cells, yielding an approximately 1.6-fold greater expansion in a Reduced Cytokine Expansion Medium (RCEM) containing only 3.3% EPO and 40% SCF of standard concentrations. During a 3-step differentiation protocol, the HuBoEP cells exhibited a 3.39-fold higher cell viability at Day 14 compared to HuEPC cells. A flow-cytometry derived relative enucleated-cell proportion was approximately 2.55-fold higher in HuBoEP cells. After additionally accounting for the relative total cell number, the derived relative total enucleated cell number was approximately 7.19-fold higher in HuBoEP cultures. Crucially, multi-omic and functional characterizations—including RT-qPCR, surface immunophenotyping, Giemsa staining, and hemoglobin-oxygen dissociation assays—confirmed that BCL-XL overexpression preserves physiological lineage maturation and functional integrity without disrupting the intrinsic erythroid differentiation program. Our findings establish BCL-XL not as a surrogate for cytokine signaling, but as an intracellular survival amplifier that mitigates bottleneck-associated cell loss during differentiation. This cellular engineering approach provides a highly translatable platform for the cost-effective and scalable manufacturing of cRBCs, representing a pivotal advancement toward clinical-grade artificial blood production.