A scalable stem cell-derived erythroid cell system for the encapsulation and delivery of therapeutic enzymes: An in...

Red blood cell (RBC)-based therapeutic enzyme delivery systems require cell sources that can support future standardized large-scale production, with the prerequisite that engineering modifications preserve the cells' inherent biocompatibility and long-circulating potential. In this study, we first demonstrated proof-of-principle by successfully engineering asparaginase (ASPG)-loaded erythroid cells in the human erythroid progenitor cell line (HUDEP-2), where efficient ASPG expression and intact enzymatic activity were confirmed. To facilitate clinical translation, the validated strategy was further applied to human induced pluripotent stem cells (iPSCs), and the differentiated products were systematically characterized. It is worth noting that the key membrane markers of iPSC-derived ASPG-loaded erythroid cells (i-ASPG-R), including CD47 and CD55, were comparable to those of human RBCs (hRBCs). The results of Annexin V staining indicated a healthy cell status. More importantly, these cells displayed ASPG activity equivalent to that of HUDEP-2-derived counterparts. Moreover, the expression of ASPG did not affect enucleation and the composition of globin. In vitro function assays showed that compared with the control group, the proliferation of CCRF-CEM leukemia cells was inhibited by 48% after 24 h of co-culture with i-ASPG-R. In summary, this study established an iPSC-derived platform for generating ASPG-loaded erythroid cells with favorable carrier properties and evident anti-leukemic activity in vitro, laying a conceptual foundation for future cell therapy manufacturing via optimized terminal maturation.