[A method for the differentiation of induced pluripotent stem cells into macrophages]

Objective To establish a simple and cost-effective protocol for the directed differentiation of induced pluripotent stem cells (iPSCs) into macrophages (Mϕ). Methods Human umbilical cord blood mononuclear cells were reprogrammed into iPSCs by introducing reprogramming factors with Sendai virus vectors. The iPSCs were then induced to differentiate into Mϕ through an embryoid body-based approach, combined with key cytokines including bone morphogenetic protein 4 (BMP4), macrophage colony-stimulating factor (M-CSF), and interleukin-3 (IL-3). Flow cytometry, real-time quantitative polymerase chain reaction and other methods were used to determine the phenotype, function, and expression of immunogenicity-related molecules of the differentiated Mϕ. Results High-purity CD14⁺CD11b⁺ Mϕ with normal phagocytic function and polarization capacity were successfully generated by this protocol, without co-culture with animal-derived feeder cells, hypoxic conditions, or cell sorting at intermediate stages. Immunophenotypic analysis revealed that the immune checkpoint CD276⁺ cells were reduced after the differentiation of iPSCs into Mϕ, which was inconsistent with the stable expression of CD276 observed during the differentiation of iPSCs into β cells or retinal pigment epithelial cells. Conclusion In this study, high-purity Mϕ were successfully generated using a feeder-free, hypoxia-free, and sorting-free system. Furthermore, the lineage-dependent downregulation of CD276 during Mϕ differentiation was observed for the first time.