Project description:Background Cell therapy based on macrophages is promising in solid tumors, but effective acquisition of macrophages is a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a good source but are time-consuming and costly. Macrophages derived from somatic cells by reprogramming technologies have the potential to enable the development of cell-based therapies for numerous malignant diseases. Methods The composition of CD45+ myeloid-like cell complex (MCC) was analyzed by flow cytometry and single-cell sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of induced macrophages were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of induced macrophages. Results Here we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in suspension culture and continuously generated functional and highly pure-induced macrophage (iMac) just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulates the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells display significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models. Conclusions Our findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts could help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy. Keywords c-Myc, fibroblast, macrophage, immunotherapy, reprogramming

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Direct reprogramming of fibroblasts into functional macrophages [scRNA-Seq]

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