Macrophage is a very promising cell type for cancer immunotherapy, yet it is difficult to obtain enough functional macrophages for clinical cell therapy. Herein, we descibe a reliable method to produce functional macrophages through the differentiation of human induced pluripotent stem cells (hiPSCs). By optimizing the size control of embryoid bodies (EBs), we accelerated the differentiation process of macrophages and increased the production of macrophages without attenuating macrophage functions. Our final yield of macrophages was close to 50-fold of starting iPSCs. The macrophages showed phagocytic capacity in vitro and a xenograft tumor model. M0 macrophages could be further polarized into M1 and M2 subtypes, and M1 cells exhibited typical proinflammatory characteristics. Moreover, we found that hematopoietic differentiation originated from the outside of EB and matured inward gradually. Taken together, our protocol provides an effective method for the generation of macrophages comparable to blood-derived macrophages, which provides potential value for cell therapy and gene editing studies.
Summary Juvenile myelomonocytic leukaemia (JMML) is an aggressive paediatric leukaemia characterized by mutations in five canonical RAS pathway genes, including the NF1 gene. JMML is driven by germline NF1 gene mutations, with additional somatic aberrations resulting in the NF1 biallelic inactivation, leading to disease progression. Germline mutations in the NF1 gene alone primarily cause benign neurofibromatosis type 1 (NF1) tumours rather than malignant JMML, yet the underlying mechanism remains unclear. Here, we demonstrate that with reduced NF1 gene dose, immune cells are promoted in anti‐tumour immune response. Comparing the biological properties of JMML and NF1 patients, we found that not only JMML but also NF1 patients driven by NF1 mutations could increase monocytes generation. But monocytes cannot further malignant development in NF1 patients. Utilizing haematopoietic and macrophage differentiation from iPSCs, we revealed that NF1 mutations or knockout (KO) recapitulated the classical haematopoietic pathological features of JMML with reduced NF1 gene dose. NF1 mutations or KO promoted the proliferation and immune function of NK cells and iMacs derived from iPSCs. Moreover, NF1‐mutated iNKs had a high capacity to kill NF1‐KO iMacs. NF1‐mutated or KO iNKs administration delayed leukaemia progression in a xenograft animal model. Our findings demonstrate that germline NF1 mutations alone cannot directly drive JMML development and suggest a potential cell immunotherapy for JMML patients.
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