Highly efficient derivation of functional human induced pluripotent stem cell-derived macrophages under serum-free conditions to study innate immune responses – macrophage interferon response

A 30-day monolayer culture system was utilized to continually generate hematopoietic progenitor cells (HPCs) from iPSCs starting on day 9, followed by the differentiation of HPCs into iMacs over 21 days. Macrophage-specific markers were evaluated using flow cytometry, while functional assays assessed phagocytosis and production of cytokines like IFNs. Transcriptomic profiling was conducted to characterize gene expression changes across differentiation stages and in response to interferon (IFN).

Results: The optimized differentiation protocol consistently generated high yields of iMacs with over 99% purity, expressing key macrophage markers such as CD14, CD16, CD163, HLADR, and CD11b. Functional assays demonstrated robust phagocytic activity and cytokine production in response to microbial stimuli, closely mimicking the behavior of primary macrophages. RNA sequencing revealed distinct gene signatures at each differentiation stage, highlighting key transitions from pluripotency to mature macrophages.

The transcriptomic signature of iMacs further aligned with tissue-resident macrophage phenotypes, suggesting their potential utility in modelling tissue-specific immune responses. Conclusion: This study establishes a highly efficient, robust protocol for generating functional human iPSC-derived macrophages, with similarities to tissue-resident macrophages offering a versatile model for investigating human innate immunity, host-pathogen interactions, and IFN signaling pathways.