Macrophages play a central role in innate immunity and homeostasis. The most common human macrophage models used are mainly monocyte-derived macrophages or immortalized macrophage cell lines such as THP-1. These cells are either difficult to access or have been transformed or have a different origin than tissue-specific macrophages. Here, we developed a feeder-free and serum-free protocol that differentiates induced pluripotent stem cells (iPSCs) into macrophages that resemble tissue-resident macrophages. The protocol includes induction of the primitive streak, generation of meso-endothelial cells and hemogenic endothelium, production of hematopoietic progenitor cells, and macrophages.
We successfully applied the protocol to five different iPSC lines, including Kolf2 iPSCs, IFNAR1 null, and IFNAR2 null iPSCs and their corresponding isogenic control iPSC lines. The IFNAR1 null and IFNAR2 null iPSCs are reprogrammed from primary immunodeficiency patients. The fully differentiated Kolf2 iPSC-derived macrophages exhibited human macrophage markers such as CD45, CD14, CD16, CD11b, HLA-DR, and CD163, and they also showed tissue-resident markers such as CD169, CD71, and CD11c by flow cytometric analyses. A PCA plot from the Myeloid Atlas on the Stemformatics website indicated that the Kolf2 iPSC-derived macrophages are very similar to colon and lung macrophages. Moreover, the Kolf2 iPSC-derived macrophages show a potent phagocytic ability and cytokine production upon LPS stimulation. The responses of Kolf2 iPSC-derived macrophages to cytokines, pathogens, commensals, and synthetic ligands are currently being analysed by transcriptomics.
Establishing this feeder-free and serum-free protocol for iPSC macrophage differentiation provides a powerful tool to study macrophage signalling in innate immunity with the advantages of unlimited expansion in vitro, the potential to be genetically manipulated and polarised to organ-specific macrophages, which recapitulate the features of human primary macrophages. The successful application of this protocol in various iPSC lines that have been reprogrammed from primary immunodeficiency patients allows us to investigate the mechanisms of key components in innate immunity.