Abstract
Background/Objectives: Macrophages play a pivotal role in various pathogenic processes, necessitating the development of efficient differentiation techniques to meet the high demand for these cells in research and therapy. Human macrophages can be obtained via culturing peripheral blood monocytes; however, this source has limited yields and requires patient contact for each proposed use. In addition, it would be difficult to perform gene editing on peripheral blood monocytes. The objectives of this study are to define a robust and consistent method for the differentiation of induced pluripotent stem cells (iPSCs) into macrophages that can address these needs for recurrent studies with high yields and the potential for gene editing. Methods: We refined the traditional embryoid body-based differentiation strategy to create a novel three-phase method that optimizes yield, consistent quality, and reproducibility. This approach incorporates microwell plates and cell filtration to standardize the production of embryoid bodies and subsequent macrophage progenitors. Using up to five independent iPSC donors, we performed several assays for macrophage functions and polarization, such as marker protein staining by flow cytometry, lipoprotein uptake, phagocytosis, cytokine release, inflammasome activation, and the effects of M1-like and M2-like polarization. RNA sequencing was performed to determine the segregation of cells at different stages of differentiation and by iPSC donor, as well as to identify marker genes for each stage of differentiation. Results: The iPSC-derived macrophages generated through this method exhibit characteristic features and cell marker proteins, as well as classical macrophage activities, including lipoprotein uptake, bacterial phagocytosis, cytokine release, and inflammasome activation. We demonstrate the effects of M1-like and M2-like polarization on cytokine release. The first three principal components of the RNA sequencing data showed clear clustering by differentiation stage. In contrast, the fourth and fifth principal components clustered the differentiated macrophages by their respective iPSC donor. Marker genes were identified for each stage of differentiation and polarization. Conclusions: The methods provide an optimized and simplified procedure to produce iPSC-derived macrophages. Our results demonstrate the reproducibility of this method in generating high-quality macrophages suitable for a variety of biomedical applications.