Optimized Method to Generate Well-Characterized Macrophages from Induced Pluripotent Stem Cells

Background: 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 of 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 differentiation of induced pluripotent stem cells (iPSCs) into macrophages can meet these needs for recurrent studies with high yields with the possibility of 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 the use of 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 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, while 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. Several iPS cell lines were subjected to differentiation into macrophage progenitors and then to M0 macrophages, followed by polarization into M1-like and M2-like macrophages. We preformed paired end bulk RNAseq on these samples in order to determine clustering by cell type and iPS donor as well as to determine marker genes for each cell state. Oligo-dT cDNA libraries were prepared and paired end 100 bp sequencing was performed on a NovaSEQ-X instrument at the University of Chicago Genomics Core. RNAseq quality control was performed using the FastQC program and alignments were performed using STAR. Normalized counts per million (CPM), differential gene expression, and principal component analysis (PCA) was determined using DESeq2. **Raw data are not provided due to donor privacy concerns**