Single-Cell Transcriptomic Profiling of Human iPSC-Derived Macrophage Differentiation Under Distinct Cytokine Conditions

The differentiation of human induced pluripotent stem cells (iPSCs) into macrophages is a complex process involving the emergence of diverse cell types and progenitors. In this study, we conducted a comprehensive single-cell transcriptomic analysis to elucidate the dynamics of iPSC-to-macrophage differentiation and explore the heterogeneity of arising cell populations. Two different cytokine conditions were employed to modulate differentiation, and we hypothesized that these conditions would yield distinct cell compartments. Our results reveal a nuanced cellular trajectory from iPSCs to macrophages, uncovering key intermediates and transitional states. Notably, the emerging cell populations remain consistent, albeit with variations in their proportions, with IL-3-only condition exhibiting a lower proportion of mature cells. This investigation advances our understanding of iPSC-to-macrophage differentiation, shedding light on the cellular heterogeneity and trajectory dynamics. The findings have implications for the development of targeted differentiation strategies and the exploration of novel therapeutic applications in regenerative medicine and immunology. Sample Types: 1. Dissociated Embryoid Bodies/Myeloid-Cell-Forming Complexes: These samples map the progenitor populations at different stages of differentiation. Experimental Conditions and Variables: 1. Embryoid Body (EB): after 5 days of Undirected Differentiation 2. Cytokine Conditions: Two distinct cytokine conditions were employed to modulate the differentiation process: • Condition 1: EBs were differentiated into macrophages in the presence of 50 ng/ml interleukin-3 (IL-3). • Condition 2: EBs were differentiated into macrophages with a combination of 25 ng/ml IL-3 and 50 ng/ml macrophage colony-stimulating factor (M-CSF). 3. Time Points: For the cytokine conditions, two time points were investigated: • Day 8 of Hematopoietic Specification: This time point provides insights into the early stages of differentiation. • Day 16 of Hematopoietic Specification: This time point represents a more advanced stage in the differentiation process.