Induced pluripotent stem cell derived monocytes and macrophages mimic human derived monocyte and macrophage phenotype and functionality and can be used to model human inflammation and vascular dysfunction

Abstract

Background Monocytes and macrophages of the innate immune system play major roles in the onset and progression of cardiovascular diseases (CVDs). However, these cells are often not included in model systems due to difficulties in consistently isolating these cells out of peripheral blood mononuclear cells (PBMCs), warranting an alternative cell source. In this report, I investigated the use of human induced pluripotent stem cell (hiPSC) derived monocytes and macrophages to model human inflammation, comparing them to PBMC-derived monocytes and macrophages based on the following 8 pre-set definitions. Monocytes are round and granulated cells (1) that can be divided into 3 subsets based on CD14 and CD16 expression (2), should be able to interact with vasculature (3) and should be able to differentiate into macrophages (4). Macrophages are granulated cell types (5) that can be divided into 3 subsets based on CD163, CD64, CD80 and CD206 marker expression (6) and should functionally be able of phagocytosis (7) and cytokine secretion (8). Materials and methods To research the similarities between hiPSC-derived and PBMC-derived monocytes and macrophages, hiPSCs from three different cell lines were differentiated to monocytes. These monocytes were phenotypically analysed using microscopy and flow cytometry analysis on monocyte markers CD14, CD16, CCR2 and CX3CR1 marker expression, and functionally analysed by determining hiPSC-derived monocyte-EC interactions after activation with TNF-α under flow. the hiPSC-derived monocytes were polarised into M0, M1 and M2 macrophages and compared to PBMC-derived macropahges using flow cytometry analysis on macrophage markers CD163, CD64, CD80 and CD206. Results I demonstrated the successful differentiation of hiPSC-derived monocytes that could be divided into classical, non-classical and intermediate monocytes based on CD14 and CD16 expression, similar to PBMC-derived monocytes. These hiPSC-derived monocytes were functional and able to interact with endothelial cells under flow, which increased after activation with TNF-α. hiPSC-derived monocytes were successfully polarised towards M0, M1 and M2 macrophages that closely resembled PBMCderived macrophages based on morphology and CD163, CD64, CD80 and CD206 marker expression. Discussion I illustrated succesfull hiPSC-derived monocyte generation which phenotypically and functionally resembled PBMC-derived monocytes and showed that these monocytes could be polarised into all three hiPSC-derived macrophage subsets that were phenotypically similar to PBMC-derived macrophages. These data suggest that hiPSC-derived monocytes and macrophages can be used to model human inflammation, however, additional macrophage functionality assays are required before I can confidenly confirm this hypothesis.