Abstract 29: Efficient Differentiation and Polarization of Macrophages Differentiated From Human Peripheral Blood Mononuclear Cells-derived iPS Cells

Abstract

Macrophages fulfill homeostatic functions beyond defense. Monocytes/macrophages derived from human induced pluripotent stem cells (hiPSCs) are useful tools to study vascular diseases and potential sources of cell-based therapies. The objective of this study is to generate PBMC-hiPSC derived monocytes, macrophages, as well as M1 and M2 type differentiated macrophages and to examine multiple macrophage characteristics of relevance to cardio-metabolic disease. We briefly cultured human adult PBMCs from healthy volunteers to enrich for the erythroid progenitors. These progenitors were used to generate hiPSCs using lentivirus-mediated transduction of Oct3/4, Sox2, Klf4 and c-Myc. A stepwise protocol was used for the differentiation of PBMC-hiPSCs to monocytes/macrophages. The protocol involves primitive streak and mesoderm induction (stage 1), hematopoietic specification (stage 2), hematopoietic cell maturation and myeloid expansion (stage 3), monocytes collection and differentiation to macrophages (stage 4). Differentiation cultures were set up in six-well plate format, from 20 embryoid bodies per well, and produced up to 30 million macrophages. hiPSC-derived macrophages (hiPSC-MΦ) exhibited spindle morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of hiPSC-MΦ revealed positive staining for CD68, CD11b, and CCL2. FACS analysis indicates >90% expression of macrophage lineage markers, including CD45, CD14, CD16, CD115, CD68, CX3CR1, CD11b, and CD18. Migration of hiPSC-MΦ in response to M-CSF was confirmed by Boyden Chamber assay. hiPSC-MΦ were polarized to M1 (classical activation stimulated by TLR ligands LPS and IFN-gamma) and M2 (alternative activation stimulated by IL-4) types. M2 macrophages were characterized by more efficient phagocytic activity of zymosan particles and greater migration capacities compared with M1 macrophages. The results demonstrated the feasibility of differentiation/polarization of macrophages from PBMC-hiPSCs. This study advanced our understanding of the suitability of subject-specific hiPSC for deriving myeloid/monocyte/macrophages for study of monocyte-macrophage functions of direct relevance to cardio-metabolic disease.