Ph.D Student Korea Research Institute of Bioscience and Biotechnology (KRIBB), Republic of Korea
Abstract: Microglia is tissue-resident macrophage that exist in the central nervous system (CNS). Microglia is involved not only immune responses in CNS, but also contributes to the development and functional regulation of neurons and other glial cells. Microglia is influenced by genetic predisposition and micro-environments, and their dysfunction is a key characteristic of neurological diseases. However, unlike pheriperal blood mononuclear cells, microglia is difficult to isolate securely. Animal model-derived microglia and cell lines only partially recapitulate human microglial characteristics due to genetic differences. In contrast, human iPSC-derived microglia provide a stable supply of cells with specific traits and replicate the microglial development process, closely resembling embryonic hematopoiesis. Therefore, we described an effective differentiation method of microglia using human iPSC and integration into cerebral organoids. Firstly, embryonic bodys (EB) were produced using human iPSC with SCF, BMP4, VEGF and 20uM ROCK inhibitor. To differentiate the hematopoietic lineage, we induced primitive macrophage progenitor (PMP) using IL-3 and M-CSF. PMP cells were expressed myeloid immune cell markers (CD11b, CD14, CD45). To induce the maturation of microglia, we cultured PMP cells in the presence of IL-34 along with low concentrations of GM-CSF and M-CSF. After maturation step, cells express typical microglia markers (IBA-1, CX3CR1, TMEM119, P2RY12). Moreover, we investigate whether iPSC-derive microglia exhibit functional properties. In the phagocytosis assay, we observed that iPSC-derived microglia internalized fluorescence latex beads in a time dependent manner after treatment. Furthermore, LPS treatment shifted microglia to an inflammatory phenotype, marked by decreased CD206 and elevated HLA-DR expression. To generate the neuro-immune organoids, microglia was co-cultured with human cerebral organoid. Notably, iPSC-derived microglia successfully integrated into the cerebral organoid and exhibited proliferation. Taken together, we established an iPSC-derived microglia differentiation protocol with functional characteristics. These microglia is expected to be applicable to the study of microglial function and their role in neuroimmunology.
