Abstract
Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer’s disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.
Highlights
Microglia play a key role in the well-being of the brain by fulfilling various functions in development, homeostasis and the first-line immune defense [1,2,3,4,5,6,7]
For the generation of iPSC microglia many protocols still mostly rely on co-culturing microglia with neurons, astrocytes or neurons and astrocytes to mimic the brain environment [25, 26, 56]
The iPSC microglia from monoculture displayed a similar morphology as the coculture iPSC microglia (Figure 1C) suggesting that the fibronectin matrix resembles aspects of the matrix found in co-cultures
Summary
Microglia play a key role in the well-being of the brain by fulfilling various functions in development, homeostasis and the first-line immune defense [1,2,3,4,5,6,7]. Genome wide association studies substantiated the long-time proposed active implication of microglia within initiation and progression of AD and other neurodegenerative diseases of the central nervous system [9, 10]. Together, this strongly supports the rationale for developing therapies that pharmacologically modulate microglia. Generation of primary cells requires either euthanizing large numbers of animals or accessing difficult to obtain highly characterized human brain samples with short postmortem delay Both approaches result in only a small number of cells, which in turn limits the throughput for compound screening campaigns or larger biological studies [14, 15]. Due to their immortalization or neoplastic-origin, cell lines show strong discrepancies compared to the desired in vivo characteristics [17, 18]
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