Alzheimer’s Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia

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.

CD22 Blockage Restores Age-Related Impairments of Microglia Surveillance Capacity

“…Since macrophages are highly phagocytic cells with similar molecular mechanisms involved in phagocytosis as in microglia and we already established a variety of functional readouts using these cells previously we decided to use these cells as surrogate for microglial phagocytosis. To assess the phagocytic activity of the macrophages, we monitored the uptake of pHrodo-labelled Aβ-coated beads ( 27 ), which were added to the respective treatments. Upon treatment with the IgG isotype control antibody the amount of red-positive cells, which reflect the amount of phagocytic cells, does not differ over time compared to the control condition, whereas the phagocytic activity is completely abolished by cytochalasin D, a known actin polymerization inhibitor ( Figure 5B ).…”

“…The following compounds were added respectively to assess their impact on phagocytosis: CD22 antibody (BioXcell, BE0011; 5 μg/ml), IgG isotype control (BioXcell, BE0083; 5 μg/ml) and Cytochalasin D (MCE, HY-N6682, 10 μM). Preparation of Aβ-coated, pHrodo-labelled beads was performed as previously described ( 27 ). After addition of the beads, phagocytosis was monitored with the IncuCyte S3 live-cell analysis system by acquiring 3-4 brightfield and red fluorescence images every 45 minutes for 12h.…”

“…For comparison with our previous work, we also used the previously described microglial gene modules (27) and compared the effect of CD22 blockage on these modules to untreated cells and isotype control antibody treated cells (Figures 6G, H). We note that CD22 itself is part of the "neurodegeneration" module analyzed by Friedman et al (33).…”

Microglia Development and Maturation and Its Implications for Induction of Microglia-Like Cells from Human iPSCs

“…These actions are mediated by the TGFβ-receptor 1 (TGFBR1), which is highly expressed by both rodent microglia cultures and hiMGLs [55]. Therefore, TGFβ1 was considered as an important maturation factor for immature hiMGLs in distinct hiMGL differentiation protocols at concentrations ranging from 2 to 50 ng/mL, and its effects have been addressed in detail (Figure 3) [55,56,62,76].…”

“…Additionally, the co-culture of neurons and hiMGLs results in a more dynamically ramified morphology and enhanced migration abilities, as well as an overall reduced secretion of chemokines and cytokines, indicating mechanisms of neuron-mediated homeostasis [58]. A recent study comparing neuron-astrocyte co-culture vs. monocultured iMGLs shows that microglial signature gene expression is barely different among these groups, indicating that both approaches can lead to fully differentiated microglia [76]. However, genes related to so-called "disease-associated microglia" (DAM) have been upregulated in the monoculture group, including TREM2 that is a "risk gene" for neurodegenerative diseases.…”