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Microglia are immune cells resident in the central nervous system (CNS) responsible for fundamental physiological and pathological processes. Microglia support neuronal homeostasis and facilitate neuronal network formation by synaptic pruning. Surveillant microglia can activate, adapt and respond accordingly to specific stimuli.
Microglia dysfunction has been implicated as a key player in neurodegeneration afflictions, including Alzheimer’s Disease (AD). The limited availability and inconsistency of primary human microglia has constrained research and therapeutic progress.
Rare variants of TREM2 (triggering receptor expressed on myeloid cells 2), an immune receptor expressed on microglia known to trigger phagocystosis of bacteria and the release of reactive oxygen species, are associated with an increased risk of developing AD. Most TREM2 mutations found in AD risk variants are heterozygous mutations that impact the binding of TREM2 ligands or the shedding of the extracellular domains. Loss‐of‐function mutations of TREM2 and adaptor protein TYROBP have also been linked to the development of Nasu–Hakola disease, an inflammatory degenerative disease of the brain and bone, leading to premature dementia and death.
To enable investigation of the functional consequences of TREM2 variants, a frameshift in TREM2 exon 2 in either one allele (heterozygous; C60MfsTer45; iCell Microglia TREM2 HZ) or both alleles (homozygous; P59AfsTer16 and P59AfsTer46; iCell Microglia TREM2 HO) near amino acid 60 was engineered in iPSCs from an otherwise apparently healthy, normal donor background, 01279.
Comparative Marker Expression iCell Microglia were labeled for the presence of cell surface (CD45, CD11b, CD11c, TREM2 and, CD33) and intracellular (P2RY12, TMEM119, CX3CR1, IBA1) antigens by flow cytometry. The specific staining is compared against matched isotype controls. A comparison of cell surface and intracellular antigens reveals similar marker expression across iCell Microglia 01279 and the two TREM2 engineered lines (Fig. 1).
Phagocytosis In Figure 2, a comparison is shown between iCell Microglia and the engineered TREM2 lines. Phagocytosis of S.aureus BioParticles® was measured over time using an IncuCyte® S3 Live-Cell Analysis System. The iCell Microglia TREM2 HZ and iCell Microglia HO exhibit phagocytic activity, but at a reduced activity relative to the control line. The reduced phagocytosis in TREM2 variants were correlated to their survival in vitro (data not shown).
TREM2 Expression Levels Soluble TREM2 levels were quantified from the iCell Microglia control and TREM2 variants using a SimpleStep ELISA (Fig. 3A). The TREM2 antibody recognizes the extracellular portion of the TREM2 protein. The iCell Microglia control and TREM2 variants were thawed and plated at the same density in maintenance media in a 96-well plate. The spent media was collected at 3 days and 7 days post thaw to determine the levels of soluble TREM2. The amount of TREM2 in total protein lysates was measured by Western blot. Cell lysates of iCell Microglia control, TREM2 and another engineered line, iCell Microglia SNCA A53T, were electrophoretically separated and detected with anti-TREM2 antibody using the ProteinSimple Wes System along with a molecular weight markers (Fig. 3B).
Cytokine Response The cytokine response of stimulated iCell Microglia and TREM2 variants were compared by multiplex assay (Fig. 4). iCell Microglia were plated in maintenance media and allowed to recover for 72 h prior to stimulation with LPS, interferon gamma or both for 24 h. Supernatants were collected and assayed using the multiplex Luminex system.
Figure 1. Schematic Representation of TREM2 Protein A frameshift mutation engineered in exon 2 of the TREM2 gene (indicated by the arrow) interferes with the signal peptide domain. Figure 2. Engineered Variants of 01279 and the Predicted Amino Acid Sequence for the TREM2 Exon 2 Domain