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Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis

Summary Dysfunction of microglia, the brain’s immune cells, is linked to neurodegeneration. Homozygous missense mutations in TREM2 cause Nasu-Hakola disease (NHD), an early-onset dementia. To study the consequences of these TREM2 variants, we generated induced pluripotent stem cell-derived microglia-like cells (iPSC-MGLCs) from patients with NHD caused by homozygous T66M or W50C missense mutations. iPSC-MGLCs expressed microglial markers and secreted higher levels of TREM2 than primary macrophages. TREM2 expression and secretion were reduced in variant lines. LPS-mediated cytokine secretion was comparable between control and TREM2 variant iPSC-MGLCs, whereas survival was markedly reduced in cells harboring missense mutations when compared with controls. Furthermore, TREM2 missense mutations caused a marked impairment in the phagocytosis of apoptotic bodies, but not in Escherichia coli or zymosan substrates. Coupled with changes in apoptotic cell-induced cytokine release and migration, these data identify specific deficits in the ability of iPSC-MGLCs harboring TREM2 missense mutations to respond to specific pathogenic signals.


Supplementary text
The karyotype of TREM2 mutant iPSC-lines was assessed with CNV analysis. Both clones of T66Mhet2 had a 47, XYY phenotype (Suppl. Fig1A). Both T66Mhom clones had large areas of loss of heterozygosity on chromosome 6 and 7 (suppl. Fig1B). Clone 3 of the T66Mhom iPSC line featured a heterozygous deletion of approximately 7MB on chromosome 3, starting at position 63348134 and ending at 69934331 (suppl. Fig. 1C). Karyotype analysis of T66Mhom clone 3 was normal (suppl. Fig. 1D). The CNV analysis data for all TREM2 mutant lines can be seen in suppl. Table 1.

Generation of human iPSC-derived microglia-like cells
TREM2 mutant primary fibroblast lines were generated from 4mm skin punch biopsies, which were obtained under informed consent. Ethical permission for this study was obtained from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology joint research ethics committee (study reference 09/H0716/64 for collection of W50C mutant fibroblasts) or approved by the Ethics Committee of Istanbul Faculty of Medicine, Istanbul University (for collection of T66M mutant fibroblasts to Dr Ebba Lohmann). iPSC were generated from fibroblast cultures using 4 factor Sendai virus reprogramming and characterised by RT-PCR for plasmid integration, immunofluorescence for expression of pluripotent markers, and 3-germ layer differentiation and at the NIHR Cambridge Biomedical Research Centre (BRC), human Induced Pluripotent Stem Cells core facility (see Brownjohn et al. 2018). Karyotypic integrity was assessed by CNV analysis using OmniExpress-24 chips run on the Illumina Infinum platform. CNVs were checked visually on GenomeStudio software. The following control iPSC lines were used: CTRL1 (kindly provided by Dr Selina Wray, UCL Institute of Neurology), ND (called CTRL2 in this paper, from Coriell Institute of Medical Research Biorepository, kindly provided by Dr Rickie Patani, UCL Institute of Neurology), SBAD03 (called CTRL3, from Stembancc), CTRL4 (SFC840, from Stembancc). iPSC colonies were maintained in E8 medium (Life Technologies), and split every 4-5 days using Versene (Lonza). iPSC colonies were differentiated into iPSC-macrophages within 5 passages.

TREM2 genotyping by Sanger sequencing
Genomic DNA was extracted from approximately 1 million cells using DNeasy Blood & Tissue Kits (QIAGEN), following the manufacturer's instructions. DNA was diluted with nuclease free water to 25ng/μl for TREM2 genotyping.
TREM2 genotyping was carried out by PCR and Sanger sequence analysis of the complete second exon of the TREM2 gene. The amplification reaction was performed in a volume of 50μl, which contained 2XKAPA HiFi HotStart ReadyMix (Kapa Biosystems), 0.3μM of each primer (Forward primer: 5'-AGTGGGTGGTTCTGCACAC-3' and Reverse primer: 5'-TCCTTCAGGGCAGGATTTTT-3'), and 50ng genomic DNA. PCR cycling conditions were as follows: Initial denaturation at 95°C for 3 minutes followed by 35 cycles with denaturation at 98°C for 20 sec, annealing at 62°C for 15 sec, extension at 72°C for 30 sec; then a final extension at 72°C for 1 minute. PCR were subsequently visualized on a 1.5% agarose gel to verify the size of the amplicon (533 bp). Finally PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and then sequenced commercially (Source Bioscience, Nottingham, UK).
Alignment of sequences and analysis was carried out using SnapGene software (GSL Biotech, version 3.3.3).

Isolation of human blood derived-monocytes
Phlebotomy was performed and blood collected into heparinised tubes. This study was approved by University College London (UCL)/UCL Hospitals Joint Research Ethics Committee (Project ID: 7451/001) and all subjects gave informed written consent. Plasma was separated by incubating the heparinized blood with 3% dextran 500 (Sigma) in an upright position for 60 minutes at room temperature. The leukocytes were pelleted from plasma by centrifugation for 6 minutes at 3 1600 rpm (461g). The cells were resuspended in PBS, layered over Histopaque (Sigma) and centrifuged with low acceleration and brake for 20 minutes at 720g. The resulting peripheral blood mononuclear cell (PBMC) layer was collected and CD14 Microbeads (Miltenyi Biotec) were used to isolate the monocytes, according to the manufacturer's instructions. Blood-derived monocytes were cultured at a density of 5x10 5 per well in X-Vivo medium (Lonza) supplemented with 100U/ml penicillin/streptomycin (Life Technologies), 1% Glutamax (Life Technologies) and matured into macrophages through continuous culture in 100 ng/ml MCSF (Preprotech) for 6 days.

Quantitative PCR
Cells were lysed and homogenised using RLT buffer (QIAGEN) and total RNA was extracted from cells using an RNeasy minikit (QIAGEN), with an on-column DNase digestion step using RNase-free DNase set (QIAGEN).
Complementary DNAs were generated using the High-Capacity RNA-cDNA kit (Life Technologies), according to the manufacturer's instructions. Quantitative PCR were conducted using TaqMan Universal Mastermix II (ThermoFisher) in the Stratagene Mx3000p qPCR system and MxPro qPCR software. Expression was normalised to GAPDH. The following primers were used (all purchased from Applied Biosystems):

Gene name
Primer ID

Immunocytochemistry
Cells were seeded at 2.5x10 4 cells/well, fixed in 4% paraformaldehyde in PBS, washed 1x with PBS, permeabilised with 0.2% Triton X in PBS for 10 minutes at room temperature (RT), blocked in 5% donkey (TREM2 AF1828 antibody) or 5% goat (all other antibodies) serum in PBS for 1h at RT, and incubated in 1% donkey or goat serum in PBS with primary antibody for 2h at RT. For double-immunofluorescence with anti-TREM2 and Golgin antibodies, cells were blocked with 3% bovine serum albumin (BSA) and incubated with 1% BSA in PBS with primary and secondary antibodies, Cells were washed 3x with PBS and incubated for 1h with Alexa Fluor secondary antibodies in 1% serum PBS. Images were taken using a Leica DM550 microscope and LAS AF software (Leica, V2.6.3). For live cell imaging of pHrodo particles, images were taken using a Zeiss Axioskop 2 microscope and Axiovison software (Zeiss, v4.8).
Confocal images were taken with a Zeiss LSM 710 confocal microscope using Zen software (Zeiss, Version 2012), and images processed with Image J1.5 (www.imagej.nih.gov/ij). The following antibodies were used for in assay buffer (0.25% BSA and 0.05% Tween 20 in PBS (pH=7.4). Plates were again washed three times with wash buffer before incubation for 1h at RT with the detector antibody monoclonal mouse anti-human TREM2 antibody (1µg/ml Santa Cruz Biotechnology; B-3, sc373828). After three additional washing steps, plates were incubated with the secondary antibody (SULFO-TAG-labeled anti-mouse secondary antibody, MSD) and incubated for 1h in the dark.
Lastly, plates were washed three times with wash buffer followed by two washing steps in PBS alone. The electrochemical signal was developed by adding MSD Read buffer (1 in 2) and the light emission measured using the MSD SECTOR Imager 6000. The concentration of sTREM2 was calculated using a five-parameter logistic curve fitting method with the MSD Workbench software package. Intra-assay cvs were < 10%, and all samples were measured on the same day using the same reagents.     Reduction of TREM2 protein (A), TREM2 mRNA (B), and TREM2 secretion (C) using TREM2 siRNA compared with non-targeting siRNA confirmed by WB, qPCR, or sTREM2 ELISA, respectively (n=3 for qPCR, n=2 for WB and sTREM2 ELISA). Phagocytosis of pHrodo zymosan particles in variant TREM2 iPSC-MGLC and controls (D, n=3).
Cytochalasin D was used as negative control. Data are presented as mean ± SEM. Fluorescent Annexin V staining to demonstrate apoptosis of SH-SY5Y cells following UV radiation and further incubation over a 24h period, compared with un-treated cells (E). FACS analysis for Annexin V staining in SH-SY5Y controls (non-heat shocked, yellow filled graph) versus heat-shock treated (green filled graph) (F). Annexin V immunocytochemistry in heat-shocked SH-SY5Y cells imaged by fluorescence microscopy (G).