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iCell Microglia AD (TREM2)

TREM2 Variants with an Isogenic Control for AD Modeling

Description

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.

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.

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.

Figure 2. Engineered Variants of 01279 and the Predicted Amino Acid Sequence for the TREM2 Exon 2 Domain

Engineered TREM2 Models

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 (Fig. 1 & 2) was engineered in iPSCs from an otherwise apparently healthy, normal donor background, 01279.

Figure 3.  Comparison of Marker Expression in iCell Microglia and the Engineered Variants

Characterization

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. 3).

Phagocytosis
In Figure 4, 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).

Phagocytosis comparison of iCell Microglia and TREM2 Engineered Mutants

Figure 4. Phagocytosis of iCell Microglia and TREM2 Variants

TREM2 Expression Levels
Soluble TREM2 levels were quantified from the iCell Microglia control and TREM2 variants using a SimpleStep ELISA (Fig. 5A). 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. 5B).

Figure 5. Measurement of TREM2 Protein by ELISA and Western Blot

Cytokine Response
The cytokine response of stimulated iCell Microglia and TREM2 variants were compared by multiplex assay (Fig. 6). 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 6. Cytokine Comparison of iCell Microglia and TREM2 Variants

  • Ordering InformationFor best pricing please log in or register
    Catalog # Description Quantity Price
    R1202 iCell Microglia Kit, TREM2 (HO), 01279

    Each kit contains:

    • C1136 Cryopreserved iCell Microglia TREM2 (HO), 01279
    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $2275
    Inquire About Pricing & Availability
    C1136 iCell Microglia, TREM2 (HO), 01279

    Product contains:

    • C1136 Cryopreserved iCell Microglia TREM2 (HO), 01279
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1975
    Inquire About Pricing & Availability
    R1201 iCell Microglia Kit, TREM2 (HT), 01279

    Each kit contains:

    • C1134 Cryopreserved iCell Microglia TREM2 (HT), 01279
    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $2275
    Inquire About Pricing & Availability
    C1134 iCell Microglia, TREM2 (HT), 01279

    Product contains:

    • Cryopreserved iCell Microglia TREM2 (HT), 01279
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1975
    Inquire About Pricing & Availability
    R1131 iCell Microglia Kit, 01279

    Each kit contains:

    • C1110 Cryopreserved iCell Microglia, 01279
    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1650
    Inquire About Pricing & Availability
    C1110 iCell Microglia, 01279

    Product contains:

    • C1110 Cryopreserved iCell Microglia, 01279
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1350
    Inquire About Pricing & Availability
    R1204 iCell Microglia Media Kit

    Each kit contains:

    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    1 Kit $365
    Inquire About Pricing & Availability
    019-19741 Anti IBA1 Polyclonal Antibody
    • Rabbit polyclonal IgG
    • 50μg supplied at 0.5mg/ml in TBS
    • Protocol included
    • Store at -20° C
    50ug $355
    Inquire About Pricing & Availability