🧫 Experiment Protocol
In VitroAlzheimer's diseaseTREM2Human iPSC-derived astrocytes + microglia, TREM2 R47H heterozygous vs isogenic WT (3 donor lines x 2 genotypes)completed
Use human iPSC-derived astrocyte-microglia co-cultures from TREM2 R47H heterozygous carriers vs isogenic controls to quantify amplified A1 astrocyte polarisation, then screen a focused panel of 24 anti-neuroinflammatory compounds for rescue of the A1 phenotype.
PRIMARY OUTCOME
Rescue of A1 astrocyte signature (C3, LCN2, H2-D1 protein) by >=50% vs R47H vehicle
EXPECTED OUTCOMES
TREM2 R47H co-cultures will show >=2x increase in C3/LCN2 vs isogenic WT. >=3 compounds will rescue A1 markers by >=50% without >20% LDH release.
SUCCESS CRITERIA
Primary: >=2 confirmed hits (>=50% C3 reduction, IC50 <10 uM, <20% LDH). Secondary: reproducibility across all 3 donor lines (CV <25%).
PROTOCOL
1. Differentiate iPSC lines (3 R47H donors + isogenic WT) to astrocytes (42-day protocol) and microglia (28-day protocol). 2. Co-culture (1:3 microglia:astrocyte) in serum-free medium; stimulate with Abeta42 oligomers (1 uM, 48 h) to induce cross-talk. 3. Multiplex ELISA (C3, LCN2, S100B) and ICC (GFAP, C3) as baseline readout. 4. Screen 24 compounds (TREM2 agonists, nSMase2 inhibitors, JAK inhibitors) at 3 doses (8-point). 5. Endpoint: C3 protein ELISA; secondary LDH cytotoxicity, microglial phagocytosis assay. 6. Hit confirmation: IC50 determination, selectivity panel (C3 vs GFAP vs LDH).
Source: task:a989715e-c687-4558-91ca-74fce1474bd2