# Differential Responses of Human iPSC-Derived Microglia to Stimulation with Diverse Inflammogens

## TL;DR

This study uses human iPSC-derived microglia to explore how these brain immune cells respond to various inflammatory triggers, revealing distinct activation patterns and signaling pathways.

## Contribution

The study introduces a robust iPSC-derived microglia model to systematically map inflammatory activation states and signaling pathways in response to diverse stimuli.

## Key findings

- MGLCs showed stimulus-specific cytokine secretion and distinct transcriptome activation signatures.
- NFκB and JAK-STAT pathways were differentially engaged depending on the inflammatory stimulus.
- MGLC supernatants activated astrocytes via a TNFα-dependent mechanism, highlighting intercellular signaling in neuroinflammation.

## Abstract

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However, access to such cells is limited, and studies systematically mapping the spectrum of their inflammatory states are scarce. Here, we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust, accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling, revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli, including pro-inflammatory cytokines (TNFα, interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1), TLR3 (Poly(I:C)), TLR4 (lipopolysaccharide, LPS), and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g., oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally, the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1, LPS, or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism, highlighting the role of soluble mediators in the signal propagation. Altogether, this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation.