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Making Sure Microglia Are Microglia

Microglial dysfunction is a factor in neurological disorders like schizophrenia and Alzheimer’s disease. To better understand any pathological contribution of microglia, you need a model that mimics what you see in humans. Currently, these models may come from human induced pluripotent stem cell(hiPSC)-derived microglia. But differentiating hiPSCs into microglia is challenging and often slow, making them unsuitable for high throughput clinical trials. There’s also the issue that induced microglia (iMGs) don’t always behave like microglia do in vivo

However, new work has generated iMGs from monocytes and validated these immunologically, behaviorally, and genetically. The iMGs were generated in under two weeks; a significant reduction from the 40 days it often takes to derive microglia from hiPSCs. The resulting iMGs could have real potential in understanding neurological diseases, such as schizophrenia and Alzheimer’s disease (AD).

A quick introduction to microglia

Microglia – the immune cells of the central nervous system – are essential for normal brain functions. When everything is working as it should, microglia play a role in neurogenesis, synaptic pruning, and phagocytosis when not undertaking immune surveillance. However, when stressed or overstimulated, microglia can become activated and shift to their proinflammatory state (M1). Hyperactive microglia release neurotoxic factors and cytokines (IL-1β, IL-6, TNF-α) and, if activated for too long, can induce neuronal apoptosis and brain damage we see in several neurological and neurodegenerative disorders. 

Microglia that look like microglia

The first step in validating these iMGs was assessing their morphology. Under phase-contrast, iMGs started branching at day 5, and by day 10–14 they looked like typical resting microglia do, with a small cell soma and multiple branches. Induced macrophages (iMacs) also had the typical “fried-egg” shape with enlarged circular cell bodies. 

Turning their attention to microglia-specific markers, the researchers looked at transmembrane protein 119 (TMEM119) and purinergic receptor P2RY12. These are deemed unique microglia markers but are absent from macrophages. Using anti-TMEM119 and Alomone’s Anti-P2Y12 Receptor (extracellular) antibody (#APR-020), it was clear that TMEM119 and P2RY12 markers were expressed by iMG cells but absent from iMacs (Figure 1) as expected. This nicely illustrates how TMEM119 and P2RY12 can be used to distinguish iMG cells from monocyte-derived macrophages.

Figure 1. iMG cells express microglia-specific markers (TMEM119 and P2RY12) and other myeloid-specific markers. Immunocytochemistry shows induced macrophages (iMacs) and induced microglia (iMGs) derived from peripheral monocytes. (A) iMG cells express unique microglial markers TMEM119 (b) and P2RY12 (d) Alomone’s Anti-P2Y12 Receptor (extracellular) antibody (#APR-020),which were absent from iMacs (a,c). (B) Myeloid lineage markers were expressed on both iMacs and iMG cells, such as Iba1 (a,b), PU.1 (c,d), CX3CR1 (e,f), and TREM2 (g,h). Image from Banerjee A, et al. Front Cell Neurosci. 2021;15:629279. doi:10.3389/fncel.2021.629279

Microglia that behave like microglia

But looks can be deceiving: the cells looked like microglia but did they behave like microglia? As immune cells, microglia are phagocytic: they remove dead or dying cells, as well as unfolded proteins, like amyloid beta (Aβ) – a hallmark of AD. 

The team used Aβ42 to mimic what we see in AD patients. With Aβ42 conjugated to a fluorophore, it was clear from both the microscopy and flow cytometry that, after four hours, the iMGs had engulfed the exogenous Aβ42 . This is the first study to show that monocyte-derived microglia can engulf exogenous Aβ proteins. 

Microglia with microglia genetics

Finally, the researchers compared iMG gene expression profiles with expression data from brain-derived microglia. Cluster analysis showed that iMG gene expression closely clusters with brain-resident microglia or hiPSC-induced microglia. 

The iMGs express microglia-specific genes, like those involved in synaptic vesicle regulation, ion transport, and synapse organization as well as several genes from the complement system. These data show that iMGs possess physiologically-relevant gene expression profiles.

Induced microglia are like microglia

The validation work here confirms that these iMGs could serve as a physiologically-relevant model to study human brain-resident microglia. An accessible and scalable model like this could prove to be invaluable for understanding the pathogenesis of psychiatric and neurological disorders.

Reagents to help your research

If you are interested in studying microglia, we have several reagents that could help.