Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer’s disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

BackgroundMicroglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD), taking on distinct transcriptional and functional states. While the ability to profile complex tissues at single‐cell resolution in postmortem brain tissue provides insight into disease‐associated cellular states within the brain, more is required to understand how these changes modulate disease pathogenesis. Building on foundational transcriptomics, the advances in multi‐modal profiling of genomics and proteomics allows for a greater understanding of neuroimmune dysfunction in neurodegenerative disease.MethodTranscriptomic datasets are often confounded by variability in collection and sequencing methodologies. We have optimized tissue dissociation and cell isolation protocols to avoid many of the pitfalls commonly found in bulk and single‐cell transcriptomic studies. Using these optimized protocols, we have begun assessing paired samples (blood, brain biopsy, and cerebrospinal fluid (CSF), from patients at risk for AD to understand neuroimmune changes in early‐phases of AD. We have also characterized the transcriptional and functional responses of human induced pluripotent stem cell (iPSC) microglia in response to a variety of neurodegenerative brain‐relevant challenges, including amyloid, apoptotic neurons and synaptic debris.ResultUsing scRNA‐seq on fresh mouse and human tissue we have identified a dissociation‐induced signature in microglia that is highly prevalent in current literature, and developed an experimental methodology to prevent this artifact. We have also identified a similar signature in post‐mortem tissue via snRNA‐seq that may be the result of acute‐pre/post‐mortem processes (Marsh et al., 2022). Powerfully, we also have cerebrospinal fluid (CSF) from the same patients, allowing us to correlate proteomic and transcriptomic analyses to determine the connections between disease‐associated transcriptomic cell states and analyte biomarkers. We can then use the iPSC microglia (iMGLs) to understand how altered transcriptomic states and biomarker profiles may alter microglial functions to elucidate mechanisms by which microglia contribute to disease pathogenesis.ConclusionOptimization of experimental and analysis methods along with extensive multi‐modal profiling of the same patients will lead to greater understanding of the neuroimmune landscape of neurodegenerative disease to better enable development of novel predictive biomarkers and therapeutic targets.