iPSC Microglia: Derivation, Function, and Neuroimmune Applications

Induced pluripotent stem cell (iPSC)-derived microglia have become a crucial tool for studying neuroimmune interactions. They offer a renewable, scalable alternative to primary human microglia, addressing challenges related to donor availability and variability. Their ability to model disease-relevant immune responses makes them highly valuable in neuroscience research.

Advancements in iPSC technology allow researchers to generate microglia-like cells that closely resemble their in vivo counterparts, opening new avenues for investigating neuroinflammatory processes, neurodegenerative diseases, and brain development.

Derivation Pathway

Generating microglia from iPSCs requires replicating embryonic developmental cues that guide these cells toward a microglial lineage. Unlike neurons or astrocytes, which originate from the neuroectoderm, microglia arise from the yolk sac early in embryogenesis and migrate into the brain. Differentiation protocols must mimic primitive hematopoiesis rather than traditional neural induction pathways. By using defined cytokine cocktails and stage-specific culture conditions, researchers direct iPSCs through mesodermal and hematopoietic intermediates before committing them to a microglial fate.

The first phase involves inducing iPSCs toward a mesodermal lineage using bone morphogenetic protein 4 (BMP4) and activin A to promote primitive streak formation. This is followed by vascular endothelial growth factor (VEGF) and stem cell factor (SCF) to drive the emergence of hemogenic endothelium, which gives rise to hematopoietic progenitors. These progenitors are then cultured with interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) to facilitate their transition into yolk sac-like macrophages.

Once macrophage-like characteristics develop, additional cues refine their identity toward microglia. Transforming growth factor-beta (TGF-β), IL-34, and granulocyte-macrophage colony-stimulating factor (GM-CSF) promote the expression of microglia-specific genes such as TMEM119, P2RY12, and CX3CR1. Co-culturing with neural progenitors or exposing them to brain-derived factors like cholesterol and fractalkine enhances their maturation, ensuring they closely resemble primary microglia.

Distinct Cellular Functions

iPSC-derived microglia perform key functions essential for brain homeostasis. They continuously survey the neural environment, extending highly motile processes that detect biochemical changes. This behavior is driven by purinergic signaling through receptors such as P2RY12, allowing them to respond swiftly to ATP and other damage-associated molecular patterns. Time-lapse imaging reveals that microglial processes reorganize within minutes of detecting ATP gradients, underscoring their role as first responders in the central nervous system.

They also contribute to synaptic remodeling by recognizing and engulfing unnecessary synapses, a process mediated by complement system components such as C1q and C3. Microglia selectively eliminate synapses tagged with complement proteins, refining neural circuits. This function is especially active during early brain development but continues throughout life, influencing synaptic plasticity and learning. Dysregulated microglial pruning has been linked to neurodevelopmental disorders like schizophrenia and autism spectrum disorder.

Additionally, iPSC-derived microglia support neuronal survival by secreting brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and TGF-β, which promote neuronal growth and resilience. BDNF, for instance, enhances dendritic spine formation in hippocampal neurons, reinforcing synaptic connectivity and supporting memory-related processes. Microglia also interact with astrocytes, modulating gliotransmission and the extracellular matrix composition, further shaping the neural microenvironment.

Molecular Markers For Identification

Characterizing iPSC-derived microglia requires assessing their molecular signature to confirm identity and functional maturity. Unlike other myeloid cells, microglia exhibit a distinct transcriptional profile shaped by their neural environment. One key marker is TMEM119, a transmembrane protein specific to microglia and absent in peripheral macrophages. Its expression, regulated by microglia-specific transcription factors like Sall1, is consistently upregulated in iPSC-derived microglia upon exposure to neural cues.

P2RY12, a purinergic receptor involved in microglial motility, distinguishes microglia from other macrophage-lineage cells, as its expression diminishes in peripheral immune populations. Single-cell RNA sequencing confirms that iPSC-derived microglia maintain robust P2RY12 expression, mirroring ex vivo human microglia. Functional assays show that these cells respond to purinergic signaling in a manner consistent with primary microglia.

Additional markers such as CX3CR1 and Sall1 further define microglial identity. CX3CR1, the fractalkine receptor, facilitates neuron-microglia communication and is crucial for homeostasis. Sall1 acts as a master regulator of microglial identity, suppressing alternative myeloid differentiation pathways. Loss of Sall1 leads to macrophage-like traits, highlighting its role in maintaining microglial specificity. The presence of these markers in iPSC-derived microglia validates their similarity to primary microglia.

Differences From Primary Microglia

Despite their similarities, iPSC-derived microglia differ from primary microglia in their epigenetic landscape, metabolic state, and environmental conditioning. Primary microglia develop in the brain’s unique milieu, where exposure to neural-derived factors establishes a distinct transcriptional program. iPSC-derived microglia, despite differentiation protocols that mimic embryonic development, often retain residual epigenetic marks from their pluripotent origins, leading to subtle deviations in gene expression.

Metabolic differences also exist. Primary microglia rely on oxidative phosphorylation under homeostatic conditions, optimizing energy production for long-term surveillance. In contrast, iPSC-derived microglia may exhibit a more glycolytic metabolic profile, a trait common in in vitro myeloid differentiation. This metabolic shift can influence cellular behavior and responses to environmental stressors. Adjusting culture conditions, such as incorporating neuron- or astrocyte-derived factors, is being explored to better replicate the brain’s metabolic environment.

Uses In Neuroimmune Research

iPSC-derived microglia have expanded neuroimmune research by providing a model to study neuroinflammation and neurodegenerative diseases. These cells enable patient-specific investigations, allowing researchers to examine microglial responses in individuals with genetic predispositions to conditions like Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. Comparing microglia from healthy and affected individuals helps identify transcriptional and functional changes contributing to disease progression. For example, iPSC-derived microglia from Alzheimer’s patients show altered cytokine profiles and impaired amyloid-beta clearance, mirroring dysfunction observed in postmortem brain tissue.

Beyond disease modeling, these cells serve as platforms for high-throughput drug screening. Traditional drug discovery often relies on rodent microglia, which do not fully replicate the human neuroimmune environment. iPSC-derived models overcome this limitation, providing a human-relevant system for testing therapeutic interventions. Small-molecule inhibitors targeting inflammatory pathways, such as NLRP3 inflammasome modulators, have been screened using iPSC-derived microglia to assess their efficacy in reducing pro-inflammatory responses. This approach accelerates drug development while reducing reliance on animal models.

Interactions In 3D Brain Cultures

Integrating iPSC-derived microglia into three-dimensional (3D) brain cultures provides a physiologically relevant context for studying their interactions with neurons, astrocytes, and the extracellular matrix. Two main approaches have emerged: direct co-culture with brain organoids and engineered microphysiological models that mimic brain complexity. These methods allow microglia to respond to endogenous signals and participate in neural network dynamics.

Brain organoids, derived from iPSCs, self-organize into multicellular structures that recapitulate aspects of cortical development and neural connectivity. Introducing microglia into these organoids enables them to migrate, integrate, and adopt a surveillance phenotype. Studies reveal that microglia influence synaptic density and circuit maturation, shedding light on neurodevelopmental disorders. Additionally, these models facilitate research into microglial responses to pathological stimuli, such as viral infections or toxic protein aggregates.

Microphysiological systems, including microfluidic devices and scaffold-based constructs, offer another avenue for studying microglia in controlled environments. These systems allow precise manipulation of biochemical gradients and mechanical properties, enabling researchers to examine microglial responses to localized injury or metabolic fluctuations. By integrating microglia into vascularized brain-on-a-chip devices, scientists are exploring their interactions with the blood-brain barrier, providing insights into immune cell trafficking and neurovascular dysfunction. These advancements enhance our understanding of neuroimmune dynamics in health and disease.