Microglia in Focus: Unraveling the Dance with Brain Organoids

An international team of researchers has discovered that microglia, immune cells that act as the brain's defense force, have a significant impact on early human brain development. Scientists were able to replicate the complex environment found in the growing human brain and get insight into the role that microglia play in brain cell growth and development by introducing microglia into lab-grown brain organoids. This work could have a profound effect on our knowledge of brain development and pathologies and marks a major advancement in the creation of human brain organoids.

The introduction of microglia into lab-grown brain organoids allowed scientists to observe their crucial role in eliminating harmful substances and promoting the formation of neural connections. This breakthrough not only enhances our understanding of brain development and disorders but also paves the way for more accurate modeling of human brain function and disease progression in the future. 

Unveiling the Guardians: Exploring the Crucial Role of Microglia in Brain Health and Disease

Microglia are a type of cell located in the brain and spinal cord. They are the primary form of active immune defense in the central nervous system (CNS). Playing a crucial role in brain homeostasis, microglia monitor the health of brain tissue and initiate an immune response if they detect damage or invaders. They constitute 10–15% of all cells found within the brain. As the brain's dedicated immune cells, they are crucial for maintaining the health of neurons. However, when they're not functioning properly, they can contribute to neurodegenerative diseases. Therefore, understanding microglia is key to developing new treatments for a range of neurological disorders.

Microglia are specialized cells that form part of the glial cells in the central nervous system (CNS). They play a pivotal role in immune defense and are considered the brain's resident macrophages. Originating from myeloid progenitor cells, microglia are scattered throughout the brain and spinal cord and account for roughly 10–15% of all cells in the CNS. Microglia are key players in neurodevelopment and neuroplasticity, contributing to synaptic pruning and phagocytosis, processes that are crucial for refining neural circuits and removing dead neurons or unnecessary synapses. They also help in the maturation and survival of neurons. The primary function of microglia is to maintain homeostasis and defend the CNS from injury and disease. They constantly scan their environment for pathogens, damaged neurons, plaques, and infectious agents. Upon detecting these harmful stimuli, microglia quickly change from a 'resting' state to an 'activated' state, where they can produce inflammatory or anti-inflammatory agents to either promote or suppress inflammation, respectively. 

Microglia: Importance for Health

Microglia are extremely important for brain health. When they function properly, they help protect the brain from injury and disease, maintain normal brain functioning, and facilitate recovery from damage. However, when microglia are dysregulated or unhealthy, they can contribute to neurodegenerative diseases and mental health disorders.

Over-activation of microglia, for instance, can lead to chronic inflammation, which is implicated in diseases such as Alzheimer's, Parkinson's, multiple sclerosis, and psychiatric disorders like depression and schizophrenia. Therefore, understanding the precise roles and functions of microglia is crucial for developing effective treatments for a wide range of neurological and psychiatric conditions. Advances in microglial research could potentially lead to breakthroughs in treating these diseases, making microglia an active area of biomedical research.

Decoding Brain Organoids: Microglial Marvels in Early Development

Researchers delved into the complexities of early human brain development in a ground-breaking study that appeared in the November 1, 2023, issue of Nature, highlighting the crucial function that microglia in brain organoids play. The researchers used induced pluripotent stem cells (iPSCs) to add microglia to lab-grown brain organoids. This fills in a big gap in current models. 

Filling the Void: Mini-Brains and Microglia Integration

Crafted by ASTAR's Singapore Immunology Network (SIgN) researchers, "mini-brains" closely resembling human brain development lacked microglia, an essential element in early brain development. ASTAR scientists bridged this gap by ingeniously introducing microglia-like cells, derived from the same human stem cells used for organoid creation.

Proteomic Insights: Deciphering the Language of Brain Organoids

Dr. Radoslaw Sobota and colleagues at A*STAR's Institute of Molecular and Cell Biology delved into the protein composition of organoids using a quantitative proteomics technique. The study uncovered variations in protein and unveiled the critical role of microglia in regulating brain cholesterol levels, vital for neuronal structure and functionality.

Molecular Ballet: Understanding Microglial Relationships

Researchers, armed with brain organoids from A*STAR and the University of Surrey, conducted proteomic and lipidomic analyses. The study illuminated the metabolic cross-talks during brain development, expanding our awareness of microglial functions. This newfound knowledge holds promise for investigating neurodevelopmental issues and potential treatments for brain disorders.

Toward Novel Treatments: The Microglia-Neuron Nexus Explored

The study, which Professor Florent Ginhoux and Professor Jerry Chan co-authored, emphasized the lack of resources for researching how microglia interact with the developing brain. Creating brain organoids from same-donor pluripotent stem cells unveils intricate relationships, potentially paving the way for groundbreaking treatments in the future.

Revolutionizing Brain Organoids: Incorporating Microglia for Enhanced Accuracy and Insight

The researchers have developed a method to create more accurate human brain organoids by incorporating microglia, crucial immune cells, into the model. Traditional organoids lacked these cells, essential for proper brain development. Using induced pluripotent stem cells, the team made organoids that were high in microglia. They saw big changes in neural rosettes, more neuronal progenitor cells, and better axonogenesis and synaptogenesis. The microglia-driven effects were attributed to cholesterol-related processes. This advancement provides a valuable tool for studying the role of microglia in human brain development and understanding communication between microglia and neuronal cells.

Fostering Neurogenesis: iMac Transformation into Microglia-Like Cells Enhances Organoid Functionality

Human induced pluripotent stem cell-derived macrophages (iMac) differentiate into microglia-like cells (iMicro) when introduced into cerebral organoids. This process mirrors the influx of microglial progenitors observed in the developing human brain. The iMicro within organoids exhibit an embryonic microglial phenotype, lacking TMEM119. Some of the things that these iMicro do are typical for microglia, like extending their dendrites to "injure" neurons and removing harmful amyloid-β peptides. Single-cell RNA sequencing shows that gene expression is different in iMac from organoid cocultures. Activity of transcription factor regulons linked to microglial identity is increased during brain development. The presence of iMicro in organoids enhances the electrophysiological maturation of neurons, indicating their role in supporting neurogenesis.

iMicro Influence: Orchestrating Neurogenesis and Axonogenesis in Cerebral Organoids

Introduction of microglia-like cells (iMicro) into cerebral organoids leads to significant changes in organoid growth and cellular composition. Organoids with iMicro exhibit reduced size and smoother, rounder morphology, affecting the total number and proportion of neuronal progenitor cells (NPCs). Single-cell RNA sequencing shows that in co-NPCs, cell proliferation genes are expressed less, which suggests that the cells are shifting their focus to axon development and neurogenesis.

Proteomic analysis confirms elevated expression of neuronal axon filament protein PRPH38 in co-NPCs, accompanied by increased neurite length. iMicro also show higher expression of genes involved in phagocytosis, suggesting their role in reducing NPC proliferative activity and promoting maturation and axonogenesis. When iMicro is present, neural rosettes change shape, which is linked to clear signs of neurogenesis, axonogenesis, and less proliferation. Overall, iMicro play a crucial role in promoting the maturation of neuronal cells and shaping the developmental trajectory of brain organoids.

Lipid Dynamics: iMicro-Mediated Cholesterol Transport and Esters Modulate Neuronal Maturation in Organoids

iMicro play a key role in lipid metabolism, showing redesigned pathways with high expression of genes like ABCA1, ABCG1, and PLIN2 that help move and store cholesterol. PLIN2, a marker of lipid droplets, is highly expressed in both iMac and iMicro. In cocultured organoids, iMicro cells are the ones that show PLIN2+ lipid droplets the most. This suggests a potential role of iMicro in exporting cholesterol to neuronal cells.

Co-NPCs in cocultured organoids show higher levels of neutral lipids, indicating cholesterol transfer. iMac can uptake fluorescent cholesterol and transfer it to co-NPCs during coculture. iMac has high levels of APOE and AIBP, which suggests that APOE+ lipoprotein-like particles (LPLs) carry cholesterol and its esters to neuronal cells. Blocking ABCA1, a cholesterol transporter, stops cholesterol from getting to co-NPCs and makes the organoid smaller. This shows how important iMac/iMicro are for getting cholesterol to neuronal cells in cocultured organoids. 

Microglial Presence and Cholesterol Dynamics in Fetal Brains and Organoid Cocultures

To validate the in vitro findings, the presence of PLIN2+ microglia was investigated in fetal mouse and human brains. Expression datasets of mouse microglia at different developmental stages revealed high expression of genes involved in cholesterol biosynthesis and storage during early brain development. Similarly, lipid droplet-rich microglial cells were detected in mouse embryonic brain and human embryos at 15-21 weeks.

By comparing scRNA-seq data from organoid coculture with human microglia datasets from embryonic and adult stages, we saw that iMicro in organoids are like embryonic microglia, only expressing PLIN2 and ABCA1 transcripts during the early stages of development. This suggests that iMicro recapitulate a key feature of embryonic microglia, acting as a major source of cholesterol stored in lipid droplets for export to neuronal cells. The study emphasizes the potential of the organoid coculture model in understanding early human brain development and the role of microglia in neurogenesis.

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• Novel model of cortical–meningeal organoid co-culture system improves human cortical brain organoid cytoarchitecture - by Jalilian, E. & Shin, S.R. (2023)

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Want to know more about the study's methods? Then keep reading and check out our summary! Let's go.

Methods | Deep Dive

Let's break down the methods the researchers used:

• Reprogramming of Fibroblasts into Induced Pluripotent Stem Cells

  • Skin samples from pediatric patients with high-grade glioma collected during surgical procedures.

  • Fibroblasts cultured in Dulbecco’s Modified Eagle’s medium (DMEM) with specific supplements.

  • Electroporation of fibroblasts using iPS cell reprogramming vector.

  • Subsequent culture in N2B27 medium with cytokines for 15 days.

  • Transition to mTeSR1 medium for long-term maintenance.

• Generation of Induced Macrophages (iMac)

  • iMac derived from human iPS cells through a series of culture steps inducing commitment to primitive streak, mesoderm, hemangioblasts, and myeloid lineage.

  • Differentiation of myeloid lineage through hematopoietic cell commitment.

  • Transition to SF-Diff medium supplemented with CSF-1 for macrophage differentiation.

  • Culture in hypoxia incubator initially and later in a conventional tissue culture incubator.

• Generation of Brain Organoids

  • Human iPS cells dissociated into single cells and seeded into ultra-low attachment plates for embryoid body formation.

  • Transfer to neural induction medium for neuro-ectoderm formation.

  • Embedding into Matrigel for neuroepithelium formation.

  • Culture in cerebral organoid medium with periodic medium changes and shaking for maturation.

• Generation of Microglia-Sufficient Brain Organoids

  • Coculture of iMac (day 26) with brain organoids (day 26) in cerebral organoid medium.

  • Subsequent medium changes and maintenance.

• Coculture of iMac with Cortical Neurons in 2D

  • iMac (200,000) seeded on cortical neurons in six-well plates.

  • Culture in neural maintenance medium with CSF-1.

• Bulk RNA Sequencing in 2D Co-iMac

  • Coculture for 14 days followed by cell dissociation.

  • Sorting of cells using flow cytometry based on specific markers.

  • RNA extraction, quality assessment, and cDNA library preparation.

  • Sequencing using HiSeq 2000.

• Data Analysis

  • Mapping to the Genome Research Consortium human build 38 using STAR aligner.

  • FeatureCounts for summarizing mapped reads to the gene level.

  • Normalization of log2 RPKM values and batch effect correction.

  • Differential expression analysis using Limma.

• Cryo-sectioning

  • Brain organoids and portions of mouse and human brains were washed and fixed in 4% PFA.

  • Organoids/tissues were embedded in a gelatin/sucrose solution and cut into 20-µm-thick slices using a cryostat.

  • Sections were placed onto polysine-coated slides for immunolabeling.

• Immunofluorescence

  • Brain or organoid sections on polysine-coated slides were blocked and labeled with primary antibodies.

  • After washing, sections were labeled with secondary antibodies and DAPI before being embedded in mounting medium.

  • For lipid droplet staining, sections were stained with LipidSpot 610.

  • Images were captured with a confocal laser scanning microscope and analyzed with Imaris Imaging software.

• Staining of E14.5 mouse fetal brain

  • Freshly dissected brains were postfixed and sliced on a vibratome.

  • Sections were blocked, incubated with primary antibodies, and labeled with secondary antibodies.

  • Hoechst was used as a nuclear counterstaining.

  • Images were acquired with a confocal microscope.

• Three-dimensional imaging of brain organoids

  • Organoids were fixed, sequentially washed in different solutions, and incubated with primary antibodies.

  • After washing, secondary antibody labeling was performed.

  • Organoids were imaged using a light sheet Ultramicroscope.

• Imaris analysis of colocalization

  • Spot and surface were created to locate iMac and Ki67+ NPCs in organoids.

  • Colocalization analysis was done using Imaris Imaging software.

• Acquisition and 3D reconstruction

  • Organoids were imaged with a confocal microscope, and 3D reconstructions were performed using Imaris Imaging software.

• Generation of EGFP-expressing iPS cells

  • HEK293T cells were transfected with plasmids for Lentiviral particle generation.

  • Human iPS cells were cultured with lentiviruses, and EGFP expression was confirmed using flow cytometry.

• Laser ablation

  • Organoids cocultured with EGFP-expressing iMac were visualized using laser ablation.

  • Time-lapse videos were generated, and image analysis was performed with Imaris Imaging software.

• Phagocytosis assay

  • Organoids cocultured with EGFP-expressing iMac were incubated with amyloid beta peptides.

  • Live images were acquired, and analysis was performed using Imaris Imaging software.

• Measurement of brain organoid size

  • Images of organoids were captured, and ImageJ software was used to measure the circumference and cross-sectional area.

• Whole-cell patch recording in organoids

  • Whole-cell patch measurements were performed on organoids infected with pAAV-CaMKIIa-EGFP.

• Digestion of brain organoids

  • Brain organoids were digested, and the cell suspension was collected for downstream analysis.

• Proteomic Analysis

  • Brain organoids cultured with or without iMac for 18 days.

  • Sorted into NPC, neuron, co-NPC, co-neuron, and iMicro populations using BD FACSARIA II/III.

  • Proteomic analysis conducted on three independent samples for each group.

  • Cells lysed, reduced, alkylated, and digested before analysis.

  • Peptides separated and analyzed using liquid chromatography and mass spectrometry.

  • MaxQuant software used for data analysis, searching against the Human Uniprot database.

  • Label-free quantification (LFQ) performed for protein quantification.

  • EdgeR used for data analysis between NPCs and co-NPCs.
    Lipidomic Analysis:

  • Brain organoids cultured with or without iMac for 18 days; culture media collected.

  • Lipid extraction from culture media using methyl tert-butyl ether/methanol.

  • Liquid chromatography and mass spectrometry used for lipid analysis.

  • Twenty-five lipid classes assessed, including CE, COH, DG, TG, PC, PE, PG, PI, and PS.

• Neural Rosettes Proteomics

  • Neural rosettes microdissected from formalin-fixed, paraffin-embedded sections.

  • Protein extraction and digestion performed.

  • Liquid chromatography and mass spectrometry used for protein analysis.

  • Proteins identified using Mascot algorithm and analyzed in Proteome Discoverer 2.5.

  • Label-free quantification of MS1 level for protein quantification.

• General Analysis

  • Statistical analysis performed using GraphPad Prism.

  • Ethical approval obtained for the use of human fetal tissues.

  • Patient-derived iPS cells used in the study.

  • C57BL/6 mice used for experiments, approved by the Institutional Animal Care and Use Committee.