@article{11478,
  abstract     = {Cerebral organoids differentiated from human-induced pluripotent stem cells (hiPSC) provide a unique opportunity to investigate brain development. However, organoids usually lack microglia, brain-resident immune cells, which are present in the early embryonic brain and participate in neuronal circuit development. Here, we find IBA1+ microglia-like cells alongside retinal cups between week 3 and 4 in 2.5D culture with an unguided retinal organoid differentiation protocol. Microglia do not infiltrate the neuroectoderm and instead enrich within non-pigmented, 3D-cystic compartments that develop in parallel to the 3D-retinal organoids. When we guide the retinal organoid differentiation with low-dosed BMP4, we prevent cup development and enhance microglia and 3D-cysts formation. Mass spectrometry identifies these 3D-cysts to express mesenchymal and epithelial markers. We confirmed this microglia-preferred environment also within the unguided protocol, providing insight into microglial behavior and migration and offer a model to study how they enter and distribute within the human brain.},
  author       = {Bartalska, Katarina and Hübschmann, Verena and Korkut, Medina and Cubero, Ryan J and Venturino, Alessandro and Rössler, Karl and Czech, Thomas and Siegert, Sandra},
  issn         = {2589-0042},
  journal      = {iScience},
  number       = {7},
  publisher    = {Elsevier},
  title        = {{A systematic characterization of microglia-like cell occurrence during retinal organoid differentiation}},
  doi          = {10.1016/j.isci.2022.104580},
  volume       = {25},
  year         = {2022},
}

@article{12117,
  abstract     = {To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia.
For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1},
  author       = {Hübschmann, Verena and Korkut, Medina and Siegert, Sandra},
  issn         = {2666-1667},
  journal      = {STAR Protocols},
  keywords     = {General Immunology and Microbiology, General Biochemistry, Genetics and Molecular Biology, General Neuroscience},
  number       = {4},
  publisher    = {Elsevier},
  title        = {{Assessing human iPSC-derived microglia identity and function by immunostaining, phagocytosis, calcium activity, and inflammation assay}},
  doi          = {10.1016/j.xpro.2022.101866},
  volume       = {3},
  year         = {2022},
}

@article{12244,
  abstract     = {Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes and overcomes feature-selection biases and biological variability. We extract spatially heterogeneous and sexually dimorphic morphological phenotypes for seven adult mouse brain regions. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration mouse models, with females showing a faster morphological shift in affected brain regions. Remarkably, microglia morphologies reflect an adaptation upon repeated exposure to ketamine anesthesia and do not recover to control morphologies. Finally, we demonstrate that both long primary processes and short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs opens a new perspective to characterize microglial morphology.},
  author       = {Colombo, Gloria and Cubero, Ryan J and Kanari, Lida and Venturino, Alessandro and Schulz, Rouven and Scolamiero, Martina and Agerberg, Jens and Mathys, Hansruedi and Tsai, Li-Huei and Chachólski, Wojciech and Hess, Kathryn and Siegert, Sandra},
  issn         = {1546-1726},
  journal      = {Nature Neuroscience},
  keywords     = {General Neuroscience},
  number       = {10},
  pages        = {1379--1393},
  publisher    = {Springer Nature},
  title        = {{A tool for mapping microglial morphology, morphOMICs, reveals brain-region and sex-dependent phenotypes}},
  doi          = {10.1038/s41593-022-01167-6},
  volume       = {25},
  year         = {2022},
}

@article{9642,
  abstract     = {Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.},
  author       = {Venturino, Alessandro and Schulz, Rouven and De Jesús-Cortés, Héctor and Maes, Margaret E and Nagy, Balint and Reilly-Andújar, Francis and Colombo, Gloria and Cubero, Ryan J and Schoot Uiterkamp, Florianne E and Bear, Mark F. and Siegert, Sandra},
  issn         = {22111247},
  journal      = {Cell Reports},
  number       = {1},
  publisher    = {Elsevier},
  title        = {{Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain}},
  doi          = {10.1016/j.celrep.2021.109313},
  volume       = {36},
  year         = {2021},
}

@article{10565,
  abstract     = {Enzymatic digestion of the extracellular matrix with chondroitinase-ABC reinstates juvenile-like plasticity in the adult cortex as it also disassembles the perineuronal nets (PNNs). The disadvantage of the enzyme is that it must be applied intracerebrally and it degrades the ECM for several weeks. Here, we provide two minimally invasive and transient protocols for microglia-enabled PNN disassembly in mouse cortex: repeated treatment with ketamine-xylazine-acepromazine (KXA) anesthesia and 60-Hz light entrainment. We also discuss how to analyze PNNs within microglial endosomes-lysosomes. For complete details on the use and execution of this protocol, please refer to Venturino et al. (2021).},
  author       = {Venturino, Alessandro and Siegert, Sandra},
  issn         = {2666-1667},
  journal      = {STAR Protocols},
  number       = {4},
  publisher    = {Elsevier ; Cell Press},
  title        = {{Minimally invasive protocols and quantification for microglia-mediated perineuronal net disassembly in mouse brain}},
  doi          = {10.1016/j.xpro.2021.101012},
  volume       = {2},
  year         = {2021},
}

@article{10655,
  abstract     = {Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.
},
  author       = {Maes, Margaret E and Wögenstein, Gabriele M. and Colombo, Gloria and Casado Polanco, Raquel and Siegert, Sandra},
  issn         = {2329-0501},
  journal      = {Molecular Therapy - Methods and Clinical Development},
  pages        = {210--224},
  publisher    = {Elsevier},
  title        = {{Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment}},
  doi          = {10.1016/j.omtm.2021.09.006},
  volume       = {23},
  year         = {2021},
}

@article{6521,
  abstract     = {Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.},
  author       = {Maes, Margaret E and Colombo, Gloria and Schulz, Rouven and Siegert, Sandra},
  issn         = {0304-3940},
  journal      = {Neuroscience Letters},
  publisher    = {Elsevier},
  title        = {{Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges}},
  doi          = {10.1016/j.neulet.2019.134310},
  volume       = {707},
  year         = {2019},
}