The authors thank Michael Ward for providing the WTC11 hNIL iPSC line for motor neuron differentiation and the Jackson Laboratories for supplying the KOLF2.1J WT clone B03 iPSC line used for microglia differentiation. We also thank Dorothy Schafer for her support during the implementation of the protocols, Anthony Giampetruzzi and John Landers for their help with the live-cell imaging system as well as Hayden Gadd for his technical contributions during revisions and Jonathan Jung for his collaboration in this study. This work was supported by the Dan and Diane Riccio Fund for Neuroscience from UMASS Chan Medical School and the Angel Fund, Inc.

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The authors have no conflicts of interest to declare.

The differentiation protocol described here provides an efficient method to obtain iPSC-derived microglia-like cells in ~6-8 weeks with high purity and in a sufficient yield to perform immunofluorescence experiments and other assays that require a higher number of cells. This protocol has yielded up to 1 &#215; 106 iMGs in 1 week, which allows for protein and RNA extraction and corresponding downstream analyses (e.g., RNASeq, qRT-PCR, western blot, mass spectrometry). That said, a limitation of this protocol is that the yield of iMGs may restrict certain applications. Additional modifications can be implemented to accumulate PMPs from different weeks and maintain a homogeneous culture in suspension until enough progenitors are collected to proceed with the differentiation process at once18. Alternatively, the differentiation of EBs can be scaled up by using higher area cell-culture plates to increase the production of PMPs.
It is advised to use iPSCs cultured in laminin 521-coated plates to initiate the differentiation process. Other coating reagents can reduce the number of PMPs produced later in the process18. Similarly, coating the plates for EB adhesion during PMP generation improves EB attachment, thus prolonging the life of the culture. EB culture has been maintained for up to 4 months, at which point, multiple EBs detach and die. However, other researchers report culturing EBs for up to 1 year12. In our hands, iMGs generated from 3-month-old EB cultures maintain similar functional properties as iMGs differentiated from young cultures; cultures older than 3 months have not been used for functional experimentation.
To optimize the final stage of microglia differentiation from the original protocols described above7,8, we included 100 ng/mL of interleukin (IL)-34,7 5 ng/mL of colony stimulation factor 1 (M-CSF)10, and 50 ng/mL of transforming growth factor-beta (TGF-&#946;) in the iMG medium. IL-34 and M-CSF stimulate CSF1 receptor-dependent pathways that are crucial for maintaining microglia fate and survival8 while TGF-&#946; supports microglia homeostasis9, thus promoting a more physiological environment for PMP commitment to microglia fate. Of note, the entire differentiation process described here is performed in serum-free conditions, which prevents possible effects of serum on iMG behavior and alterations in downstream applications. For functional experimentation with iMGs, it is recommended to conduct experiments between 10 and 12 days of terminal differentiation. iMGs have been cultured for up to 14 days with a modest reduction of cell viability, after which point, cell health is significantly compromised.
Since one of the canonical functions of microglia is phagocytosis, an assay to investigate phagocytic activity in vitro was included in this protocol.&#160;To develop a human-cell-based system, synaptosomes derived from human i3LMNs were used. The quality of the synaptosome preparation was assessed by western blot analysis of synaptic markers in this study. Additionally, other structural and functional properties of the synaptosome preparation can be investigated by electron microscopy or immunostaining before the phagocytosis assay. The protocol described here to obtain human synaptosomes heavily relies on i3LMNs, which yield a relatively high number of neurons. However, neuronal yields will likely be lower when using other differentiation methods (i.e., protocols requiring cocktails of small molecules). Notably, the phagocytosis assay can be performed with multiple other substrates such as mouse-brain derived synaptosomes, aggregated proteins, or dead cells, all of which can be labeled with pH-sensitive dyes to monitor phagocytosis in a similar manner. It is proposed to use pH-sensitive dyes because the fluorescent signal is mainly emitted when the synaptosomes are localized within acid compartments during phagosome processing and degradation in the lysosomes21. This property reduces the background signal from synaptosomes outside the cells and facilitates the use of automated methods of quantification. Additionally, pH-sensitive dyes allow for the detection of true phagocytosis events as opposed to substrate adherence or docking onto the cell membrane.
While herein it is suggested to use ~35 &#181;g of synaptosomes per well, the specific amount of human synaptosomes and other substrates should be optimized, as optimal conditions depend on the iPSC line and the experimental conditions. An ideal range of synaptosomes should result in a dose-response in the phagocytosis assay. Too much synaptosomes can saturate the system, making it difficult to detect meaningful differences as a function of genotype or treatment group. Too little material will result in a weak output signal in the assay. For this reason, it is advised to test multiple concentrations of each substrate to establish a working assay. Additionally, it is important to maintain the % DMSO within a safe range to preserve cell health.
In this protocol, phagocytosis was monitored using a live-cell imaging system (Table of Materials) because it provides kinetic information about the engulfment capacity of the cells. Microscopes and imaging systems that can support live-cell imaging and maintain stable temperature, humidity and CO2 concentrations are also suitable for this assay. All the phagocytosis experiments described here were imaged at 37 &#176;C with 85%-95% humidity and 5% CO2. Other detection methods such as imaging of fixed cells or flow cytometry can also be used to assess phagocytosis using the same materials and cell culture protocols (up to and including the generation of iMGs and labeled synptosomes) when live-cell imaging is not feasible or desired. Similarly, other open-source software can be utilized for data analysis such as ImageJ or CellProfiler; the latter will allow for an automated analysis comparable to the software used here.
In summary, implementation of a robust protocol to differentiate microglia-like cells from human iPSCs is illustrated, thus overcoming the limited resource of human microglia. Differentiated microglia can be used for a variety of applications in the study of neurodevelopmental and neurodegenerative diseases. Moreover, a fully &#34;humanized&#34; phagocytosis assay is included, which will further aid the study of microglia function and dysfunction in the context of human development and disease.

Microglia are the resident immune cells in the central nervous system (CNS) and play a crucial role in developing the CNS. Microglia are also important in the adult brain for maintaining homeostasis and actively responding to trauma and disease processes. Cumulative evidence shows that microglia are key contributors to the pathogenesis of multiple neurodevelopmental and neurodegenerative diseases1,2. Although current knowledge about microglial biology has been predominately derived from mouse models, recent studies have elucidated important differences between murine and human microglia, underscoring the need for developing technologies to study the genetics and biological functions of human microglia3,4. Isolation of microglia from dissected primary tissue can severely modify microglia properties5, potentially confounding results acquired with such cells. The overall goal of this method is to differentiate human iPSCs into iMGs, thereby providing a cell culture system to study human microglia under basal conditions. Furthermore, a phagocytosis assay using a fully human model system is included herein as a means to study the functionality of iMGs, both as a quality control measure and to assess iMG dysfunction in the context of disease.
Multiple protocols for microglia differentiation from iPSCs have recently emerged in the literature6,7,8,9,10. Potential disadvantages of some protocols include extended or long periods of differentiation, the addition of multiple growth factors, and/or complex experimental procedures6,9,10. Here, a &#34;user-friendly&#34; differentiation method is demonstrated that recapitulates aspects of microglia ontogeny through differentiation of iPSCs into precursor cells termed primitive macrophage precursors (PMPs)7,11. PMPs are generated as described previously, with some optimizations presented herein12. The PMPs mimic MYB-independent yolk-sac-derived macrophages, which give rise to microglia during embryonic development by invading the brain before blood-brain barrier closure13. To terminally differentiate PMPs into iMGs, we used a fast and simplified monoculture method based on protocols by Haenseler et al. and Brownjohn et al., with some modifications to generate an efficient microglia differentiation method in which iMGs robustly express microglia-enriched markers7,8. This differentiation method can be reproduced in laboratories with expertise in the culture of iPSCs and with research goals aiming to study microglia biology using a human model system.
iPSC-derived microglia represent a biologically relevant source of human microglia for in vitro experimentation and are an important tool to investigate microglial canonical functions, including phagocytosis. Microglia are the professional phagocytes of the brain and CNS, where they clear cell debris, aggregated proteins, and degraded myelin14. Microglia also function in synaptic remodeling by engulfing synapses and in the defense against external infections through phagocytosis of pathogens15,16. In this protocol, phagocytosis by iMGs is assessed using human synaptosomes as material for iMG engulfment. To this end, a description for isolating synaptosomes derived from human i3LMNs is described. The i3LMN-derived human synaptosomes are labeled with a pH-sensitive dye that allows for quantification of synaptosomes localized within acidic compartments during phagosome processing and degradation in vitro. A phagocytosis assay using live-cell microscopy is shown for monitoring the dynamic process of microglia engulfment in real-time. This functional assay establishes a basis to investigate possible defects in microglial phagocytosis in health and disease using a complete human system.

<table><tbody><tr><td>&lt;strong&gt;Antibodies for immunofluorescence analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-IBA1 rabbit antibody</td><td>Wako Chemical USA</td><td>NC9288364</td><td>1:350 dilution</td></tr><tr><td>anti-P2RY12 rabbit antibody</td><td>Sigma-Aldrich</td><td>HPA014518</td><td>1:50 dilution</td></tr><tr><td>anti-TMEM119 rabbit antibody</td><td>Sigma-Aldrich</td><td>HPA051870</td><td>1:100 dilution</td></tr><tr><td>&lt;strong&gt;Antibodies for Western blot analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-&amp;beta;-Tubulin rabbit antibody</td><td>Abcam</td><td>ab6046</td><td>1:500 dilution</td></tr><tr><td>anti-Synaptophysin (SYP) rabbit antibody</td><td>Abclonal</td><td>A6344</td><td>1:1,000 dilution</td></tr><tr><td>anti-PSD95 mouse antibody</td><td>Millipore</td><td>MAB1596</td><td>1:500 dilution</td></tr><tr><td>&lt;strong&gt;Borate buffer components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Boric acid (100 mM)</td><td>Sigma</td><td>B6768</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;) BioXtra</td><td>Sigma-Aldrich</td><td>S6297-250G</td><td></td></tr><tr><td>Sodium chloride (75 mM)</td><td>Sigma</td><td>&amp;nbsp;S7653</td><td></td></tr><tr><td>Sodium tetraborate (25 mM)</td><td>Sigma</td><td>221732</td><td></td></tr><tr><td>&lt;strong&gt;Cell culture materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6-well plates</td><td>Greiner Bio-One</td><td>657160</td><td></td></tr><tr><td>40 &amp;mu;m Cell Strainers</td><td>&amp;nbsp;Falcon</td><td>352340</td><td></td></tr><tr><td>100 mm x 20 mm Tissue Culture Treated</td><td>CELLTREAT</td><td>229620</td><td></td></tr><tr><td>Cell Lifter, Double End, Flat Blade &amp;amp; Narrow Blade, Sterile</td><td>CELLTREAT</td><td>229305</td><td></td></tr><tr><td>low adherence round-bottom 96-well plate</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>Primaria 24-well Flat Bottom Surface Modified Multiwell Cell Culture Plate</td><td>Corning</td><td>353847,</td><td></td></tr><tr><td>Primaria 6-well Cell Clear Flat Bottom Surface-Modified Multiwell Culture Plate</td><td>Corning</td><td>353846</td><td></td></tr><tr><td>Primaria 96-well Clear Flat Bottom Microplate</td><td>Corning</td><td>353872</td><td></td></tr><tr><td>&lt;strong&gt;Cell dissociation reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>&amp;nbsp;Corning</td><td>25058CI</td><td>dissociation reagents used for lower motor neuron differentiation</td></tr><tr><td>TrypLE reagent</td><td>Life Technologies</td><td>12-605-010</td><td>dissociation reagents used for microglia differentiation</td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>&lt;strong&gt;Coating reagents for cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matrigel GFR Membrane Matrix</td><td>Corning&amp;trade;</td><td>354230</td><td>Referred as to extracellular matrix coating reagent</td></tr><tr><td>CellAdhere Laminin-521</td><td>STEMCELL Technology</td><td>77004</td><td>Referred as to laminin 521</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P7405</td><td>Reconstitute to 0.1 mg/mL in borate buffer</td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma</td><td>&amp;nbsp;P3655</td><td>Reconstitute to 1 mg/mL in borate buffer</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;iPSC media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;mTeSR Plus Kit</td><td>STEMCELL Technology</td><td>100-0276</td><td>To prepare &lt;em&gt;iPSC media &lt;/em&gt;mixed the components to 1x</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;EB media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BMP-4</td><td>Fisher Scientific</td><td>PHC9534</td><td>final concentration 50 ng/mL</td></tr><tr><td>&lt;em&gt;iPSC media&lt;/em&gt;</td><td></td><td></td><td>final concentration 1x</td></tr><tr><td>ROCK inhibitor Y27632</td><td>Fisher Scientific</td><td>BD 562822</td><td>final concentration 10 &amp;micro;M</td></tr><tr><td>SCF</td><td>PeproTech</td><td>300-07</td><td>final concentration 20 ng/mL</td></tr><tr><td>VEGF</td><td>PeproTech</td><td>100-20A</td><td>final concentration 50 ng/mL</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;PMP base media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>final concentration 1x</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td>final concentration 100 U/mL</td></tr><tr><td>X-VIVO 15</td><td>Lonza</td><td>12001-988</td><td>final concentration 1x</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;PMP complete media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>55 mM 2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>final concentration 55 &amp;micro;M</td></tr><tr><td>IL-3</td><td>PeproTech</td><td>200-03</td><td>final concentration 25 ng/mL</td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td>final concentration 100 ng/mL</td></tr><tr><td>&lt;em&gt;PMP base media&lt;/em&gt;</td><td></td><td></td><td>final concentration 1x</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;iMG base media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634010</td><td>final concentration 1x</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>final concentration 1x</td></tr><tr><td>N2 supplement, 100x</td><td>Gibco</td><td>17502-048</td><td>final concentration 1x</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td>final concentration 100 U/mL</td></tr><tr><td>&lt;strong&gt;Components of &lt;em&gt;iMG complete media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>55 mM 2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>final concentration 55 &amp;micro;M</td></tr><tr><td>IL-34</td><td>PeproTech or Biologend</td><td>200-34 or 577904</td><td>final concentration 100 ng/mL</td></tr><tr><td>&lt;em&gt;iMG base media&lt;/em&gt;</td><td></td><td></td><td>final concentration 1x</td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td>final concentration 5 ng/mL</td></tr><tr><td>TGF-&amp;beta;</td><td>PeproTech</td><td>100-21</td><td>final concentration 50 ng/mL</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Components of Induction base media&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 with HEPES</td><td>Gibco</td><td>11330032</td><td>final concentration 1x</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>final concentration 1x</td></tr><tr><td>N2 supplement, 100x</td><td>Gibco</td><td>17502-048</td><td>final concentration 1x</td></tr><tr><td>Non-essential amino acids (NEAA), 100x</td><td>Gibco</td><td>11140050</td><td>final concentration 1x</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Components of Complete induction media&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Compound E</td><td>Calbiochem</td><td>565790</td><td>final concentration 0.2 &amp;mu;M and reconstitute stock reagent to 2 mM in 1:1 ethanol and DMSO</td></tr><tr><td>Doxycycline</td><td>Sigma</td><td>D9891</td><td>final concentration 2 &amp;mu;g/mL and reconstitute stock reagent to 2 mg/mL in DPBS</td></tr><tr><td>&lt;em&gt;Induction base media&lt;/em&gt;</td><td></td><td></td><td>final concentration 1x</td></tr><tr><td>ROCK inhibitor Y27632</td><td>Fisher Scientific</td><td>BD 562822</td><td>final concentration 10 &amp;mu;M</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Components of Neuron media&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>B-27 Plus Neuronal Culture System</td><td>Gibco</td><td>A3653401</td><td>final concentration 1x for media and suplemment</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>final concentration 1x</td></tr><tr><td>N2 supplement, 100x</td><td>Gibco</td><td>17502-048</td><td>final concentration 1x</td></tr><tr><td>Non-essential amino acids (NEAA), 100x</td><td>Gibco</td><td>11140050</td><td>final concentration 1x</td></tr><tr><td>&lt;strong&gt;iPSC lines used in this study&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KOLF2.1J: WT clone B03</td><td>The Jackson Laboratories</td><td></td><td></td></tr><tr><td>WTC11 hNIL</td><td>National Institute of Health</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Synaptosome isolation reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Scientific Pierce</td><td>23227</td><td></td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Syn-PER Synaptic Protein Extraction Reagent</td><td>Thermo Scientific</td><td>87793</td><td>Referred as to cell lysis reagent for isolation of synaptosomes</td></tr><tr><td>&lt;strong&gt;Phagocytosis assay dyes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NucBlue Live Ready reagent</td><td>Invitrogen</td><td>&amp;nbsp;R37605</td><td></td></tr><tr><td>pHrodo Red, succinimidyl ester</td><td>ThermoFisher Scientific</td><td>&amp;nbsp;P36600</td><td>Referred as to pH-sensitive dye</td></tr><tr><td>&lt;strong&gt;Other cell-culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypan Blue, 0.4% Solution</td><td>AMRESCO INC</td><td>K940-100ML</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>22144-77-0</td><td></td></tr><tr><td>BrdU</td><td>Sigma</td><td>B9285</td><td>Reconstitute to 40 mM in sterile water</td></tr><tr><td>Cytochalasin D</td><td>Sigma</td><td></td><td>final concentration 10 &amp;micro;M</td></tr><tr><td>DPBS with Calcium and magnesium</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>DPBS without calcium and magnesium</td><td>Corning</td><td>21-031-CV</td><td>Referred as to DPBS</td></tr><tr><td>KnockOut&amp;nbsp; DMEM/F-12</td><td>Gibco</td><td>12660012</td><td>Referred as to DMEM-F12 optimized for growth of human embryonic and induced pluripotent stem cells</td></tr><tr><td>Laminin Mouse Protein, Natural</td><td>Gibco</td><td>23017015</td><td>Referred as to laminin</td></tr><tr><td>&lt;strong&gt;Software and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model 5810R</td><td></td></tr><tr><td>Cytation 5 live cell imaging reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 Microplate Reader and Imager Software</td><td>Biotek</td><td>version 3.03</td><td></td></tr><tr><td>Multi-Therm Heat-Shake</td><td>Benchmark</td><td></td><td>refer as tube shaker</td></tr><tr><td>Water sonicator</td><td>Elma</td><td>Mode Transsonic 310</td><td></td></tr></tbody></table>

human microglia-like cells: differentiation from induced pluripotent stem cells and in vitro live-cell phagocytosis assay using human synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

To generate iMGs using this protocol, it is important to start with undifferentiated iPSCs that show compact colony morphology with well-defined edges (Figure 2A). Dissociated iPSCs maintained as described in the EB formation section will form spherical aggregates, termed EBs, which will grow in size until day 4 of differentiation (Figure 2B). Once the EBs are collected and plated in the appropriate conditions for PMP generation, they attach to the Matrigel-coated plates, and a layer of cells will spread and surround the spherical aggregates (Figure 2C). Unhealthy EBs will detach from the plate and should be eliminated. From day 14 to 21 of PMP generation, potentially different cell types can emerge and will float in the medium; these cells should be discarded during media changes12. On day 28, round cells with a large cytoplasm-to-nucleus ratio will appear in suspension (Figure 2D), termed PMPs. These cells are produced continuously and can be harvested weekly during medium changes for up to 3 months. The number of PMPs harvested every week varies and depends on the iPSC line and the age of the culture; the yield generally declines with time. It is strongly recommended to culture iPSCs in laminin 521-coated plates instead of Matrigel, as the PMP yield is higher when iPSCs are maintained in the former conditions as reported previously and in this study (Figure 2E)18.
After PMPs have been exposed to iMG medium for 10-12 days, the cells acquire a microglia-like-morphology with a small cytoplasm and the presence of elongated processes (Figure 3A). To verify microglia identity, we performed immunofluorescence staining with antibodies against the myeloid marker ionized calcium-binding adapter molecule 1 (IBA1) and the microglia-enriched markers purinergic receptor P2RY12 and transmembrane protein 119 (TMEM119) as described previously20 (also see the Table of Materials). Typically, &#62;95% of the cells express IBA1 and approximately 90% of the cells express P2RY12 and TMEM119 (Figure 3B).
To assess the phagocytic capacity of iMGs, i3LMN-derived human synaptosomes labeled with a pH-sensitive dye were used. To verify that the synaptosome preparation retains canonical structural properties, the enrichment of the presynaptic marker, synaptophysin (SYP), and the postsynaptic marker, postsynaptic density protein 95 (PSD95)19 were confirmed by Western blot analysis (Figure 4) performed as described before20 (also see the Table of Materials). Synaptosomes labeled with a pH-sensitive dye become fluorescent after being engulfed by iMGs and once they are localized within intracellular acidic (i.e., low pH) compartments.
At the initial time point, the red fluorescent signal was minimal, as most of the synaptosomes were not yet phagocytosed. After 30 min, a red fluorescent signal was detected and increased further over time. By 10 h, the red fluorescent signal was robust, as most of the synaptosomes had been engulfed and were localized to acidic intracellular compartments via the process of phagocytosis (Figure 5A). Cytochalasin D is an actin polymerization inhibitor widely used to block phagocytosis. As expected, we observed a strong reduction of the red fluorescent signal in cytochalasin D-treated iMGs, indicating that the detected signal results from phagocytic events. We also noticed that iMGs show changes in morphology after 10 h of phagocytosis that were not detected in the cytochalasin D-treated iMGs, possibly due to the lack of actin remodeling in the cytochalasin D-treated iMGs. Changes in microglia morphology are usually associated with activation status2.
By using the described automated quantification method, we measured the total area of red signal at each time point and normalized it to the cell number obtained by counting the nuclei. The results indicate that the area of engulfed synaptosomes increased with time until a plateau was reached at 16 h. As expected, the area of red signal from cytochalasin D-treated cells did not increase with time, since phagocytosis was inhibited (Figure 5B). Together, these results indicate that iMGs are capable of phagocytosing brain-disease-relevant material and are suitable for functional studies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64323/64323fig01.jpg" /> 
Figure 1: Schematic of the protocol to differentiate iPSCs into microglia-like cells. On day 0, once iPSCs have reached 80% confluency, the colonies are dissociated into single cells and cultured in EB medium to induce EB formation. On day 4, EBs are collected and plated in PMP medium to induce PMP generation. After 28 days, PMPs are collected and terminally differentiated into iMGs by using iMG medium. PMPs can be collected weekly for up to 3 months. Abbreviations: iPSCs = induced pluripotent stem cells; iMGs = microglia-like cells; EB = embryoid body; PMP = primitive macrophage precursor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64323/64323fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64323/64323fig02.jpg" /> 
Figure 2: Cell morphology assessed by light microscopy at the differentiation stages from iPSCs to PMPs. (A) iPSCs maintained in iPSC medium until 80% confluency. (B) An embryoid body after 4 days of differentiation in EB medium. (C) EBs attached to coated plates and (D) PMPs emerging in suspension from EBs after 28 days of culture in PMP medium. (E) Number of PMPs collected from ~48 EBs at week 1 (after 28 days of PMP differentiation) and week 12. Bar graphs show the average of six independent differentiations (data points) from two different iPSC lines. Statistics were obtained by two-way ANOVA and &#352;&#237;d&#225;k&#39;s multiple comparisons test. Scale bars = 1,000 &#181;m (A), 400 &#181;m (B-D). Abbreviations: iPSCs = induced pluripotent stem cells; iMGs = microglia-like cells; EB = embryoid body; PMP = primitive macrophage precursor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64323/64323fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64323/64323fig03.jpg" /> 
Figure 3: iMGs differentiated by this protocol display bona fide microglia characteristics after 10 days of terminal differentiation. (A) Representative brightfield image of iMGs presenting microglia-like morphology. Scale bar= 400 &#181;m. (B) Representative immunofluorescence images of iMGs expressing the myeloid/microglia markers IBA1, P2RY12, and TMEM119. Scale bars= 400 &#181;m (A), 50 &#181;m (B). Abbreviations: iMGs = microglia-like cells; IBA1 = ionized calcium-binding adapter molecule 1; P2RY12 = purinergic receptor P2RY12; TMEM119 = transmembrane protein 119; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64323/64323fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64323/64323fig04.jpg" /> 
Figure 4: i3LMN-derived human synaptosomes expressed synaptic markers by western blot analysis. Representative western blot analysis of a human synaptosome preparation derived from i3LMN after 28 days of culture probed with the postsynaptic marker, PSD95, the presynaptic marker, SYP, and &#946;-tubulin. Abbreviations: i3LMN = iPSC-derived lower motor neurons; PSD95 = postsynaptic density protein 95; SYP = synaptophysin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64323/64323fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64323/64323fig05.jpg" /> 
Figure 5: Phagocytosis assay of iMGs using human synaptosomes labeled with a pH-sensitive dye and monitored by live-cell, longitudinal imaging. (A) Representative brightfield and fluorescence montages of iMGs with nuclear staining (blue channel) exposed to labeled human synaptosomes (red channel) with and without cytoD treatment at the indicated time points. The montages were created by postacquisition processing of 16 tiles acquired at 20x. Scale bars= 400 &#181;m. (B) Quantification of the area of the synaptosome fluorescent signal normalized to the cell number. Data points indicate the average &#177; SEM of three technical replicates. Each curve shows the quantification for two independent differentiations (iMGs 1 and 2) from one iPSC line with and without cytoD treatment. Abbreviations: iMGs = microglia-like cells; cytoD = cytochalasin D. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64323/64323fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number of 10 cm dishes of DIF28 i3LMN</td>
			<td>Total protein (&#181;g)</td>
		</tr>
		<tr>
			<td>2</td>
			<td>143.2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>34.9</td>
		</tr>
		<tr>
			<td>5</td>
			<td>613.8</td>
		</tr>
		<tr>
			<td>7</td>
			<td>1,159</td>
		</tr>
	</tbody>
</table>
Table 1: Total protein yield after synaptosome isolation from iPSC-derived lower-motor neurons at 28 days of differentiation. Total protein amount obtained from four independent differentiations after synaptosomes were extracted. Different numbers of 10 cm dishes containing i3LMN were harvested in each differentiation. The protein yield was measured by BCA protein assay. Abbreviations: iPSCs = induced pluripotent stem cells; i3LMN = iPSC-derived lower motor neurons; DIF28 = 28 days of differentiation; BCA = bicinchoninic acid.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Registration channel</td>
			<td>Bright field, DAPI, and RFP</td>
		</tr>
		<tr>
			<td>Fusion method</td>
			<td>Linear blend</td>
		</tr>
		<tr>
			<td>Crop Stitched Image to Remove Black Rectangles on the Borders</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Downsize Final Image</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Parameters</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Threshold</td>
			<td>Determined by user</td>
		</tr>
		<tr>
			<td>Background</td>
			<td>Dark</td>
		</tr>
		<tr>
			<td>Split touching objects</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Fill holes in masks</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Min. object size</td>
			<td>7 &#181;m</td>
		</tr>
		<tr>
			<td>Max. object size</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>Include primary edge objects</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Analyze entire image</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Parameters</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Threshold</td>
			<td>Determined by user</td>
		</tr>
		<tr>
			<td>Background</td>
			<td>Dark</td>
		</tr>
		<tr>
			<td>Split touching objects</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Fill holes in masks</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Min. object size</td>
			<td>5 &#181;m</td>
		</tr>
		<tr>
			<td>Max. object size</td>
			<td>100 &#181;m</td>
		</tr>
		<tr>
			<td>Include primary edge objects</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Analyze entire image</td>
			<td>Checked</td>
		</tr>
	</tbody>
</table>
Table 2: Parameters used for imaging acquisition and data analysis. Suggested parameters to consider for imaging acquisition during the phagocytosis assay and for analysis of the acquired images are listed.

Watch this Scientific Journal Video about Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes at JoVE.com

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

human-microglia-like-cells-differentiation-from-induced-pluripotent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.8K Views.  University of Massachusetts Chan Medical School. iPS-afgeleide microglia vormen een biologisch relevante bron van menselijke microglia voor in vitro experimenten en zijn een belangrijk hulpmiddel om microgliabiologie in gezondheid en ziekte te onderzoeken. We presenteren een eenvoudig microglia differentiatieprotocol dat minimaal gebruik van groeifactoren vereist en een hoge zuiverheid en opbrengst bereikt. Ook demonstreren we een volledig gehumaniseerde fagocytosetest.iPSC-afgeleide microglia-achtige cellen zijn erg gevoelig voor me...

Video: Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Developing Blood-Induced Microglia-Like Cells for Studying Human Brain Disorders

Engineering of Human Blood-Induced Microglia-like Cells for Reverse-Translational Brain Research

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various neuropsychiatric and physical diseases. Postmortem brain analysis and positron emission tomography imaging are representative research methods that evaluate microglial activation in human patients; the findings have revealed the activation of microglia in the brains of patients presenting with various neuropsychiatric disorders and chronic pain. Nonetheless, the aforementioned technique merely facilitates the assessment of limited aspects of microglial activation. 
In lieu of brain biopsy and the induced pluripotent stem cell technique, we initially devised a technique to generate directly induced microglia-like (iMG) cells from freshly derived human peripheral blood monocytes by supplementing them with granulocyte-macrophage colony-stimulating factor and interleukin 34 for 2 weeks. These iMG cells can be employed to perform dynamic morphological and molecular-level analyses concerning phagocytic capacity and cytokine releases following cellular-level stress stimulation. Recently, comprehensive transcriptome analysis has been used to verify the similarity between human iMG cells and brain primary microglia. 
The patient-derived iMG cells may serve as key surrogate markers for predicting microglial activation in human brains and have aided in the unveiling of previously unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease, fibromyalgia, bipolar disorder, and Moyamoya disease. Therefore, the iMG-based technique serves as a valuable reverse-translational tool and provides novel insights into elucidating dynamic the molecular pathophysiology of microglia in a variety of mental and physical diseases.

Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various ...

Immunology and Infection

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Source: Hall-Roberts, H. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62217/in-vitro-quantitative-imaging-assay-for-phagocytosis-dead">In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.</a> J. Vis. Exp. (2021)
In this video, we perform the microglial phagocytosis assay for neuroblastoma using induced pluripotent stem cells. The assay helps assess the impairment of microglial function in neurodegenerative diseases.

Source: Hall-Roberts, H. et al. In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages. J. Vis. Exp. (2021)
...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vitro studies

Live-cell Phagocytosis Assay of Microglia-Like Cells Using Human Synaptosomes

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/64323/human-microglia-like-cells-differentiation-from-induced-pluripotent'>Source: Funes, S., et. al.,&#160; Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes. J. Vis. Exp. (2022)</a>This video demonstrates an assay to study the phagocytic capacity of microglia-like cells using pH-sensitive fluorescent dye-labeled synaptosomes.

Source: Funes, S., et. al.,&#160; Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis ...

Neuropathology

Infectious Diseases

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

Source: Funes, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64323">Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the procedure of the differentiation of human pluripotent cells into microglia-like cells. The process involves forming embryoid bodies, transferring them to coated wells, and using specific growth factors to guide differentiation into primitive macrophage precursors, which finally differentiate into microglia-like cells.

1. Microglia differentiation
NOTE: An overview of the protocol is summarized in&#160;Figure 1.

	Induced pluripotent stem cell (iPSC) culture
	NOTE: ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

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