This work was partially supported by the following Grants-in-Aid for Scientific Research: (1) The Japan Society for the Promotion of Science (KAKENHI; JP18H04042, JP19K21591, JP20H01773, and JP22H00494 to TAK, JP22H03000 to M.O.); (2) The Japan Agency for Medical Research and Development (AMED; JP21wm0425010 to TAK, JP22dk0207065 to M.O.) and (3) The Japan Science and Technology Agency CREST (JPMJCR22N5 to TAK). The funding bodies assumed no roles in the study design, data collection and analysis, decision to publish, or manuscript preparation. We would like to thank Editage (www.editage.jp) for English language editing.

The study protocol was approved by the Ethics Committee of Kyushu University and complied with all the provisions of the Declaration of Helsinki. Written informed consent was obtained from all participants, including healthy volunteers and patients, to analyze their blood and publish their data. Materials and equipment are listed in the Table of Materials, and the compositions of the solutions are detailed in Table 1.
1. Preparation of media and buffers for experiments
<ol>
	<li>Prepare the isolation medium by supplementing RPMI-1640 with 10% heat-inactivated fetal bovine serum (56 &#176;C, 30 min) and 1 % penicillin-streptomycin.</li>
	<li>Prepare the isolation buffer by supplementing the basic buffer solution with 0.5% bovine serum albumin (BSA) stock solution and sterilize it via filtration (0.22 &#181;m).</li>
</ol>
2. Isolation of mononuclear cells from whole blood
<ol>
	<li>Transfer 15 mL of the density gradient medium to a 50-mL density gradient centrifugation tube and centrifuge at 1,000 &#215; g for 1 min at 20 &#176;C. 
	NOTE: Isolate 15 mL of blood per 50-mL density gradient centrifugation tube. Increase the number of tubes when blood volume is high.</li>
	<li>Homogenize the blood collected in heparin tubes by inversion and remove the lid from the blood collection tube with fire-sterilized tweezers.</li>
	<li>Dispense an equal volume (10-15 mL) of blood to the density gradient medium in the density gradient centrifugation tube.</li>
	<li>Set the deceleration level of the centrifuge to 1 of 4 levels and centrifuge at 1,000 &#215; g for 10 min at room temperature (RT). 
	NOTE: At this deceleration rate, a rotational speed of 180 &#215; g (1,000 rpm) arrests in 3 min.</li>
	<li>Prepare an equal number of 50-mL centrifuge tubes as the 50-mL density gradient centrifugation tubes and fill each tube with 10 mL of PBS (&#8722;).</li>
	<li>Remove the top layer of plasma up to approximately 1 cm above the buffy coat following centrifugation. 
	NOTE: Insert an aspirating pipette vertically into the tube and aspirate carefully from the center of the liquid surface. Do not overaspirate to avoid disturbing the buffy coat.</li>
	<li>Decant all of the remaining supernatant from the 50 mL density gradient centrifugation tube into a centrifuge tube containing 10 mL of PBS (&#8722;).</li>
	<li>Reset the deceleration level of the centrifuge to 3 of 4 levels, and centrifuge at 250 &#215; g for 10 min at RT. During this step, prepare one 50-mL and two 15-mL centrifuge tubes and a 100-&#181;m cell strainer.</li>
	<li>After centrifugation, establish the temperature of the centrifuge at 4 &#176;C. Search for a white pellet with a red periphery at the bottom of the centrifuge tube. Remove the supernatant by aspiration and loosen the pellet gently by tapping.</li>
	<li>Position a 100-&#181;m cell strainer on the 50-mL conical tube using fire-sterilized tweezers.</li>
	<li>Incorporate 13 mL of isolation medium into one of the centrifuged tubes and wash the inner wall to collect the cells properly. Take the cell suspension and wash the remaining centrifuged tubes in a consecutive manner. After collecting the cells from all the tubes, filter the cell suspension through a cell strainer into the 50-mL conical tube. 
	NOTE: The isolation medium needs to be chilled throughout the isolation process (steps 2.11-2.16).</li>
	<li>Wash each tube again with 13 mL of isolation medium as described in step 2.11. Collect a total of 26 mL of cell suspension.</li>
	<li>Dispense equal amounts into two 15-mL conical tubes, enumerate the cells, and calculate the appropriate concentration of isolation buffer (60 &#181;L of buffer per 107 total cells) for step 2.16. 
	NOTE: The 15-mL conical tube is better to work with because the liquid volume is small and the liquid bubbles easily when pipetting in step 2.16.</li>
	<li>Centrifuge at 250 &#215; g for 10 min at 4 &#176;C.</li>
	<li>During the centrifugation, thoroughly disinfect the magnetic stand (especially the part in contact with the column) with 70% ethanol and place it on the bench to air dry. Keep the isolation buffer on ice.</li>
	<li>Remove the supernatant by aspiration (at this stage, look for red pellets ~1 mm thick because of the presence of red blood cells), add the appropriate amount of isolation buffer, and loosen the pellet gently by pipetting ~20x.</li>
</ol>
3. Isolation of monocytes using CD11b microbeads
<ol>
	<li>Vortex human FcR blocking reagent for 10 s prior to application. Incorporate the required amount of reagent (20 &#181;L of FcR Blocking Reagent per 107 total cells) and incubate in a chamber at 4 &#176;C for 5 min.</li>
	<li>Vortex the CD11b microbeads for 10 s prior to application. Incorporate the required amount of reagent (20 &#181;L of CD11b microbeads per 107 total cells), and incubate in a 4 &#176;C chamber for 20 min while agitating by hand every 5 min.</li>
	<li>After incubation, add 10 mL of isolation buffer, suspend by gentle pipetting, and centrifuge at 300 &#215; g for 10 min at 4 &#176;C.</li>
	<li>During centrifugation, place the magnetic column on the magnetic stand and rinse it with 3 mL of isolation buffer. 
	NOTE: Refrain from touching the magnetic part of the column and position the column correctly. If bubbles enter the column, remove them with a pipette. The isolation buffer is mandated to be chilled throughout the isolation process (steps 3.4-3.9).</li>
	<li>Remove the post-centrifugation supernatant by aspiration, incorporate 1 mL of isolation buffer, mix by pipetting gently, and apply the suspension onto the column.</li>
	<li>Wash the column 3x by adding 3 mL of isolation buffer each time the column reservoir is empty.</li>
	<li>Place the column in a 15-mL conical tube without touching the magnetic part; incorporate 5 mL of isolation buffer into the column and force the liquid out through the column with a plunger inserted into the tube.</li>
	<li>Centrifuge the cell suspension collected in the tube at 300 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cells in isolation medium. As ~5-10% of the number of cells counted in step 2.13 will be recovered, adjust the suspension volume according to the number of cells. 
	NOTE: Isolation medium should be used at RT and not warmed to avoid rapid temperature change from 4 &#176;C.</li>
	<li>Enumerate the cells and aliquot 500 &#181;L of cell suspension into each well of 24-well plates at a concentration of 40 &#215; 104 cells/mL and incubate overnight. 
	NOTE: The cell suspensions should be pipetted appropriately in accordance with the adjusted seeding volume required for different plates. For example, 1 mL for one well in a 12-well plate and 250 &#181;L for one well in an 8-well chamber slide.</li>
	<li>Finally, dispose of or disinfect the contaminated equipment in accordance with the policy for handling infectious waste established by the facility.</li>
</ol>
4. Induction of iMG cells from monocytes
<ol>
	<li>Prepare the induction medium by supplementing basal induction medium with 1% antibiotic-antimycotic solution, 10 ng/mL recombinant human GM-CSF, and 100 ng/mL recombinant human IL-34.</li>
	<li>Replace the isolation medium from the seeding of the previous day with induction medium. Tilt the plate forward and promptly aspirate the depleted medium from the front edge of the well bottom with a Pasteur pipette. Replace the medium at the rate of a maximum of 1 plate (= 24 wells) at a time, and immediately incorporate 500 &#181;L of induction medium gently. 
	NOTE: Adhered monocytes should be selected in this step.</li>
	<li>Incubate the cells for 14 days to complete the induction.</li>
	<li>Replace the medium again with 500 &#181;L of induction medium after 14 days. Subsequently, for post-induction maintenance of the cells, replace the induction medium with 500 &#181;L of fresh induction medium every week.</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Remove the medium from the 8-well chamber slide seeded with cells in step 3.10 by aspiration and rinse with PBS (&#8722;).</li>
	<li>Fix the cells for 20 min at RT with 4% paraformaldehyde.</li>
	<li>Remove the fixative reagent and rinse the cells for 3 x 5 min with PBS (&#8722;).</li>
	<li>Permeabilize the cells with PBS (&#8722;) containing 0.1% Triton X-100 at RT.</li>
	<li>Incubate the permeabilized cells with a blocking solution (3% BSA/PBS (&#8722;)) for 1 h at RT.</li>
	<li>Incubate the cells overnight with primary antibodies (Table of Materials) at 4 &#176;C. 
	NOTE: All antibodies are diluted in blocking solution.</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;) for 5 min each.</li>
	<li>Incubate the rinsed cells with the appropriate fluorescently tagged secondary antibodies for 1 h at RT (Table of Materials).</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;) to remove the secondary antibodies.</li>
	<li>Incubate the cells with DAPI (1:10,000) solution diluted with PBS (&#8722;) for 5 min at RT.</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;).</li>
	<li>Mount the well using the mounting medium and visualize the cells under a fluorescence microscope.</li>
</ol>

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

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The authors have nothing to disclose.

Analytical techniques employing iMG cells may serve as potent reverse-translational research tools5,6. To generate sufficient quantities of human iMG cells, experimenters should design their studies taking certain issues into consideration. Blood samples derived from human beings are extremely sensitive; consequently, the obtained samples warrant prompt processing, and meticulous handling to avoid contamination. Specifically, blood samples should be separated immediately after being drawn and not left unattended for extended periods. Experiments are conventionally commenced within 3 h of blood collection to confirm their validity; however, it is possible to store blood samples at 4&#176;C for over 3 h and bring them to room temperature prior to starting separation protocols.
To ensure stable results, reagents such as culture media are recommended to be used within a month of unsealing and discarded upon expiration. When separating peripheral blood mononuclear cells using density gradient centrifugation, cell aggregation may be observed in certain specimens. In such cases, the whole blood sample must be diluted with PBS (&#8722;) to prevent aggregation. During the induction of differentiation into microglia, the culture medium is not replaced for 14 days to avoid unnecessary stimulation of the cells. After the completion of differentiation induction, medium replacement with fresh medium may restore the weakened cells and maintain viability for a while.
The iMG technique established in this study presents a limitation; the experimentally induced &#34;microglia-like cells&#34; are comparable but not completely identical to the actual microglial cells in the human brain. Additionally, prior research has documented methodology for the induction of human monocyte-derived microglia-like cells, and different research groups have proposed their own distinct protocols and names contingent upon the original iMG methods28. For instance, certain protocols use M-CSF29,30,31,32 or TGF-&#946;33, which are reported to promote a microglia-like phenotype in iPSC-derived microglia cultures or on coatings such as Geltrex24,34; these factors may prove to be effective for inducing microglia-like cells.
The original iMG technology is advantageous owing to its simple and easy-to-reproduce protocol with merely two cytokines and no coatings, which enables a variety of reverse-translational experiments using human blood. Conversely, advancements in technology have facilitated the development of many products. Magnetic separation methods include column-based positive selection, similar to our protocol, as well as negative selection and column-free methods. Moreover, in lieu of magnetic separation, an alternative method of separation using plastic adhesion may be considered. Column-based positive selection ensures purity, but the technique is time-consuming, and stimulation by magnetic beads substantially affects the cells35. Contrarily, negative selection and plastic adhesion may be less detrimental to the cells; nevertheless, these approaches may be less pure35,36. Further modification may be needed based on other protocols in the future.
The aforementioned findings substantiate that the predominant advantage of this iMG technology is its brief duration, cost-effective methodology, and ability to process a large number of samples for high-throughput studies. Therefore, this technology can be employed to acquire knowledge from human samples and then conduct further validation studies using different animal models. Moreover, the findings obtained from investigations conducted on cellular and animal models by applying the iMG technique may be beneficial. In this way, our iMG technique can be applied to bi-directional translational research in a bedside-to-bench and bench-to-bedside manner.

In recent years, brain inflammation has been suggested to assume pivotal roles in the pathophysiology of various brain and neuropsychiatric disorders; the microglia have been highlighted as key immunomodulatory cells by human postmortem brain analysis and positron emission tomography (PET)-based bio-imaging techniques1,2,3,4. Postmortem brain and PET imaging analyses reveal significant findings; nevertheless, however, these approaches are inefficient in terms of capturing the dynamic molecular activities of human microglia in the brain in their entirety. Therefore, novel strategies are required to enable the comprehensive evaluation of human microglial functions and dysfunctions at the molecular and cellular levels.
In 2014, we originally engineered a novel technique to produce directly induced microglia-like(iMG) cells5,6, prior to the first publication of human-induced pluripotent stem cell (iPSC)-derived microglia-like cells in 20167. In just 2 weeks, we successfully converted human peripheral blood monocytes into iMG cells by optimizing the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 34 (IL-34). When we developed this technique, the innovative reprogramming method of inducing neuronal cells from iPS cells or fibroblasts was just beginning to prevail in the world8,9,10,11. However, at that time, a method for inducing iPS-derived microglial cells had not yet been reported, and the generation of a human somatic cell-derived microglial model was desired. Since cytokines such as GM-CSF and IL-34, macrophage colony-stimulating factor, were reported to be necessary for the development and maintenance of microglia12,13,14,15, we hypothesized that a combination of these cytokines could be applied directly to generate a microglial cellular model from blood monocytes. Finally, we succeeded in developing a model of microglia derived from monocytes by combining GM-CSF and IL-345. In addition, some of these combinations of cytokines are also employed to induce microglia from iPS cells7,16 and are assumed to be an important factor in acquiring microglial characteristics.
In contrast to iPSC methods, iMG cells do not require any genetic modification and can be generated in a very short time by simple chemical induction, resulting in lower time and financial costs. Furthermore, iMG cells do not require genetic reprogramming, so we believe that iMG cells are potent surrogate cells to evaluate not only the traits but also the states of human microglia. In the initial paper on the iMG technique in 2014, we confirmed that iMG cells exhibit a phenotype of human microglia, which can be distinct from monocytes and macrophages. For example, iMG cells exhibited an overexpression ratio of CX3C chemokine receptor 1 (CX3CR1) and C-C chemokine receptor type 2 (CCR2) than monocytes and typical microglia markers, including transmembrane protein 119(TMEM 119) and purinergic receptor P2RY125,17. Recently, we validated that peripheral blood-derived iMG cells resemble brain microglia in their gene expression profile of well-known microglial markers in the same patient who underwent brain surgeries18. The iMG cells can be analyzed for dynamic functions at the molecular level, such as phagocytosis and cytokine production, and are expected to compensate for the disadvantages of postmortem brain research and PET studies.
We have discovered previously unknown dynamic pathophysiological mechanisms involving microglia in patients diagnosed with Nasu-Hakola disease5, fibromyalgia19, bipolar disorder20,21, or Moyamoya disease22. Furthermore, based on our original methodology, various laboratories have employed the iMG cells (certain laboratories have designated alternative names to these cells) as a crucial reverse-translational research tool23,24,25,26,27. Sellgren et al. successfully generated iMG cells in compliance with our recommendations and conducted a microarray analysis, which revealed that these cells closely resemble human brain microglia23. Recently, we confirmed the resemblance between human iMG cells and brain primary microglia using RNA sequencing18.
This study aimed to document the methodology to generate iMG cells from human peripheral blood to facilitate reverse-translational research focused on neuropsychiatric diseases. This technique presents potential as a reasonable analytical tool that can effortlessly produce microglial cellular models in a brief duration, even in ill-equipped laboratories that lack gene transfer apparatus or proficient personnel.

<table><tbody><tr><td>0.1% Triton X-100</td><td>Sigma-Aldrich</td><td>30-5140-5</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Nacalai Tesque</td><td>09154-14</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>gibco</td><td>15240-062</td><td>described as &quot;antibiotic-antimycotic solution&quot;</td></tr><tr><td>autoMACS Rinsing Solution</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>described as &quot;basic buffer solution&quot; and used for &quot;isolation buffer&quot;</td></tr><tr><td>CD11b MicroBeads</td><td>Miltenyi Biotec</td><td>130&amp;ndash;049-601</td><td></td></tr><tr><td>DAPI solution</td><td>DOJINDO</td><td>28718-90-3</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Nacalai Tesque</td><td>14249-24</td><td>described as &quot;PBS (&amp;minus;)&quot;</td></tr><tr><td>Fetal Bovine Serum</td><td>biowest</td><td>S1760-500</td><td></td></tr><tr><td>Human FcR Blocking Reagent</td><td>Miltenyi Biotec</td><td>130&amp;ndash;059-901</td><td></td></tr><tr><td>Leucosep</td><td>Greiner Bio-One</td><td>227290</td><td>described as &quot;density gradient centrifugation tube&quot;</td></tr><tr><td>MACS LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>described as &quot;magnetic column&quot;</td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>described as &quot;bovine serum albumin (BSA) stock solution&quot;</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td>described as &quot;magnetic stand&quot;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Nacalai Tesque</td><td>26253&amp;ndash;84</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen</td><td>P10144</td><td>described as &quot;mounting media&quot;</td></tr><tr><td>recombinant human GM-CSF</td><td>R&amp;amp;D Systems</td><td>215-GM</td><td></td></tr><tr><td>recombinant human IL-34</td><td>R&amp;amp;D Systems</td><td>5265-IL</td><td></td></tr><tr><td>RPMI 1640 Medium + GlutaMAX Supplement (pre-supplemented medium)</td><td>Thermo Fisher Scientific</td><td>61870036</td><td>described as &quot;basal induction medium&quot;</td></tr><tr><td>RPMI-1640</td><td>Nacalai Tesque</td><td>30264-56</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-P2RY12 antibody</td><td>Sigma-Aldrich</td><td>HPA014518</td><td>primary antibody, rabbit, 1:100</td></tr><tr><td>anti-TMEM119 antibody</td><td>Sigma-Aldrich</td><td>HPA051870</td><td>primary antibody, rabbit, 1:100</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td><td>invitrogen</td><td>A-11011</td><td>secondary antibody, rabbit, 1:1000</td></tr></tbody></table>

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.

Importantly, there is a great deal of person-level and timing-level heterogeneity in the characters of iMG cells including morphologies and gene expressions. The iMG cells in certain individuals assume a numerous branching appearance (Figure 1A), while in others they remain spherical (Figure 1B). The iMG characteristics may differ even within a single individual, rendering iMG cells as a pivotal tool for detecting disease state biomarkers. Conversely, the examination of such inter-individual differences warrants the reduction of technical errors. The iMG cells are defined as monocytes induced with GM-CSF and IL-34 for a minimum of 14 days. To minimize technical errors, it is recommended to standardize the induction time, utilize the same amounts of reagents, and refrain from using expired reagents.
Validation of immunostaining also confirms that iMG cells express microglial markers, including P2RY12 and TMEM119 (Figure 2). Prior research has demonstrated that the morphological and gene-expression characteristics of iMG cells are associated with the pathophysiology of bipolar disorder20,21. The earliest branched cells were observed on DAY 3 after GM-CSF and IL-34 treatment, and iMG cells in optimum condition can survive for more than 1 month when medium change is done once a week. The iMG cells can be used for gene expression analysis. For example, under stimulations with interferon gamma or IL-4, iMG cells changed the gene expression pattern, which can be used for the functional evaluation of M1 and M2 polarized microglia5,22. Moreover, FITC beads trigger amoeboid-like morphological modifications and upregulate the expression of TNF&#945; and other genes5. Furthermore, phagocytosis can be evaluated by measuring the fluorescence intensity of the beads5,22. Moreover, as iMG cells are viable cells, their actual movement and morphological alterations can be observed by taking cell pictures (Figure 3) or through time-lapse photography (Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66819/66819fig01.jpg" /> 
Figure 1: Morphology of iMG cells. (A) Typical ramified iMG exhibited minute soma bodies and numerous branched collaterals. (B) Certain samples varied in appearance such as the ameboid type. Scale bar, 50&#8197;&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66819/66819fig01large.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/66819/66819fig02.jpg" /> 
Figure 2: Immunocytostaining of iMG cells. iMG cells express typical microglial markers: (A) TMEM 119, (B) P2RY12. Nuclei were stained with DAPI (blue). Scale bar =10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66819/66819fig02large.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/66819/66819fig03.jpg" /> 
Figure 3: Morphological modifications in iMG cells. iMG alter their morphology during the induction process. Cell protrusions appear (A) around day 3 and elongate on days (B) 7 and (C) 14 as induction proceeds. Scale bar = 50&#8197;&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66819/66819fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Time-lapse photography of iMG cells. The ramified iMG cell in the center appeared to establish contact with the surrounding cells while extending and contracting their processes. Furthermore, the cell performed phagocytosis of damaged cells. The image acquisition intervals were 46 sec, and the filming time was approximately 14 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66819/Mov.1.zip" target="_blank">Please click here to download this Video.</a>
Table 1: Composition of solutions <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66819/66819_Table 1.zip" target="_blank">Please click here to download this Table.</a>

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.

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 ...

engineering-human-blood-induced-microglia-like-cells-for-reverse

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.1K Views.  Kyushu University. 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.

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

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Neuroscience

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Developmental Biology

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

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 ...

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 ...

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.

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.

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

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

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

Summary

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Chapters in this video

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Derivation of a Human Brain Organoid with Microglia Development

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Obtaining Human Microglia from Adult Human Brain Tissue

GM-Free Generation of Blood-Derived Neuronal Cells

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

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

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

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons