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

<ol>
	<li>Steiner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+aspects+in+the+neurobiology+of+suicide:+elevated+microglial+density+in+schizophrenia+and+depression+is+associated+with+suicide.">Immunological aspects in the neurobiology of suicide: elevated microglial density in schizophrenia and depression is associated with suicide.</a> J Psychiatr Res. 42 (2), 151-157 (2008).</li><li>Setiawan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+translocator+protein+total+distribution+volume+with+duration+of+untreated+major+depressive+disorder:+a+cross-sectional+study.">Association of translocator protein total distribution volume with duration of untreated major depressive disorder: a cross-sectional study.</a> Lancet Psychiatry. 5 (4), 339-347 (2018).</li><li>Holmes, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+translocator+protein+in+anterior+cingulate+in+major+depression+and+a+role+for+inflammation+in+suicidal+thinking:+a+positron+emission+tomography+study.">Elevated translocator protein in anterior cingulate in major depression and a role for inflammation in suicidal thinking: a positron emission tomography study.</a> Biol Psychiatry. 83 (1), 61-69 (2018).</li><li>Meyer, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+in+psychiatric+disorders:+PET+imaging+and+promising+new+targets.">Neuroinflammation in psychiatric disorders: PET imaging and promising new targets.</a> Lancet Psychiatry. 7 (12), 1064-1074 (2020).</li><li>Ohgidani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+induction+of+ramified+microglia-like+cells+from+human+monocytes:+dynamic+microglial+dysfunction+in+Nasu-Hakola+disease.">Direct induction of ramified microglia-like cells from human monocytes: dynamic microglial dysfunction in Nasu-Hakola disease.</a> Sci Rep. 4, 4957 (2014).</li><li>Kato, T. A., Ohgidani, M., Kanba, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+producing+microglial+cells.">Method of producing microglial cells.</a> France patent EP. , (2023).</li><li>Muffat, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+microglia-like+cells+from+human+pluripotent+stem+cells.">Efficient derivation of microglia-like cells from human pluripotent stem cells.</a> Nat Med. 22 (11), 1358-1367 (2016).</li><li>Mattis, V. B., Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+a+new+revolution+for+clinical+neurology.">Induced pluripotent stem cells: a new revolution for clinical neurology.</a> Lancet Neurol. 10 (4), 383-394 (2011).</li><li>Dimos, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321 (5893), 1218-1221 (2008).</li><li>Pfisterer, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+human+fibroblasts+to+dopaminergic+neurons.">Direct conversion of human fibroblasts to dopaminergic neurons.</a> Proc Natl Acad Sci U S A. 108 (25), 10343-10348 (2011).</li><li>Pang, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+human+neuronal+cells+by+defined+transcription+factors.">Induction of human neuronal cells by defined transcription factors.</a> Nature. 476 (7359), 220-223 (2011).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-34+is+a+tissue-restricted+ligand+of+CSF1R+required+for+the+development+of+Langerhans+cells+and+microglia.">IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia.</a> Nat Immunol. 13 (8), 753-760 (2012).</li><li>Aloisi, F., De Simone, R., Columba-Cabezas, S., Penna, G., Adorini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+adult+mouse+resting+microglia+into+an+APC+is+promoted+by+granulocyte-macrophage+colony-stimulating+factor+and+interaction+with+Th1+cells.">Functional maturation of adult mouse resting microglia into an APC is promoted by granulocyte-macrophage colony-stimulating factor and interaction with Th1 cells.</a> J Immunol. 164 (4), 1705-1712 (2000).</li><li>Erblich, B., Zhu, L., Etgen, A. M., Dobrenis, K., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+colony+stimulation+factor-1+receptor+results+in+loss+of+microglia,+disrupted+brain+development+and+olfactory+deficits.">Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.</a> PLoS One. 6 (10), e26317 (2011).</li><li>Nandi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CSF-1+receptor+ligands+IL-34+and+CSF-1+exhibit+distinct+developmental+brain+expression+patterns+and+regulate+neural+progenitor+cell+maintenance+and+maturation.">The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation.</a> Dev Biol. 367 (2), 100-113 (2012).</li><li>Douvaras, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+to+microglia.">Directed differentiation of human pluripotent stem cells to microglia.</a> Stem Cell Rep. 8 (6), 1516-1524 (2017).</li><li>Yonemoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+mitochondrial+vulnerability+configurate+the+divergent+immunoreactivity+of+human+induced+microglia-like+cells.">Heterogeneity and mitochondrial vulnerability configurate the divergent immunoreactivity of human induced microglia-like cells.</a> Clin Immunol. 255, 109756 (2023).</li><li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD206+expression+in+induced+microglia-like+cells+from+peripheral+blood+as+a+surrogate+biomarker+for+the+specific+immune+microenvironment+of+neurosurgical+diseases+including+glioma.">CD206 expression in induced microglia-like cells from peripheral blood as a surrogate biomarker for the specific immune microenvironment of neurosurgical diseases including glioma.</a> Front Immunol. 12, 2424-2424 (2021).</li><li>Ohgidani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibromyalgia+and+microglial+TNF-alpha:+Translational+research+using+human+blood+induced+microglia-like+cells.">Fibromyalgia and microglial TNF-alpha: Translational research using human blood induced microglia-like cells.</a> Sci Rep. 7 (1), 11882 (2017).</li><li>Ohgidani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+CD206+gene+has+potential+as+a+state+marker+of+bipolar+disorder.">Microglial CD206 gene has potential as a state marker of bipolar disorder.</a> Front Immunol. 7, 676 (2016).</li><li>Ohgidani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+bipolar+disorder+with+AIF1+(coding+gene+of+Iba-1)+deletion:+A+pilot+in+vitro+analysis+using+blood-derived+microglia-like+cells.">A case of bipolar disorder with AIF1 (coding gene of Iba-1) deletion: A pilot in vitro analysis using blood-derived microglia-like cells.</a> Psychiatry Clin Neurosci. 77 (2), 128-130 (2023).</li><li>Shirozu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+and+inflammatory+responses+in+human+induced+microglia-like+(iMG)+cells+from+patients+with+Moyamoya+disease.">Angiogenic and inflammatory responses in human induced microglia-like (iMG) cells from patients with Moyamoya disease.</a> Sci Rep. 13 (1), 14842 (2023).</li><li>Sellgren, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+models+of+microglia-mediated+engulfment+of+synapses+and+neural+progenitors.">Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors.</a> Mol Psychiatry. 22 (2), 170-177 (2017).</li><li>Sellgren, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+synapse+elimination+by+microglia+in+schizophrenia+patient-derived+models+of+synaptic+pruning.">Increased synapse elimination by microglia in schizophrenia patient-derived models of synaptic pruning.</a> Nat Neurosci. 22 (3), 374-385 (2019).</li><li>Banerjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+induced+microglia-like+cells+(iMG+Cells)+for+future+studies+of+brain+diseases.">Validation of induced microglia-like cells (iMG Cells) for future studies of brain diseases.</a> Front Cell Neurosci. 15, 629279 (2021).</li><li>You, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+characterization+and+clinical+relevance+of+microglia-like+cells+derived+from+patients+with+panic+disorder.">A molecular characterization and clinical relevance of microglia-like cells derived from patients with panic disorder.</a> Transl Psychiatry. 13 (1), 48 (2023).</li><li>Rocha, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+activation+in+basal+ganglia+is+a+late+event+in+Huntington+disease+pathophysiology.">Microglia activation in basal ganglia is a late event in Huntington disease pathophysiology.</a> Neurol Neuroimmunol Neuroinflamm. 8 (3), e984 (2021).</li><li>Sargeant, T. J., Fourrier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocyte-derived+microglia-like+cell+models:+A+review+of+the+benefits,+limitations+and+recommendations.">Human monocyte-derived microglia-like cell models: A review of the benefits, limitations and recommendations.</a> Brain Behav Immun. 107, 98-109 (2023).</li><li>Lannes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+microglia+cells+with+Japanese+encephalitis+virus.">Interactions of human microglia cells with Japanese encephalitis virus.</a> Virol J. 14 (1), 8 (2017).</li><li>Ryan, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+microglia-like+cellular+model+for+assessing+the+effects+of+neurodegenerative+disease+gene+variants.">A human microglia-like cellular model for assessing the effects of neurodegenerative disease gene variants.</a> Sci Transl Med. 9 (421), 7635 (2017).</li><li>Etemad, S., Zamin, R. M., Ruitenberg, M. J., Filgueira, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+human+microglia+model:+characterization+of+human+monocyte-derived+microglia.">A novel in vitro human microglia model: characterization of human monocyte-derived microglia.</a> J Neurosci Methods. 209 (1), 79-89 (2012).</li><li>Bortolotti, D., Gentili, V., Rotola, A., Caselli, E., Rizzo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HHV-6A+infection+induces+amyloid-beta+expression+and+activation+of+microglial+cells.">HHV-6A infection induces amyloid-beta expression and activation of microglial cells.</a> Alzheimers Res Ther. 11 (1), 104 (2019).</li><li>Ormel, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+characterization+of+the+molecular+phenotype+and+inflammatory+response+of+schizophrenia+patient-derived+microglia-like+cells.">A characterization of the molecular phenotype and inflammatory response of schizophrenia patient-derived microglia-like cells.</a> Brain Behav Immun. 90, 196-207 (2020).</li><li>Akiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+HIV-1+intron-containing+RNA+in+microglia+induces+inflammatory+responses.">Expression of HIV-1 intron-containing RNA in microglia induces inflammatory responses.</a> J Virol. 95 (5), e01386-e01420 (2021).</li><li>Nielsen, M. C., Andersen, M. N., Moller, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+isolation+techniques+significantly+impact+the+phenotype+of+both+isolated+monocytes+and+derived+macrophages+in+vitro.">Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro.</a> Immunology. 159 (1), 63-74 (2020).</li><li>Kelly, A., Grabiec, A. M., Travis, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+monocyte-derived+macrophages.">Culture of human monocyte-derived macrophages.</a> Methods Mol Biol. 1784, 1-11 (2018).</li></ol>

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 imm...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;antibiotic-antimycotic solution&#34;</td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>described as &#34;basic buffer solution&#34; and used for &#34;isolation buffer&#34;</td>
		</tr>
		<tr>
			<td>CD11b MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130&#8211;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&#39;s Phosphate Buffered Saline</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td>described as &#34;PBS (&#8722;)&#34;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>biowest</td>
			<td>S1760-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>described as &#34;density gradient medium&#34;</td>
		</tr>
		<tr>
			<td>Human FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>130&#8211;059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucosep</td>
			<td>Greiner Bio-One</td>
			<td>227290</td>
			<td>described as &#34;density gradient centrifugation tube&#34;</td>
		</tr>
		<tr>
			<td>MACS LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>described as &#34;magnetic column&#34;</td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>described as &#34;bovine serum albumin (BSA) stock solution&#34;</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>described as &#34;magnetic stand&#34;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Nacalai Tesque</td>
			<td>26253&#8211;84</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td>described as &#34;mounting media&#34;</td>
		</tr>
		<tr>
			<td>recombinant human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human IL-34</td>
			<td>R&#38;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 &#34;basal induction medium&#34;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Nacalai Tesque</td>
			<td>30264-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</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 examin...

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

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

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

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

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Chapters in this video

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins