Name: Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders
Uploaded: 2024-09-06T13:30:00
Description: Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various ...

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 ...

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>

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.

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

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

Our lab at Kyushu University aimed to clarify the roles of microglial dysfunction in various neuropsychiatric disorders. We believe that our iMG cell technology will shed new light on microglial dynamic function and dysfunction at the cellular level in human beyond the limitation of human brain analysis, such as postmortem study and also pet brain studies. Even now, animal experiments are essential to clarify the brain pathophysiology of mental disorders, but it is unclear how well rodent data can be applied to human pathophysiology.Our human blood-induced iMG cell technology will this gap between rodent data, clinical data, and microglial function and/or dysfunction. Other human-derived cellular disease model, human iPS cell-derived microglia cells are also developed, which need much time and much money to develop and can assess trait markers but not state markers. The advantage of our iMG method is that it is much simpler, less time-consuming, and cost-effective.We have already revealed several candidate molecular factors by our reverse translational iMG research focusing on neuropsychiatric disorders such as dementia disease, bipolar disorder, fibromyalgia, glioma, and disease. We believe that our iMG cell technology will shed new light on microglial dynamic function and dysfunction at the cellular levels in various neuropsychiatric disorders.

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...

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.