obtaining induced microglia-like cells from whole-blood derived monocytes for probing microglial dynamics in human diseases 

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

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.

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.

Obtaining Induced Microglia-Like Cells from Whole-Blood Derived Monocytes for Probing Microglial Dynamics in Human Diseases 

To begin, obtain human mononuclear cells from whole blood. Vortex human FcR blocking reagent, and add the required amount of reagent to the cells. Incubate the tube with cells at four degrees Celsius for five minutes.Add 20 microliters of CD11b MicroBeads per 10 to the power of seven total cells, and incubate at four degrees Celsius for 20 minutes. Thoroughly disinfect the magnetic stand with 70%ethanol. After incubation, add 10 milliliters of isolation buffer.Mix, and centrifuge the tube at 300G for 10 minutes at four degrees celsius. Once the stand dries, position the magnetic column on the magnetic stand. After removing the supernatant, add one milliliter of isolation buffer and mix gently with the pipette.Then load the cell suspension onto the column and wash it three times with three milliliters of isolation buffer each time the column reservoir is empty. Place the column in a 15-milliliter conical tube without touching the magnetic part. After adding five milliliters of isolation buffer, push the plunger into the tube to force the liquid out through the column.Centrifuge the cell suspension collected in the tube. Aspirate the supernatant and re-suspend the cells in isolation medium. Following cell counting, aliquot 500 microliters of cell suspension into each well of a 24-well plate, and incubate overnight.To induce microglia-like cells, replace the isolation medium from the seeding of the previous day with an induction medium and incubate the cells for 14 days. The induced microglia-like cells exhibited minute soma bodies and numerous branched collaterals.