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

ヒト脳疾患研究のための血液誘導性ミクログリア様細胞の開発

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.

著者スポットライト:神経精神疾患におけるミクログリア機能障害の役割を明らかにするための誘導ミクログリア様細胞技術

ヒト血液誘導性ミクログリア様細胞の改変による脳の逆翻訳研究

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.

ヒト疾患におけるミクログリア動態を調べるための全血由来単球からの誘導ミクログリア様細胞の取得 

まず、全血からヒト単核細胞を入手します。Vortex Human FcR ブロッキング試薬を添加し、必要な量の試薬を細胞に添加します。チューブを細胞と4°Cで5分間インキュベートします。10個あたり20マイクロリットルのCD11b MicroBeadsを合計7個の細胞の累乗に添加し、摂氏4度で20分間インキュベートします。マグネティックスタンドを70%エタノールで十分に消毒します。インキュベーション後、10ミリリットルのアイソレーションバッファーを添加します。混合し、チューブを300Gで摂氏4度で10分間遠心分離します。スタンドが乾いたら、磁気カラムを磁気スタンドに置きます。上清を取り除いた後、1ミリリットルのアイソレーションバッファーを加え、ピペットで穏やかに混合します。次に、細胞懸濁液をカラムにロードし、カラムリザーバーが空になるたびに3ミリリットルの分離バッファーで3回洗浄します。カラムを磁気部分に触れないように15ミリリットルの円錐管に入れます。5ミリリットルのアイソレーションバッファーを追加した後、プランジャーをチューブに押し込み、液体をカラムから押し出します。チューブに回収した細胞懸濁液を遠心分離します。上清を吸引し、細胞を分離培地に再懸濁します。細胞計数後、500マイクロリットルの細胞懸濁液を24ウェルプレートの各ウェルに分注し、一晩インキュベートします。ミクログリア様細胞を誘導するには、前日の播種から分離した培地を誘導培地に交換し、細胞を14日間インキュベートします。誘導されたミクログリア様細胞は、微細な体細胞体と多数の分岐した側副体を示しました。

Research

JoVE Journal

Immunology and Infection

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

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

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

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

免疫・感染学

Isolation of Mononuclear Cells from Whole Blood to Obtain Induced Microglia-Like Cells for Brain Research

脳研究のための誘導ミクログリア様細胞を得るための全血からの単離