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

인간의 뇌 질환을 연구하기 위한 혈액 유도 미세아교세포 유사 세포 개발

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.

저자 스포트라이트: 신경정신 질환에서 미세아교세포 기능 장애의 역할을 밝히기 위한 유도된 미세아교세포와 유사한 세포 기술

역번역 뇌 연구를 위한 인간 혈액 유도 미세아교세포 유사 세포 엔지니어링

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.

인간 질병의 미세아교세포 역학을 조사하기 위해 전혈 유래 단핵구에서 유도된 미세아교세포 유사 세포 획득 

시작하려면 전혈에서 인간 단핵 세포를 얻습니다. Vortex human FcR 차단 시약을 넣고 필요한 양의 시약을 세포에 추가합니다. 섭씨 4도에서 5분 동안 세포가 있는 튜브를 배양합니다.10개당 20마이크로리터의 CD11b MicroBeads를 총 7개의 세포의 거듭제곱에 추가하고 섭씨 4도에서 20분 동안 배양합니다. 마그네틱 스탠드를 70% 에탄올로 철저히 소독하십시오. 배양 후 10mL의 분리 완충액을 추가합니다.튜브를 혼합하고 섭씨 4도에서 300G에서 10분 동안 원심분리합니다. 스탠드가 마르면 마그네틱 스탠드에 자기 기둥을 놓습니다. 상층액을 제거한 후 1mL의 분리 완충액을 추가하고 피펫과 부드럽게 혼합합니다.그런 다음 셀 현탁액을 컬럼에 로드하고 컬럼 저장소가 비워질 때마다 3mL의 격리 버퍼로 세 번 세척합니다. 자기 부분을 건드리지 않고 15밀리리터 원추형 튜브에 컬럼을 놓습니다. 5mL의 분리 완충액을 추가한 후 플런저를 튜브에 밀어 넣어 액체가 컬럼을 통해 강제로 배출되도록 합니다.튜브에 수집된 셀 현탁액을 원심분리합니다. 상층액을 흡인하고 세포를 분리 배지에 다시 부유시킵니다. 세포 계수 후 500마이크로리터의 세포 현탁액을 24웰 플레이트의 각 웰에 분양하고 하룻밤 동안 배양합니다.미세아교세포(microglia-like cell)를 유도하기 위해, 전날의 파종에서 추출한 분리 배지를 유도 배지로 교체하고 세포를 14일 동안 배양합니다. 유도된 미세아교세포(microglia-like cells)는 미세한 체세포(soma body)와 수많은 분지된 측부(branched collaterals)를 나타냈다.

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 ...

연구

면역학 및 감염병학

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

뇌 연구를 위한 유도 미세아교세포(induced microglia-like cells)를 얻기 위해 전혈(whole blood)에서 단핵 세포를 분리합니다.