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

मानव मस्तिष्क विकारों के अध्ययन के लिए रक्त-प्रेरित माइक्रोग्लिया जैसी कोशिकाओं का विकास करना

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.

लेखक स्पॉटलाइट: न्यूरोसाइकिएट्रिक विकारों में माइक्रोग्लियल डिसफंक्शन की भूमिका पर प्रकाश डालने के लिए प्रेरित माइक्रोग्लिया-जैसी सेल टेक्नोलॉजी

रिवर्स-ट्रांसलेशनल मस्तिष्क अनुसंधान के लिए मानव रक्त-प्रेरित माइक्रोग्लिया-जैसी कोशिकाओं की इंजीनियरिंग

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.

मानव रोगों में माइक्रोग्लियल गतिशीलता की जांच के लिए पूरे रक्त व्युत्पन्न मोनोसाइट्स से प्रेरित माइक्रोग्लिया जैसी कोशिकाएं प्राप्त करना 

शुरू करने के लिए, पूरे रक्त से मानव मोनोन्यूक्लियर कोशिकाएं प्राप्त करें। भंवर मानव एफसीआर अवरुद्ध अभिकर्मक, और कोशिकाओं के लिए अभिकर्मक की आवश्यक मात्रा में जोड़ें। पांच मिनट के लिए चार डिग्री सेल्सियस पर कोशिकाओं के साथ ट्यूब सेते हैं.सात कुल कोशिकाओं की शक्ति के लिए 10 प्रति सीडी 11 बी माइक्रोबीड्स के 20 माइक्रोलीटर जोड़ें, और 20 मिनट के लिए चार डिग्री सेल्सियस पर सेते हैं। 70% इथेनॉल के साथ चुंबकीय स्टैंड को अच्छी तरह से कीटाणुरहित करें। इनक्यूबेशन के बाद, 10 मिलीलीटर आइसोलेशन बफर डालें।मिक्स करें, और ट्यूब को 300 ग्राम पर 10 मिनट के लिए चार डिग्री सेल्सियस पर अपकेंद्रित्र करें। एक बार स्टैंड सूख जाने के बाद, चुंबकीय स्तंभ को चुंबकीय स्टैंड पर रखें। सतह पर तैरनेवाला हटाने के बाद, अलगाव बफर के एक मिलीलीटर जोड़ें और विंदुक के साथ धीरे मिश्रण.फिर स्तंभ पर सेल निलंबन लोड और अलगाव बफर के तीन मिलीलीटर के साथ यह तीन बार धोने हर बार स्तंभ जलाशय खाली है. चुंबकीय भाग को छूने के बिना स्तंभ को 15-मिलीलीटर शंक्वाकार ट्यूब में रखें। पांच मिलीलीटर अलगाव बफर जोड़ने के बाद, स्तंभ के माध्यम से तरल को बाहर निकालने के लिए ट्यूब में सवार को धक्का दें।ट्यूब में एकत्र सेल निलंबन अपकेंद्रित्र। सतह पर तैरनेवाला महाप्राण और अलगाव माध्यम में कोशिकाओं को फिर से निलंबित करें। सेल गिनती के बाद, एक 24 अच्छी तरह से थाली के प्रत्येक अच्छी तरह से में सेल निलंबन के 500 माइक्रोलीटर विभाज्य, और रात भर सेते हैं.माइक्रोग्लिया जैसी कोशिकाओं को प्रेरित करने के लिए, पिछले दिन के बीजारोपण से अलगाव माध्यम को एक प्रेरण माध्यम से बदलें और कोशिकाओं को 14 दिनों के लिए इनक्यूबेट करें। प्रेरित माइक्रोग्लिया जैसी कोशिकाओं ने मिनट सोमा निकायों और कई शाखित संपार्श्विक का प्रदर्शन किया।

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 ...

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

मस्तिष्क अनुसंधान के लिए प्रेरित माइक्रोग्लिया जैसी कोशिकाओं को प्राप्त करने के लिए पूरे रक्त से मोनोन्यूक्लियर कोशिकाओं का अलगाव