isolation of mononuclear cells from whole blood to obtain induced microglia-like cells for brain research

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.

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

शुरू करने के लिए, 15-मिलीलीटर सेंट्रीफ्यूजेशन ट्यूब में घनत्व ढाल माध्यम के 15 मिलीलीटर डालें और इसे 20 डिग्री सेल्सियस पर एक मिनट के लिए 1, 000 ग्राम पर स्पिन करें। हेपरिन में एकत्र मानव रक्त युक्त ट्यूब पलटना, और आग निष्फल चिमटी का उपयोग कर, रक्त संग्रह ट्यूब से ढक्कन हटा दें. ट्यूब में घनत्व-ढाल माध्यम में लगभग 10 से 15 मिलीलीटर रक्त का वितरण करें।अपकेंद्रित्र के मंदी स्तर को एक पर सेट करें और कमरे के तापमान पर 10 मिनट के लिए 1, 000 ग्राम पर रक्त के साथ ट्यूब को अपकेंद्रित्र करें। बफी कोट के ऊपर एक सेंटीमीटर तक प्लाज्मा की ऊपरी परत निकालें। सेंट्रीफ्यूजेशन ट्यूब से शेष सतह पर तैरनेवाला को एक और अपकेंद्रित्र ट्यूब में छान लें जिसमें पीबीएस के 10 मिलीलीटर होते हैं।अपकेंद्रित्र के मंदी स्तर को तीन पर रीसेट करें और पीबीएस और सतह पर तैरनेवाला के साथ ट्यूब को अपकेंद्रित्र करें। सतह पर तैरनेवाला महाप्राण और धीरे गोली ढीला करने के लिए ट्यूब नल. अगला, अपकेंद्रित्र ट्यूब में अलगाव माध्यम के 13 मिलीलीटर शामिल हैं और कोशिकाओं को ठीक से इकट्ठा करने के लिए भीतरी दीवार धो लें।आग निष्फल चिमटी का प्रयोग, 15 मिलीलीटर शंक्वाकार ट्यूब पर एक 100 माइक्रोमीटर सेल छलनी स्थिति. फिर सेल स्ट्रेनर के माध्यम से सेल सस्पेंशन को 15-मिलीलीटर शंक्वाकार ट्यूब में फ़िल्टर करें। दो 15 मिलीलीटर शंक्वाकार ट्यूबों में सेल निलंबन के बराबर मात्रा बांटना.अलगाव बफर की उचित एकाग्रता की गणना करने के लिए कोशिकाओं की गणना करें। चार डिग्री सेल्सियस पर 10 मिनट के लिए 250 ग्राम पर कोशिकाओं को गोली मारें। सतह पर तैरनेवाला हटाने के बाद, अलगाव बफर और विंदुक की उचित मात्रा में जोड़ें गोली ढीला करने के लिए लगभग 20 बार.

Research

JoVE Journal

Immunology and Infection

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

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 ...

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

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