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

Sviluppo di cellule simili alla microglia indotte dal sangue per lo studio dei disturbi cerebrali umani

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.

Autore in primo piano: La tecnologia cellulare microglia-simile indotta per far luce sul ruolo della disfunzione microgliale nei disturbi neuropsichiatrici

Ingegnerizzazione di cellule simili alla microglia indotte dal sangue umano per la ricerca cerebrale traslazionale inversa

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.

Isolamento di cellule mononucleate dal sangue intero per ottenere cellule simili alla microglia indotte per la ricerca sul cervello

Per iniziare, metti 15 millilitri del mezzo del gradiente di densità in una provetta da centrifugazione da 15 millilitri e girala a 1.000 G per un minuto a 20 gradi Celsius. Capovolgere la provetta contenente il sangue umano raccolto nell'eparina e, utilizzando una pinzetta sterilizzata a fuoco, rimuovere il coperchio dalla provetta di raccolta del sangue. Erogare circa 10-15 millilitri di sangue nel mezzo di gradiente di densità nella provetta.Impostare il livello di decelerazione della centrifuga su uno e centrifugare la provetta con sangue a 1.000 G per 10 minuti a temperatura ambiente. Rimuovere lo strato superiore di plasma fino a un centimetro sopra il buffy coat. Decantare il surnatante rimanente dalla provetta da centrifugazione in un'altra provetta da centrifuga contenente 10 millilitri di PBS.Reimpostare il livello di decelerazione della centrifuga a tre e centrifugare la provetta con PBS e surnatante. Aspirare il surnatante e picchiettare delicatamente il tubo per allentare il pellet. Quindi, incorporare 13 millilitri di terreno isolante nella provetta da centrifuga e lavare la parete interna per raccogliere correttamente le cellule.Utilizzando una pinzetta sterilizzata a fuoco, posizionare un colino cellulare da 100 micrometri sul tubo conico da 15 millilitri. Quindi filtrare la sospensione cellulare attraverso il filtro cellulare nel tubo conico da 15 millilitri. Erogare quantità uguali di sospensione cellulare in due provette coniche da 15 millilitri.Enumerare le celle per calcolare la concentrazione appropriata del tampone di isolamento. Pellettare le celle a 250 G per 10 minuti a quattro gradi Celsius. Dopo aver rimosso il surnatante, aggiungere la quantità appropriata di tampone isolante e pipettare circa 20 volte per allentare il pellet.

isolation-mononuclear-cells-from-whole-blood-to-obtain-induced

Research

JoVE Journal

Immunology and Infection

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

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

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various neuropsychiatric and physical diseases. Postmortem brain analysis and positron emission tomography imaging are representative research methods that evaluate microglial activation in human patients; the findings have revealed the activation of microglia in the brains of patients presenting with various neuropsychiatric disorders and chronic pain. Nonetheless, the aforementioned technique merely facilitates the assessment of limited aspects of microglial activation. 
In lieu of brain biopsy and the induced pluripotent stem cell technique, we initially devised a technique to generate directly induced microglia-like (iMG) cells from freshly derived human peripheral blood monocytes by supplementing them with granulocyte-macrophage colony-stimulating factor and interleukin 34 for 2 weeks. These iMG cells can be employed to perform dynamic morphological and molecular-level analyses concerning phagocytic capacity and cytokine releases following cellular-level stress stimulation. Recently, comprehensive transcriptome analysis has been used to verify the similarity between human iMG cells and brain primary microglia. 
The patient-derived iMG cells may serve as key surrogate markers for predicting microglial activation in human brains and have aided in the unveiling of previously unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease, fibromyalgia, bipolar disorder, and Moyamoya disease. Therefore, the iMG-based technique serves as a valuable reverse-translational tool and provides novel insights into elucidating dynamic the molecular pathophysiology of microglia in a variety of mental and physical diseases.

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

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

Ricerca

Immunologia e infezioni

To begin, put 15 milliliters of the density gradient medium in a 15-milliliter centrifugation tube and spin it at 1, 000 G for one minute at 20 degrees celsius. Invert the tube containing human blood collected in heparin, and using fire sterilized tweezers, remove the lid from the blood collection tube. Dispense around 10 to 15 milliliters of blood to the density-gradient medium in the tube.Set the deceleration level of the centrifuge to one and centrifuge the tube with blood at 1, 000 G for 10 minutes at room temperature. Remove the top layer of plasma up to one centimeter above of the buffy coat. Decant the remaining supernatant from the centrifugation tube into another centrifuge tube containing 10 milliliters of PBS.Reset the deceleration level of the centrifuge to three and centrifuge the tube with PBS and supernatant. Aspirate the supernatant and gently tap the tube to loosen the pellet. Next, incorporate 13 milliliters of isolation medium into the centrifuge tube and wash the inner wall to collect the cells properly.Using fire sterilized tweezers, position a 100-micrometer cell strainer on the 15-milliliter conical tube. Then filter the cell suspension through the cell strainer into the 15-milliliter conical tube. Dispense equal amounts of cell suspension into two 15-milliliter conical tubes.Enumerate the cells to calculate the appropriate concentration of isolation buffer. Pellet the cells at 250 G for 10 minutes at four degrees celsius. After removing the supernatant, add the appropriate amount of isolation buffer and pipette around 20 times to loosen the pellet.

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.

Ottenimento di cellule simili alla microglia indotte da monociti derivati da sangue intero per sondare le dinamiche della microglia nelle malattie umane 