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

Summary

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.

Abstract

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.

Introduction

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 techniques^1,2,3,4. 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) cells^5,6, 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 world^8,9,10,11. 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 microglia^12,13,14,15, 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 cells^7,16 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 P2RY12^5,17. 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 disorder^20,21, 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 tool^{23,24,25,26,27}. 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.

Protocol

The study protocol was approved by the Ethics Committee of Kyushu University and complied with all the provisions of the Declaration of Helsinki. Written informed consent was obtained from all participants, including healthy volunteers and patients, to analyze their blood and publish their data. Materials and equipment are listed in the Table of Materials, and the compositions of the solutions are detailed in Table 1.

1. Preparation of media and buffers for experiments

1. Prepare the isolation medium by supplementing RPMI-1640 with 10% heat-inactivated fetal bovine serum (56 °C, 30 min) and 1 % penicillin-streptomycin.
2. Prepare the isolation buffer by supplementing the basic buffer solution with 0.5% bovine serum albumin (BSA) stock solution and sterilize it via filtration (0.22 µm).

2. Isolation of mononuclear cells from whole blood

1. Transfer 15 mL of the density gradient medium to a 50-mL density gradient centrifugation tube and centrifuge at 1,000 × g for 1 min at 20 °C.
   NOTE: Isolate 15 mL of blood per 50-mL density gradient centrifugation tube. Increase the number of tubes when blood volume is high.
2. Homogenize the blood collected in heparin tubes by inversion and remove the lid from the blood collection tube with fire-sterilized tweezers.
3. Dispense an equal volume (10-15 mL) of blood to the density gradient medium in the density gradient centrifugation tube.
4. Set the deceleration level of the centrifuge to 1 of 4 levels and centrifuge at 1,000 × g for 10 min at room temperature (RT).
   NOTE: At this deceleration rate, a rotational speed of 180 × g (1,000 rpm) arrests in 3 min.
5. Prepare an equal number of 50-mL centrifuge tubes as the 50-mL density gradient centrifugation tubes and fill each tube with 10 mL of PBS (−).
6. Remove the top layer of plasma up to approximately 1 cm above the buffy coat following centrifugation.
   NOTE: Insert an aspirating pipette vertically into the tube and aspirate carefully from the center of the liquid surface. Do not overaspirate to avoid disturbing the buffy coat.
7. Decant all of the remaining supernatant from the 50 mL density gradient centrifugation tube into a centrifuge tube containing 10 mL of PBS (−).
8. Reset the deceleration level of the centrifuge to 3 of 4 levels, and centrifuge at 250 × g for 10 min at RT. During this step, prepare one 50-mL and two 15-mL centrifuge tubes and a 100-µm cell strainer.
9. After centrifugation, establish the temperature of the centrifuge at 4 °C. Search for a white pellet with a red periphery at the bottom of the centrifuge tube. Remove the supernatant by aspiration and loosen the pellet gently by tapping.
10. Position a 100-µm cell strainer on the 50-mL conical tube using fire-sterilized tweezers.
11. Incorporate 13 mL of isolation medium into one of the centrifuged tubes and wash the inner wall to collect the cells properly. Take the cell suspension and wash the remaining centrifuged tubes in a consecutive manner. After collecting the cells from all the tubes, filter the cell suspension through a cell strainer into the 50-mL conical tube.
    NOTE: The isolation medium needs to be chilled throughout the isolation process (steps 2.11-2.16).
12. Wash each tube again with 13 mL of isolation medium as described in step 2.11. Collect a total of 26 mL of cell suspension.
13. Dispense equal amounts into two 15-mL conical tubes, enumerate the cells, and calculate the appropriate concentration of isolation buffer (60 µL of buffer per 10⁷ total cells) for step 2.16.
    NOTE: The 15-mL conical tube is better to work with because the liquid volume is small and the liquid bubbles easily when pipetting in step 2.16.
14. Centrifuge at 250 × g for 10 min at 4 °C.
15. During the centrifugation, thoroughly disinfect the magnetic stand (especially the part in contact with the column) with 70% ethanol and place it on the bench to air dry. Keep the isolation buffer on ice.
16. Remove the supernatant by aspiration (at this stage, look for red pellets ~1 mm thick because of the presence of red blood cells), add the appropriate amount of isolation buffer, and loosen the pellet gently by pipetting ~20x.

3. Isolation of monocytes using CD11b microbeads

1. Vortex human FcR blocking reagent for 10 s prior to application. Incorporate the required amount of reagent (20 µL of FcR Blocking Reagent per 10⁷ total cells) and incubate in a chamber at 4 °C for 5 min.
2. Vortex the CD11b microbeads for 10 s prior to application. Incorporate the required amount of reagent (20 µL of CD11b microbeads per 10⁷ total cells), and incubate in a 4 °C chamber for 20 min while agitating by hand every 5 min.
3. After incubation, add 10 mL of isolation buffer, suspend by gentle pipetting, and centrifuge at 300 × g for 10 min at 4 °C.
4. During centrifugation, place the magnetic column on the magnetic stand and rinse it with 3 mL of isolation buffer.
   NOTE: Refrain from touching the magnetic part of the column and position the column correctly. If bubbles enter the column, remove them with a pipette. The isolation buffer is mandated to be chilled throughout the isolation process (steps 3.4-3.9).
5. Remove the post-centrifugation supernatant by aspiration, incorporate 1 mL of isolation buffer, mix by pipetting gently, and apply the suspension onto the column.
6. Wash the column 3x by adding 3 mL of isolation buffer each time the column reservoir is empty.
7. Place the column in a 15-mL conical tube without touching the magnetic part; incorporate 5 mL of isolation buffer into the column and force the liquid out through the column with a plunger inserted into the tube.
8. Centrifuge the cell suspension collected in the tube at 300 × g for 10 min at 4 °C.
9. Aspirate the supernatant and resuspend the cells in isolation medium. As ~5-10% of the number of cells counted in step 2.13 will be recovered, adjust the suspension volume according to the number of cells.
   NOTE: Isolation medium should be used at RT and not warmed to avoid rapid temperature change from 4 °C.
10. Enumerate the cells and aliquot 500 µL of cell suspension into each well of 24-well plates at a concentration of 40 × 10⁴ cells/mL and incubate overnight.
    NOTE: The cell suspensions should be pipetted appropriately in accordance with the adjusted seeding volume required for different plates. For example, 1 mL for one well in a 12-well plate and 250 µL for one well in an 8-well chamber slide.
11. Finally, dispose of or disinfect the contaminated equipment in accordance with the policy for handling infectious waste established by the facility.

4. Induction of iMG cells from monocytes

1. Prepare the induction medium by supplementing basal induction medium with 1% antibiotic-antimycotic solution, 10 ng/mL recombinant human GM-CSF, and 100 ng/mL recombinant human IL-34.
2. Replace the isolation medium from the seeding of the previous day with induction medium. Tilt the plate forward and promptly aspirate the depleted medium from the front edge of the well bottom with a Pasteur pipette. Replace the medium at the rate of a maximum of 1 plate (= 24 wells) at a time, and immediately incorporate 500 µL of induction medium gently.
   NOTE: Adhered monocytes should be selected in this step.
3. Incubate the cells for 14 days to complete the induction.
4. Replace the medium again with 500 µL of induction medium after 14 days. Subsequently, for post-induction maintenance of the cells, replace the induction medium with 500 µL of fresh induction medium every week.

5. Immunocytochemistry

1. Remove the medium from the 8-well chamber slide seeded with cells in step 3.10 by aspiration and rinse with PBS (−).
2. Fix the cells for 20 min at RT with 4% paraformaldehyde.
3. Remove the fixative reagent and rinse the cells for 3 x 5 min with PBS (−).
4. Permeabilize the cells with PBS (−) containing 0.1% Triton X-100 at RT.
5. Incubate the permeabilized cells with a blocking solution (3% BSA/PBS (−)) for 1 h at RT.
6. Incubate the cells overnight with primary antibodies (Table of Materials) at 4 °C.
   NOTE: All antibodies are diluted in blocking solution.
7. Rinse the cells for 3 x 5 min with PBS (−) for 5 min each.
8. Incubate the rinsed cells with the appropriate fluorescently tagged secondary antibodies for 1 h at RT (Table of Materials).
9. Rinse the cells for 3 x 5 min with PBS (−) to remove the secondary antibodies.
10. Incubate the cells with DAPI (1:10,000) solution diluted with PBS (−) for 5 min at RT.
11. Rinse the cells for 3 x 5 min with PBS (−).
12. Mount the well using the mounting medium and visualize the cells under a fluorescence microscope.

Results

Importantly, there is a great deal of person-level and timing-level heterogeneity in the characters of iMG cells including morphologies and gene expressions. The iMG cells in certain individuals assume a numerous branching appearance (Figure 1A), while in others they remain spherical (Figure 1B). The iMG characteristics may differ even within a single individual, rendering iMG cells as a pivotal tool for detecting disease state biomarkers. Conversely, the examination of such inter-individual differences warrants the reduction of technical errors. The iMG cells are defined as monocytes induced with GM-CSF and IL-34 for a minimum of 14 days. To minimize technical errors, it is recommended to standardize the induction time, utilize the same amounts of reagents, and refrain from using expired reagents.

Validation of immunostaining also confirms that iMG cells express microglial markers, including P2RY12 and TMEM119 (Figure 2). Prior research has demonstrated that the morphological and gene-expression characteristics of iMG cells are associated with the pathophysiology of bipolar disorder^20,21. The earliest branched cells were observed on DAY 3 after GM-CSF and IL-34 treatment, and iMG cells in optimum condition can survive for more than 1 month when medium change is done once a week. The iMG cells can be used for gene expression analysis. For example, under stimulations with interferon gamma or IL-4, iMG cells changed the gene expression pattern, which can be used for the functional evaluation of M1 and M2 polarized microglia^5,22. Moreover, FITC beads trigger amoeboid-like morphological modifications and upregulate the expression of TNFα and other genes⁵. Furthermore, phagocytosis can be evaluated by measuring the fluorescence intensity of the beads^5,22. Moreover, as iMG cells are viable cells, their actual movement and morphological alterations can be observed by taking cell pictures (Figure 3) or through time-lapse photography (Video 1).

Figure 1: Morphology of iMG cells. (A) Typical ramified iMG exhibited minute soma bodies and numerous branched collaterals. (B) Certain samples varied in appearance such as the ameboid type. Scale bar, 50 µm. Please click here to view a larger version of this figure.

Figure 2: Immunocytostaining of iMG cells. iMG cells express typical microglial markers: (A) TMEM 119, (B) P2RY12. Nuclei were stained with DAPI (blue). Scale bar =10 µm. Please click here to view a larger version of this figure.

Figure 3: Morphological modifications in iMG cells. iMG alter their morphology during the induction process. Cell protrusions appear (A) around day 3 and elongate on days (B) 7 and (C) 14 as induction proceeds. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Video 1: Time-lapse photography of iMG cells. The ramified iMG cell in the center appeared to establish contact with the surrounding cells while extending and contracting their processes. Furthermore, the cell performed phagocytosis of damaged cells. The image acquisition intervals were 46 sec, and the filming time was approximately 14 h. Please click here to download this Video.

Table 1: Composition of solutions Please click here to download this Table.

Discussion

Analytical techniques employing iMG cells may serve as potent reverse-translational research tools^5,6. To generate sufficient quantities of human iMG cells, experimenters should design their studies taking certain issues into consideration. Blood samples derived from human beings are extremely sensitive; consequently, the obtained samples warrant prompt processing, and meticulous handling to avoid contamination. Specifically, blood samples should be separated immediately after being drawn and not left unattended for extended periods. Experiments are conventionally commenced within 3 h of blood collection to confirm their validity; however, it is possible to store blood samples at 4°C for over 3 h and bring them to room temperature prior to starting separation protocols.

To ensure stable results, reagents such as culture media are recommended to be used within a month of unsealing and discarded upon expiration. When separating peripheral blood mononuclear cells using density gradient centrifugation, cell aggregation may be observed in certain specimens. In such cases, the whole blood sample must be diluted with PBS (−) to prevent aggregation. During the induction of differentiation into microglia, the culture medium is not replaced for 14 days to avoid unnecessary stimulation of the cells. After the completion of differentiation induction, medium replacement with fresh medium may restore the weakened cells and maintain viability for a while.

The iMG technique established in this study presents a limitation; the experimentally induced "microglia-like cells" are comparable but not completely identical to the actual microglial cells in the human brain. Additionally, prior research has documented methodology for the induction of human monocyte-derived microglia-like cells, and different research groups have proposed their own distinct protocols and names contingent upon the original iMG methods²⁸. For instance, certain protocols use M-CSF^29,30,31,32 or TGF-β³³, which are reported to promote a microglia-like phenotype in iPSC-derived microglia cultures or on coatings such as Geltrex^24,34; these factors may prove to be effective for inducing microglia-like cells.

The original iMG technology is advantageous owing to its simple and easy-to-reproduce protocol with merely two cytokines and no coatings, which enables a variety of reverse-translational experiments using human blood. Conversely, advancements in technology have facilitated the development of many products. Magnetic separation methods include column-based positive selection, similar to our protocol, as well as negative selection and column-free methods. Moreover, in lieu of magnetic separation, an alternative method of separation using plastic adhesion may be considered. Column-based positive selection ensures purity, but the technique is time-consuming, and stimulation by magnetic beads substantially affects the cells³⁵. Contrarily, negative selection and plastic adhesion may be less detrimental to the cells; nevertheless, these approaches may be less pure^35,36. Further modification may be needed based on other protocols in the future.

The aforementioned findings substantiate that the predominant advantage of this iMG technology is its brief duration, cost-effective methodology, and ability to process a large number of samples for high-throughput studies. Therefore, this technology can be employed to acquire knowledge from human samples and then conduct further validation studies using different animal models. Moreover, the findings obtained from investigations conducted on cellular and animal models by applying the iMG technique may be beneficial. In this way, our iMG technique can be applied to bi-directional translational research in a bedside-to-bench and bench-to-bedside manner.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% Triton X-100	Sigma-Aldrich	30-5140-5
4% paraformaldehyde	Nacalai Tesque	09154-14
Antibiotic-Antimycotic (100x)	gibco	15240-062	described as "antibiotic-antimycotic solution"
autoMACS Rinsing Solution	Miltenyi Biotec	130-091-222	described as "basic buffer solution" and used for "isolation buffer"
CD11b MicroBeads	Miltenyi Biotec	130–049-601
DAPI solution	DOJINDO	28718-90-3
Dulbecco's Phosphate Buffered Saline	Nacalai Tesque	14249-24	described as "PBS (−)"
Fetal Bovine Serum	biowest	S1760-500
Human FcR Blocking Reagent	Miltenyi Biotec	130–059-901
Leucosep	Greiner Bio-One	227290	described as "density gradient centrifugation tube"
MACS LS columns	Miltenyi Biotec	130-042-401	described as "magnetic column"
MACS BSA Stock Solution	Miltenyi Biotec	130-091-376	described as "bovine serum albumin (BSA) stock solution"
MACS MultiStand	Miltenyi Biotec	130-042-303	described as "magnetic stand"
Penicillin-Streptomycin	Nacalai Tesque	26253–84
ProLong Gold Antifade Mountant	Invitrogen	P10144	described as "mounting media"
recombinant human GM-CSF	R&D Systems	215-GM
recombinant human IL-34	R&D Systems	5265-IL
RPMI 1640 Medium + GlutaMAX Supplement (pre-supplemented medium)	Thermo Fisher Scientific	61870036	described as "basal induction medium"
RPMI-1640	Nacalai Tesque	30264-56
Antibodies
anti-P2RY12 antibody	Sigma-Aldrich	HPA014518	primary antibody, rabbit, 1:100
anti-TMEM119 antibody	Sigma-Aldrich	HPA051870	primary antibody, rabbit, 1:100
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	invitrogen	A-11011	secondary antibody, rabbit, 1:1000