Name: differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (cb-sc)-derived exosomes
Uploaded: 2020-11-12T13:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes at JoVE.com

We are grateful to Mr. Poddar and Mr. Ludwig for generous funding support via Hackensack UMC Foundation. We appreciate Laura Zhao for English editing.

The protocol follows the guidelines of institutional human research ethics committee at Center for Discovery and Innovation, Hackensack Meridian Health. Human buffy coat blood units were purchased from the New York Blood Center (New York, NY). Human umbilical cord blood units were collected from healthy donors and purchased from Cryo-Cell International blood bank (Oldsmar, FL). Both New York Blood Center and Cryo-Cell have received all accreditations for blood collections and distributions, with IRB approval and signed Consent Forms from donors.
1. Cell culture and preparation of CB-SC-derived conditioned medium
<ol>
	<li>Transfer 25 mL of cord blood (Table of Materials) over 20 mL of density gradient medium (&#947; = 1.077) into a 50 mL conical tube.</li>
	<li>Centrifuge at 1,690 x g for 20 min at 20 &#176;C in a swinging-bucket rotor without brake.</li>
	<li>Carefully transfer the mononuclear cell layer (buffy coat) to a new 50 mL conical tube. Fill the conical tube with phosphate buffered saline (PBS) to 40 mL. Mix and centrifuge to pellet cells at 751 x g for 10 min at 20 &#176;C.</li>
	<li>Discard the supernatant and add 15 mL of ACK lysis buffer (Table of Materials) to the cell pellet. Re-suspend cells through pipetting. Then incubate for 10 min at room temperature. 
	NOTE: This step removes the red blood cells.</li>
	<li>Fill the conical tube with 25 mL of PBS. Centrifuge at 751 x g for 5 min and discard supernatant to obtain pelleted mononuclear cells.</li>
	<li>Wash 2x with 40 mL of PBS to remove the remaining lysis buffer.</li>
	<li>Centrifuge at 751 x g for 5 min to pellet the cells.</li>
	<li>Discard the supernatant and re-suspend cord blood mononuclear cells with 10 mL of chemical-defined serum-free medium (Table of Materials) per tube.</li>
	<li>Combine cord blood mononuclear cells to one tube.</li>
	<li>Take 20 &#181;L cell suspension and mix with 20 &#181;L of 0.4% trypan blue solution (Table of Materials) in a 1.5 mL tube.</li>
	<li>Load into the chamber slide and quantify the cell number and cell viability with an automated cell counter. 
	NOTE: Cell suspension is diluted at 1:10 if cell concentration is above 1 x 107 cells/mL.</li>
	<li>Seed mononuclear cells in 150 mm x 15 mm Petri dishes at 1 x 106 cells/mL, 25 mL/dish in chemical-defined serum-free cell culture medium.</li>
	<li>Incubate at 37 &#176;C under 8% CO2 conditions for 10&#8210;14 days until CB-SC reach more than 80% confluence.</li>
	<li>Discard the supernatant and wash with 15 mL of PBS per Petri dish; then, remove the PBS. 
	NOTE: CB-SC are attached to Petri dishes tightly.</li>
	<li>Repeat step 1.14 two times.</li>
	<li>Add 25 mL of chemical-defined serum free medium per Petri dish.</li>
	<li>Incubate at 37 &#176;C under 8% CO2 conditions for 3&#8210;4 days.</li>
	<li>Collect the CB-SC-derived conditioned medium into 50 mL conical tubes.</li>
</ol>
2. Characterization of CB-SC
<ol>
	<li>Detach CB-SC by pipetting 10 mL of PBS-based cell dissociation buffer up and down with a 5 mL pipette tip (Table of Materials).</li>
	<li>Centrifuge at 1,690 x g for 5 min to pellet cells and re-suspend in 200 &#181;L of PBS.</li>
	<li>Fix and permeabilize cells for intracellular staining via staining preparation kit (Table of Materials).</li>
	<li>Add 5 &#181;L of Fc blocker (Table of Materials) per sample and incubate for 15 min at room temperature. 
	NOTE: Fc blocker inhibits non-specific binding when staining with antibodies.</li>
	<li>Add fluorescence-conjugated mouse anti-human monoclonal antibodies including CD34, CD45, SOX2, OCT3/4, CD270, and Galectin 9 at 25 &#181;g/mL (Table of Materials) to 100 &#956;L volume of cells. Incubate for 30 min at room temperature with light protection.</li>
	<li>After staining, wash cells with 1 mL of PBS and centrifuge at 751 x g for 10 min to pellet cells.</li>
	<li>Re-suspend cells with 200 &#956;L of PBS and transfer into a 5 mL tube.</li>
	<li>Perform flow cytometry to validate the expression of CB-SC-associated above specific markers.</li>
</ol>
3. Isolation of CB-SC-derived exosomes
<ol>
	<li>Centrifuge the conditioned medium collected from step 1.18 at 300 x g for 10 min at 4 &#176;C. Transfer the supernatant to a new 50 mL conical tube.</li>
	<li>Centrifuge the supernatant collected from step 3.1 at 2,000 x g for 20 min at 4 &#176;C. Transfer the supernatant to a new 50 mL conical tube.</li>
	<li>Centrifuge the supernatant collected from step 3.2 at 10,000 x g for 30 min at 4 &#176;C. Transfer the supernatant to a new 50 mL conical tube. 
	NOTE: The fixed angle rotor is used so that the cell pellets are precipitated to the side of the tube. Mark the side of the cap and draw a circle on the side of the tube where the pellet is expected.</li>
	<li>Filter supernatants collected from step 3.3 with a 0.22 &#181;m filter (Table of Materials).</li>
	<li>Transfer 15 mL of media to each 10 kDa centrifugal filter unit (Table of Materials).</li>
	<li>Centrifuge at 4,000 x g for 30 min to isolate the concentrated exosome media.</li>
	<li>Transfer the concentrated exosomes to an ultracentrifuge tube. Then, pellet exosomes at 100,000 x g for 80 min at 4 &#176;C.</li>
	<li>Discard the supernatant and re-suspend the pellet exosomes in 10 mL of PBS.</li>
	<li>Centrifuge at 100,000 x g for 80 min at 4 &#176;C to collect the exosomes pellet.</li>
	<li>Re-suspend the exosomes pellet in 200 &#181;L of PBS by pipetting up and down.</li>
</ol>
4. Characterization of CB-SC-derived exosomes
<ol>
	<li>Quantifying total protein concentration of exosome preparation by bicinchoninic acid assay (BCA) kit
	<ol>
		<li>Pipette 10 &#181;L of each albumin standard and isolated exosome sample prepared in step 3.10 into a 96-well plate in duplicate.</li>
		<li>Add 200 &#181;L of the working reagent from the BCA kit to each well. Mix contents of the plate thoroughly on the plate shaker for 10 s. 
		NOTE: Working reagent (WR): 50-part reagent A with 1-part reagent B.</li>
		<li>Cover the plates with foil and incubate them at 37 &#176;C for 30 min.</li>
		<li>Cool the plates to room temperature (RT).</li>
		<li>Measure sample absorbance at 562 nm via a plate reader.</li>
	</ol>
	</li>
	<li>Preparation and staining of exosomes for flow cytometry
	<ol>
		<li>Capture exosomes by adding 20 &#956;L of anti-human CD63 magnetic beads (4.5 &#956;m size) (Table of Materials) into 25 &#956;g of CB-SC-derived exosomes prepared in step 3.10 in total 100 &#956;L volume of PBS.</li>
		<li>Incubate the tube overnight (18&#8210;22 h) at 4 &#176;C on the shaker at 800 rpm.</li>
		<li>Centrifuge the tube at 300 x g for 30 s to collect the sample at the bottom of the tube.</li>
		<li>Add 300 &#181;L of isolation buffer (0.1% bovine serum albumin (BSA) in PBS) and mix gently by pipetting. 
		NOTE: This step washes the bead-bound exosomes.</li>
		<li>Place the tube on a magnet stand for 1 min (Table of Materials) and discard the supernatant.</li>
		<li>Repeat steps 4.2.4&#8210;4.2.5.</li>
		<li>Re-suspend the bead-bound exosomes with 400 &#181;L of isolation buffer.</li>
		<li>Aliquot 100 &#181;L of bead-bound exosomes to each tube.</li>
		<li>Add fluorescence-conjugated antibodies (CD9-FITC, CD81-PE, and CD63-FITC at 25 &#181;g/mL, respectively) to each flow tube with CD63 bead-captured exosomes. 
		NOTE: Isotype-matched IgGs serve as negative controls.</li>
		<li>Incubate for 45 min at room temperature with light protection on the shaker at 800 rpm.</li>
		<li>Repeat steps 4.2.4&#8210;4.2.5.</li>
		<li>Re-suspend the bead-bound exosomes in 200 &#181;L of isolation buffer and transfer to 5 mL flow tubes.</li>
		<li>Place the tubes in the sample carousel of the flow cytometer.</li>
		<li>Open the protocol for exosome testing.</li>
		<li>Run the sample automatically by flow cytometer.</li>
	</ol>
	</li>
	<li>Exosome detection by western blot 
	NOTE: Western blot is a well-established method and we will not go into details of the method itself.
	<ol>
		<li>Lyse the pellets of CB-SC-derived exosomes from step 3.10&#160;with 100 &#181;L of RIPA buffer, pipette 20x, then place on ice for 5 min.</li>
		<li>Quantify the protein concentration of exosome lysate by BCA kit and load 25 &#181;g of protein per well.</li>
		<li>Separate the proteins by gel electrophoresis for 40 min at 150 V.</li>
		<li>Transfer the protein to polyvinylidene fluoride (PVDF) membrane using semi-dry transferring method11.</li>
		<li>Block the membrane with 5% non-fat milk for 30 min.</li>
		<li>Incubate with 2 &#181;g/mL anti-human Alix&#160;(Table of Materials) and 1 &#181;g/mL anti-human Calnexin&#160;antibodies (Table of Materials).</li>
		<li>Detect the protein by chemiluminescence with a digital imaging system.</li>
	</ol>
	</li>
	<li>Exosome validation by dynamic light scattering (DLS)
	<ol>
		<li>Dilute 10 &#181;g of CB-SC-derived exosome samples in 1 mL of PBS.</li>
		<li>Transfer the 1 mL of diluted sample into disposable semi-micro cuvette (Table of Materials).</li>
		<li>Place the cuvette in the DLS instrument. Set the refractive index (RI) as 1.39 for all the sample monitor.</li>
		<li>Run samples at 25 &#176;C and acquire three measurements per fraction to get an average size distribution.</li>
	</ol>
	</li>
	<li>Exosome validation by transmission electron microscopy (TEM)
	<ol>
		<li>Coat formvar on 300 mesh copper grids12 (Table of Materials).</li>
		<li>Strengthen the formvar with the additional layer of evaporated carbon on copper grids12. 
		NOTE: Such a coating approach is excellent for specimen support.</li>
		<li>Load 10 &#181;L of exosome samples onto grids and leave to air-dry.</li>
		<li>Negatively stain samples with uranyl acetate for 5 min.</li>
		<li>Wash three times with DI water and leave to air-dry.</li>
		<li>Observe and photograph the samples under TEM. Set the accelerating voltage at 200 kV and spot size at 2.</li>
	</ol>
	</li>
</ol>
5. Measure Dio-labeled CB-SC-derived exosomes up taken by different subpopulations of PBMC
<ol>
	<li>Label CB-SC-derived exosomes with green fluorescent lipophilic dye Dio
	<ol>
		<li>Transfer 100 &#181;g of CB-SC-derived exosomes (prepared in step 3.10) into a 15 mL centrifuge tube.</li>
		<li>Dilute sample with PBS to 5 mL.</li>
		<li>Add green fluorescent lipophilic dye Dio (Table of Materials) until working concentration reaches 5 &#181;M.</li>
		<li>Incubate for 15 min at room temperature protected from light.</li>
		<li>Transfer the sample into an ultracentrifuge tube.</li>
		<li>Centrifuge at 100,000 x g for 80 min to pellet Dio-labeled CB-SC-derived exosomes.</li>
		<li>Re-suspend the labeled exosomes in 200 &#181;L of PBS.</li>
	</ol>
	</li>
	<li>Preparation of human PBMC
	<ol>
		<li>Transfer 25 mL of human buffy coat (Table of Materials) over 20 mL of density gradient medium (&#947; = 1.077) into a 50 mL conical tube.</li>
		<li>Repeat steps 1.2 to 1.11.</li>
		<li>Transfer 1 x 106 PBMC into a non-tissue-treated hydrophobic 24-well plate (1 mL/well). 
		NOTE: A non-tissue-treated plate was used to avoid adhering of monocytes.</li>
	</ol>
	</li>
	<li>Co-culture Dio-labeled exosomes with PBMC
	<ol>
		<li>Transfer 40 &#181;L Dio-labeled CB-SC-derived exosomes prepared in step 5.1.7 to each PBMC-containing well in a 24-well plate using a 200 &#181;L pipette. Add the same volume of PBS to control wells.</li>
		<li>Mix by pipetting 10x. Incubate for 4 h.</li>
		<li>Collect 200 &#956;L exosome-treated PBMC and label with Hoechst 33342 for 10 min at room temperature.</li>
		<li>Centrifuge at 300 x g for 10 min at room temperature. Discard the supernatant and re-suspend the cell pellet in 100 &#181;L of PBS.</li>
		<li>Mount cells onto microscope slides.</li>
		<li>Observe and photograph the interaction of Dio-labeled CB-SC-derived exosomes with Hoechst 33342-labeled PBMC using a microscope.</li>
		<li>Transfer the remaining cells from step 5.3.3 into a 1.5 mL tube.</li>
		<li>Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant and re-suspend the cell pellet in 200 &#181;L of PBS.</li>
		<li>Add 5 &#181;L of Fc blocker&#160;per sample. Incubate for 15 min at room temperature.</li>
		<li>Add antibodies (CD3, CD4, CD8, CD11c, CD14, CD19, and CD56 at 25 &#181;g/mL) (Table of Materials) to stain PBMC. 
		NOTE: Isotype-matched IgGs serve as negative controls.</li>
		<li>Incubate for 30 min at room temperature with light protection.</li>
		<li>Add 1 mL of PBS and centrifuge at 300 x g for 10 min at 4 &#176;C to pellet the cells.</li>
		<li>Re-suspend the cells with 200 &#181;L PBS. Add 5 &#181;L of propidium iodide.</li>
		<li>Use flow cytometry to evaluate the level of Dio-labeled exosome uptake in different subpopulation of PBMC.</li>
	</ol>
	</li>
</ol>
6. Examine the action of CB-SC-derived exosomes on monocytes
<ol>
	<li>Isolation human CD14-positive monocytes
	<ol>
		<li>Transfer 3 x 107 human PBMC into a 15 mL tube.</li>
		<li>Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
		<li>Place the separation column (Table of Materials) in the magnet separator (Table of Materials).</li>
		<li>Wash separation columns three times with 2 mL of cold running buffer (Table of Materials).</li>
		<li>Re-suspend the cells in 300 &#181;L of cold PBS. Add 60 &#181;L of CD14 microbeads. Mix well and incubate on ice for 15 min.</li>
		<li>Add 6 mL of cold PBS. Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
		<li>Re-suspend the pelleted cells in 500 &#181;L of cold running buffer.</li>
		<li>Transfer cells into the separation column (prepared in step 6.14) and let them pass through.</li>
		<li>Wash the separation column three times with 2 mL of running buffer per wash. Lift the column from the magnet separator and place it in a 15 mL centrifuge tube. 
		NOTE: The 15 mL tube should be placed on ice due to the adherence of CD14-positive monocytes to the tube at room temperature.</li>
		<li>Transfer 2 mL of cold running buffer to the top of the column and isolate the CD14-positive cells into the 15 mL tube.</li>
		<li>Centrifuge at 300 x g for 10 min at 4 &#176;C to pellet the CD14-positive cells.</li>
		<li>Re-suspend the cells with 2 mL of cold chemical-defined serum free medium (Table of Materials).</li>
		<li>Transfer 50 &#181;L of cells into a 1.5 mL tube.</li>
		<li>Stain with 10 &#181;L of Krome Orange-conjugated anti-human CD14 mAb (Table of Materials) for 20 min. 
		NOTE: Isotype-matched IgGs serve as negative controls.</li>
		<li>Add 1 mL PBS to the cells. Centrifuge at 300 x g for 10 min to pellet cells.</li>
		<li>Re-suspend cells in 200 &#181;L of PBS and transfer it to a 5 mL tube. Determine the purity of CD14-positive monocytes by flow cytometry.</li>
	</ol>
	</li>
	<li>Treatment of monocytes with CB-SC-derived exosomes
	<ol>
		<li>Seed 1 x 106 purified monocytes with chemical-defined serum-free culture medium (Table of Materials) in tissue culture-treated 6-well plate (2 mL/well).</li>
		<li>Incubate for 2 h at 37 &#176;C under 5% CO2.</li>
		<li>Discard the supernatant with 1 mL pipette. Add 2 mL of 37 &#176;C pre-warmed chemical-defined serum-free culture medium (Table of Materials) gently. 
		NOTE: Monocytes were adhered to the plate within 2 h. Floating cells were identified as dead or other cell contaminations.</li>
		<li>Add 80 &#181;g CB-SC-derived exosomes isolated from step 3.10 to monocyte cultures in a 6-well plate with total volume of 2 mL. 
		NOTE: The same volume of PBS was added to control wells.</li>
		<li>Incubate at 37 &#176;C under 5% CO2 for 3&#8210;4 days.</li>
		<li>Photograph the cell morphology using an inverted microscope at 200&#215; magnification (Table of Materials).</li>
		<li>Detach cells by pipetting up and down in 1 mL of a PBS-based cell dissociation buffer with 1 mL pipette tip.</li>
		<li>Harvest the remaining attached cells via a cell scraper. 
		NOTE: Since primary monocytes or differentiated macrophages attach tightly, some cells remain adhered to the bottom after the treatment with dissociation buffer. Therefore, these cells are harvested with a cell scraper.</li>
		<li>Collect cells at 1,690 x g for 5 min. Re-suspend cells in 200 &#181;L of PBS.</li>
		<li>Add 5 &#181;L of Fc blocker (25 &#181;g/mL) to block non-specific binding.</li>
		<li>Add antibodies (CD14, CD80, CD86, CD163, CD206, and CD209 at 25 &#181;g/mL, Table of Materials) to cells. Incubate for 30 min at room temperature. 
		NOTE: Isotype-matched IgGs serve as negative control</li>
		<li>Add 1 mL of PBS to cells and centrifuge at 300 x g for 10 min. Discard the supernatant and re-suspend with 200 &#181;L of PBS.</li>
		<li>Add 5 &#181;L of propidium iodide per sample (200 &#181;L) and transfer cells to a new 5 mL flow tube.</li>
		<li>Perform the flow cytometry and evaluate the levels of CD14, CD80, CD86, CD163, CD206, and CD209 expressions.</li>
	</ol>
	</li>
</ol>

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Dr. Zhao is a founder of Tianhe Stem Cell Biotechnology Inc. Dr. Zhao is an inventor of Stem Cell Educator technology. All other authors have no financial interests that may be relevant to the submitted work.

Application of exosomes is an emerging field for clinical diagnosis, drug developments and regenerative medicine. Here, we present a detailed protocol regarding the preparation of CB-SC-derived exosomes and the functional study of exosomes on the differentiation of human monocytes. The current protocol demonstrated that functional CB-SC-derived exosomes are isolated by sequential centrifugation and ultracentrifugation with high purity and exhibiting the immune modulation on monocytes.
As compared with other conventional protocols, ultrafiltration is an established approach for the isolation and purification of exosomes from different cells or media, based upon the molecular weight and exclusion sizes that are different from other extracellular vesicles (EVs). While ultrafiltration isolation is more time-saving than the ultracentrifugation-based separation, it may cause structural damage to vesicles at large sizes. Exosomes can also be collected by polyethylene glycol (PEG)-mediated precipitation at low cost, though this method risks the exosome purity due to the protein contaminations13,14. Therefore, the current protocol was cost-effective to produce exosomes at high purity. Based on the immune modulations of CB-SC-derived exosomes10, characterization of CB-SC-derived exosomes may offer a valuable biomarker to evaluate the potency of Stem Cell Educator with CB-SC before clinical applications.
Macrophages are professional antigen-presenting cells against viral and bacterial infections, with varied biological functions and heterogeneities. Based on their differences in surface markers and immune function, macrophages are categorized with two sub-populations: type 1 macrophages (M1, conventional macrophages causing inflammation) and type 2 macrophages (M2, displaying anti-inflammation)15. This study established that purified human monocytes were differentiated into type 2 macrophages after the treatment with CB-SC-derived exosomes, displaying an anti-inflammation phenotype10. CB-SC-derived exosome-treated monocytes exhibited the elongated morphology and expressed the M2-associated surface markers (e.g., CD163, CD206, and CD209), with the similar phenotype as the conventional M2 macrophages generated using cytokines M-CSF + IL-4. Such phenotypic changes of monocytes highlight the new mechanism underlying the immune modulation of CB-SC for the treatment of type 1 diabetes and other autoimmune diseases. During the SCE therapy, patients&#8217; immune cells were co-cultured with CB-SC for around 8&#8210;9 h. The SCE-treated monocytes carried the CB-SC-derived exosomes back into the body, which contributed to the M2 differentiation and the expansion of the induction of immune tolerance, leading to the improvement of clinical outcomes after the treatment with SCE therapy.

Cord blood stem cells (CB-SC) are unique type of stem cells identified from human cord blood and are distinguished from other known types of stem cells such as mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC)1. Based on their unique properties of immune modulation and their ability to tightly adhere to the surface of Petri dishes, we developed a new technology designated as Stem Cell Educator (SCE) therapy in clinical trials2,3. During SCE therapy, a patient&#8217;s peripheral blood mononuclear cells (PBMC) are collected and circulated through a cell separator and co-cultured with adherent CB-SC in vitro. These &#8220;educated&#8221; cells (CB-SC-treated PBMC) are then returned to the patient&#8217;s circulation in a closed-loop system. Clinical trials have already demonstrated the clinical safety and efficacy of SCE therapy for the treatment of autoimmune diseases including type 1 diabetes (T1D)2,4 and alopecia areata (AA)5.
Exosomes are a family of nanoparticles with diameters ranging 30&#8210;150 nm and exist in all biofluid and cell culture media6. Exosomes are enriched with many bioactive molecules including lipids, mRNAs, proteins, and microRNAs (miRNA), and play an important role in cell-to-cell communications. Of late, exosomes have become more attractive for researchers and pharmaceutical companies due to their therapeutic potentials in clinics7,8,9. Recently, our mechanistic studies demonstrated that CB-SC-released exosomes contribute to the immune modulation of SCE therapy10.
Here, we describe the protocol to explore the mechanism of SCE therapy targeting monocytes by CB-SC-released exosomes. First, CB-SC-released exosomes were isolated from CB-SC-derived conditioned media using ultracentrifugation methods and validated by flow cytometry, western blot (WB) and dynamic light scattering (DLS). Second, CB-SC-derived exosomes were labeled with a green fluorescent lipophilic dye: Dio. Third, they were co-cultured with PBMC to examine the positive percentages of Dio-labeled CB-SC-derived exosomes at the different subpopulations of PBMC by flow cytometry. This protocol provides guidance to study the action of exosomes underlying the immune modulation of stem cells.

<table><tbody><tr><td>1.5 ml Microcentrifuge tube</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352196</td><td></td></tr><tr><td>24-well plate</td><td>Falcon</td><td>351147</td><td>Non-tissue culture&lt;br/&gt; plate</td></tr><tr><td>3,3&#039;-Diostadecyloxacarbocyanine perchlorate(Dio)</td><td>Millipore sigma</td><td>D4292-20MG</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>300 Mesh Grids</td><td>Ted Pella</td><td>1GC300</td><td></td></tr><tr><td>50 mL conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>6-well plate</td><td>Falcon</td><td>353046</td><td>Tissue culture plate</td></tr><tr><td>96-well plate</td><td>Falcon</td><td>353072</td><td>Tissue culture plate</td></tr><tr><td>ACK Lysis Buffer</td><td>Lonza</td><td>10-548E</td><td>100 ml</td></tr><tr><td>Amicon-15 10kDa Centrifuge Fliter Unit</td><td>Millipore sigma</td><td>UFC901024</td><td></td></tr><tr><td>Anti-Human Alix</td><td>Biolegend</td><td>634501</td><td>store at 4 &amp;deg;C, RRID: AB_2268110</td></tr><tr><td>Anti-Human Calnexin</td><td>Biolegend</td><td>699401</td><td>store at 4 &amp;deg;C, RRID: AB_2728519</td></tr><tr><td>Anti-Human CD11c antibody, Pe-Cy7</td><td>BD Bioscience</td><td>561356</td><td>store at 4 &amp;deg;C, RRID: AB_10611859</td></tr><tr><td>Anti-Human CD14 antibody, Karma Orange</td><td>Beckman Coulter</td><td>B36294</td><td>store at 4 &amp;deg;C, RRID: AB_2728099</td></tr><tr><td>Anti-Human CD163 antibody, PE</td><td>BD Bioscience</td><td>556018</td><td>store at 4 &amp;deg;C, RRID: AB_396296</td></tr><tr><td>Anti-Human CD19 antibody, PC5</td><td>Beckman Coulter</td><td>IM2643U</td><td>store at 4 &amp;deg;C, RRID: AB_131160</td></tr><tr><td>Anti-Human CD206 antibody, FITC</td><td>BD Bioscience</td><td>551135</td><td>store at 4 &amp;deg;C, RRID: AB_394065</td></tr><tr><td>Anti-Human CD209 antibody, Brilliant Violet 421</td><td>BD Bioscience</td><td>564127</td><td>store at 4 &amp;deg;C, RRID: AB_2738610</td></tr><tr><td>Anti-Human CD270 antibody, PE</td><td>Biolegend</td><td>318806</td><td>store at 4 &amp;deg;C, RRID: AB_2203703</td></tr><tr><td>Anti-Human CD3 antibody, Pacfic Blue</td><td>Biolegend</td><td>300431</td><td>store at 4 &amp;deg;C, RRID: AB_1595437</td></tr><tr><td>Anti-Human CD34 antibody, APC</td><td>Beckman Coulter</td><td>IM2427U</td><td>store at 4 &amp;deg;C, RRID: N/A</td></tr><tr><td>Anti-Human CD4 antibody, APC</td><td>BD Bioscience</td><td>555349</td><td>store at 4 &amp;deg;C, RRID: AB_398593</td></tr><tr><td>Anti-Human CD45 antibody, Pe-Cy7</td><td>Beckman Coulter</td><td>IM3548U</td><td>store at 4 &amp;deg;C, RRID: AB_1575969</td></tr><tr><td>Anti-Human CD56 antibody, PE</td><td>Beckman Coulter</td><td>IM2073U</td><td>store at 4 &amp;deg;C, RRID: AB_131195</td></tr><tr><td>Anti-Human CD63 antibody,PE</td><td>BD Bioscience</td><td>561925</td><td>store at 4 &amp;deg;C, RRID: AB_10896821</td></tr><tr><td>Anti-Human CD8 antibody, APC-Alexa Fluor 750</td><td>Beckman Coulter</td><td>A94686</td><td>store at 4 &amp;deg;C, RRID: N/A</td></tr><tr><td>Anti-Human CD80 antibody, APC</td><td>Beckman Coulter</td><td>B30642</td><td>store at 4 &amp;deg;C, RRID: N/A</td></tr><tr><td>Anti-Human CD81 antibody, FITC</td><td>BD Bioscience</td><td>561956</td><td>store at 4 &amp;deg;C, RRID: AB_394049</td></tr><tr><td>Anti-Human CD86 antibody, APC-Alexa Fluor 750</td><td>Beckman Coulter</td><td>B30646</td><td>store at 4 &amp;deg;C, RRID: N/A</td></tr><tr><td>Anti-Human CD9 antibody, FITC</td><td>ThermoScientic</td><td>MA5-16860</td><td>store at 4 &amp;deg;C, RRID: AB_2538339</td></tr><tr><td>Anti-Human Galectin 9 antibody, Brilliant Violet 421</td><td>Biolegend</td><td>348919</td><td>store at 4 &amp;deg;C, RRID: AB_2716134</td></tr><tr><td>Anti-Human OCT3/4 antibody, eFluor660</td><td>ThermoScientic</td><td>50-5841-82</td><td>store at 4 &amp;deg;C, RRID: AB_11218882</td></tr><tr><td>Anti-Human SOX2 antibody, Alexa Fluor 488</td><td>ThermoScientic</td><td>53-9811-82</td><td>store at 4 &amp;deg;C, RRID: AB_2574479</td></tr><tr><td>BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23227</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A1933</td><td></td></tr><tr><td>Buffy coat</td><td>New York Blood Center</td><td></td><td>40-60 ml/unit</td></tr><tr><td>Cell scraper</td><td>Falcon</td><td>353085</td><td></td></tr><tr><td>Disposable semi-micro cuvette</td><td>VWR</td><td>97000590</td><td></td></tr><tr><td>Dissociation buffer</td><td>Gibco</td><td>131510014</td><td>100 ml</td></tr><tr><td>Dual-Chanber cell counting slides</td><td>Bio-Rad</td><td>1450015</td><td></td></tr><tr><td>Exosome-Human CD63 Detection reagent</td><td>ThermoFisher Scientific</td><td>10606D</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Ficoll-Paque PLUS density grandient media</td><td>GE Health</td><td>17-1440-03</td><td>500 ml</td></tr><tr><td>Fixed-Angle Rotor(25&amp;deg;)</td><td>Thermo Scientifc</td><td>75003698</td><td>Maxium 24,700 x g</td></tr><tr><td>Gallios flow cytometer</td><td>Beckman Coulter</td><td></td><td>3 lasers&lt;br/&gt; 10 Color Max</td></tr><tr><td>Human cord blood</td><td>Cryo-Cell International</td><td></td><td>40-100 ml/unit</td></tr><tr><td>Human Fc Block</td><td>BD Bioscience</td><td>564220</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Immun-Blot PVDF membrane</td><td>Bio-Rad</td><td>1620177</td><td></td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22 &amp;micro;m</td><td>Millipore Sigma</td><td>SLGP033RS</td><td></td></tr><tr><td>Optima XE-90 Ultracentriguge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Orbital Shaker MP4</td><td>BioExpress</td><td>S-3500-1</td><td></td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>10010049</td><td>500 ml</td></tr><tr><td>Propidium Iodide</td><td>BD Bioscience</td><td>56-66211E</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Nikon Eclipse Ti2 microscope</td><td>Nikon instruments Inc</td><td>Eclipse Ti2</td><td>NIS-Elements software version 5.11.02</td></tr><tr><td>Hochest 33342</td><td>Thermo Scientifc</td><td>62249</td><td>5 ml</td></tr><tr><td>Revert microsopy</td><td>Fisher scientific</td><td>12563518</td><td></td></tr><tr><td>Rotor 41 Ti</td><td>Beckman Coulter</td><td>331362</td><td>Maxium 41,000 rpm</td></tr><tr><td>Sorvall St 16R Centrifuge</td><td>Thermo Scientifc</td><td>75004381</td><td></td></tr><tr><td>Swinging Bucket Rotor</td><td>Thermo Scientifc</td><td>75003655</td><td></td></tr><tr><td>TC-20 cell counter</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>ThermoScientific Forma 380 Steri Cycle CO2 Incubator</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Transmission electron microscopy</td><td>JEOL</td><td>JEM-2100 PLUS</td><td></td></tr><tr><td>Ultracentrifuge tube</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>X&#039;VIVO 15 Serum-free medium</td><td>Lonza</td><td>BEBP04-744Q</td><td>1000 ml Culture medium, store at 4 &amp;deg;C</td></tr></tbody></table>

differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (cb-sc)-derived exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Initially, the phenotype and purity of CB-SC were examined by flow cytometry with CB-SC-associated markers such as leukocyte common antigen CD45, ES cell-specific transcription factors OCT3/4, and SOX2. CB-SC display high levels of CD45, OCT3/4, SOX2, CD270, and galectin 9 expression, but no expression of CD34 (Figure 1A). Flow cytometry analysis confirmed the expression of exosome-specific markers including CD9, CD81, and CD63 were on CB-SC-derived exosomes (Figure 1B). Morphology and size distribution of exosomes were characterized by TEM and DLS (Figure 1C,D), with the size of 79.38&#160;&#177; 20.07&#160;nm. Western blot further proved the expression of the exosome-associated marker Alix, without expression of the ER-associated marker Calnexin (Figure 1E).
PBMC were treated with Dio-labeled CB-SC-Exo. The microscopy observation demonstrated the direct interaction of Dio-labeled CB-SC-Exo with PBMC (Figure 2A). To better define which cell population interacted with the Dio-labeled CB-SC-Exo, different cell compartments were gated with cell-specific markers such as CD3 for T cells, CD11c for myeloid dendritic cells (DC), CD14 for monocytes, CD19 for B cells, and CD56 for NK cells (Figure 2B). After an incubation for 4 hr, flow cytometry demonstrated that different blood cell compartments displayed at different median fluorescence intensity (MFI) of Dio-positive exosomes (Figure 2C). Notably, monocytes exhibited higher median fluorescence intensity of Dio-positive CB-SC-Exo than those of other immune cells (Figure 2C), highlighting that monocytes were primarily targeted by the CB-SC-derived exosomes.
To explore the direct effects of CB-SC-derived exosomes on monocytes, the purified CD14+ monocytes were co-cultured with CB-SC-derived exosomes for 3 days. The exosome-treated monocyte successfully differentiated into spindle-like morphologies (Figure 3A). Next, phenotypes of the CB-SC-Exo treated or untreated monocytes were tested, revealing the expressions of M2-associated markers including CD163, CD206, CD209 were markedly increased among the exosome-treated group (Figure 3B, red histogram). Comparing with the conventional M2 macrophages generated by M-CSF + IL-4, CB-SC-Exo-treated monocytes expressed similar levels of M2-associated markers such as CD163, CD206, CD209, with no significant differences (Figure 3C). Therefore, the data indicates that monocytes differentiate into macrophages with M2 phenotype after the treatment with CB-SC-derived exosomes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61562/61562fig01v2.jpg" /> 
Figure 1: Characterization of CB-SC-derived exosomes.&#160;(A) Phenotypic characterization of CB-SC, high expression of CD45, OCT3/4, SOX2, CD270 and Galectin-9, no expression of CD34. (B) Expressions of exosome-associated markers (CD63, CD9, CD81) on CB-SC-derived exosomes. Isotype-matched IgGs served as controls for flow cytometry (gray histogram). (C) Transmission Electron Microscopy (TEM) image of the CB-SC-derived exosomes. (D) Size distribution of CB-SC-derived exosomes using Dynamitic Light Scattering (DLS). (E) Western blots show that CB-SC-derived exosomes display the exosome-specific marker Alix, but negative for endoplasmic reticulum (ER)-associated marker Calnexin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61562/61562fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61562/61562fig02.jpg" /> 
Figure 2: Interaction of CB-SC-derived exosomes with different populations of PBMC.&#160;(A) The interaction of Dio-labeled CB-SC-Exo (green) with PBMC (blue, nuclear staining with Hoechst 33342) was photographed with Nikon Eclipse Ti2 microscope with NIS-Elements software version 5.11.02, with a high magnification showing the distribution of Dio-labeled exosomes (green) in the PBMC cells after the co-incubation for 4 h 5% CO2 in the non-tissue culture-treated 24-well plate. n = 2. (B) Gating strategy for flow cytometry analysis with cell-specific surface markers for different subpopulations in PBMC, including CD3 for T cells, CD14 for monocytes, CD19 for B cells, CD56 for NK cells, and CD11c for DCs. (C) Display different median fluorescence intensity (MFI) of Dio-labeled exosome among different PBMC subpopulations (e.g., T cells, Monocytes, B cells, NK cells, DCs). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61562/61562fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61562/61562fig03v2.jpg" /> 
Figure 3: Effects of CB-SC-derived exosomes on monocytes.&#160;(A) Morphological change of monocytes into the spindle-like cells after treatment with CB-SC-derived exosomes. (B) Up-regulated the level of M2-associate markers&#8217; expression after the treatment with CB-SC-derived exosomes, such as CD163, CD206, and CD209 (red line). Untreated monocytes (green line) served as control. Isotype-matched IgGs served as negative controls (gray line). (C) Phenotypic comparison between conventional M2 macrophages and the CB-SC-Exo-induced M2 macrophages. To generate the conventional M2 macrophages, the purified CD14+ monocytes were treated with 50 ng/mL macrophage colony-stimulating factor (M-CSF) at 37 &#176;C, 5% CO2 conditions for 7 days, and followed by the overnight treatment with 10 ng/mL IL-4. M2-associated markers including CD14. CD80, CD86, CD163, CD206, and CD209 were evaluated by flow cytometry. Isotype-matched immunoglobulin G (IgG) serve as control. The data is presented as mean &#177; SD; N = 3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61562/61562fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes at JoVE.com

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...

differentiation-monocytes-into-phenotypically-distinct-macrophages

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

5.7K Views.  Hackensack Meridian Health Center. Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

Video: Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Developmental Biology

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Source: Plaisance-Bonstaff, K., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF).

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

Source: Lopez-Yrigoyen, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61038">Production and Characterization of Human Macrophages from Pluripotent Stem Cells.</a> J. Vis. Exp. (2020).
This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional macrophages.

Source: Lopez-Yrigoyen, M., et al. Production and Characterization of Human Macrophages from Pluripotent Stem Cells. J. Vis. Exp. (2020).
This video ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Summary

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Chapters in this video

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Pan-myeloid Differentiation of Human Cord Blood Derived CD34⁺ Hematopoietic Stem and Progenitor Cells

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14⁺ Monocytes

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages