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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	<li>Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+educator+therapy+and+induction+of+immune+balance.">Stem cell educator therapy and induction of immune balance.</a> Current Diabetes Reports. 12 (5), 517-523 (2012).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+type+1+diabetes+via+islet+beta+cell+regeneration+following+immune+modulation+by+cord+blood-derived+multipotent+stem+cells.">Reversal of type 1 diabetes via islet beta cell regeneration following immune modulation by cord blood-derived multipotent stem cells.</a> BMC Medicine. 10 (1), 3 (2012).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+insulin+resistance+in+type+2+diabetes+via+immune+modulation+of+cord+blood-derived+multipotent+stem+cells+(CB-SCs)+in+stem+cell+educator+therapy:+phase+I/II+clinical+trial.">Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.</a> BMC Medicine. 11, 160 (2013).</li><li>Delgado, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+autoimmune+T-cell+memory+by+stem+cell+educator+therapy:+phase+1/2+clinical+trial.">Modulation of autoimmune T-cell memory by stem cell educator therapy: phase 1/2 clinical trial.</a> EBioMedicine. 2 (12), 2024-2036 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+regrowth+in+alopecia+areata+patients+following+Stem+Cell+Educator+therapy+BMC.">Hair regrowth in alopecia areata patients following Stem Cell Educator therapy BMC.</a> Medicine. 13 (1), 87 (2015).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annual Review of Cell And Developmental Biology. 30, 255-289 (2014).</li><li>Abak, A., Abhari, A., Rahimzadeh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+cancer:+small+vesicular+transporters+for+cancer+progression+and+metastasis,+biomarkers+in+cancer+therapeutics.">Exosomes in cancer: small vesicular transporters for cancer progression and metastasis, biomarkers in cancer therapeutics.</a> PeerJ. 6, 4763 (2018).</li><li>Adamiak, M., Sahoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+myocardial+repair:+advances+and+challenges+in+the+development+of+next-generation+therapeutics.">Exosomes in myocardial repair: advances and challenges in the development of next-generation therapeutics.</a> Molecular Therapy. 26 (7), 1635-1643 (2018).</li><li>Akyurekli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+preclinical+studies+on+the+therapeutic+potential+of+mesenchymal+stromal+cell-derived+microvesicles.">A systematic review of preclinical studies on the therapeutic potential of mesenchymal stromal cell-derived microvesicles.</a> Stem Cell Review and Report. 11 (1), 150-160 (2015).</li><li>Hu, W., Song, X., Yu, H., Sun, J., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Released+exosomes+contribute+to+the+immune+modulation+of+cord+blood-derived+stem+cells+(CB-SC).">Released exosomes contribute to the immune modulation of cord blood-derived stem cells (CB-SC).</a> Frontiers in Immunology. (11), 165 (2020).</li><li>Jacobson, G., K&#229;rsn&#228;s, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+parameters+in+semi-dry+electrophoretic+transfer.">Important parameters in semi-dry electrophoretic transfer.</a> Electrophoresis. 11 (1), 46-52 (1990).</li><li>Dykstra, M. J., Reuss, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological Electron Microscopy: Theory, Techniques, and Troubleshooting. , (2011).</li><li>Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V., Laktionov, P. 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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 compa...

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 
			plate</td>
		</tr>
		<tr>
			<td>3,3&#39;-Diostadecyloxacarbocyanine perchlorate(Dio)</td>
			<td>Millipore sigma</td>
			<td>D4292-20MG</td>
			<td>Store at 4 &#176;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 &#176;C, RRID: AB_2268110</td>
		</tr>
		<tr>
			<td>Anti-Human Calnexin</td>
			<td>Biolegend</td>
			<td>699401</td>
			<td>store at 4 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;C, RRID: AB_2738610</td>
		</tr>
		<tr>
			<td>Anti-Human CD270 antibody, PE</td>
			<td>Biolegend</td>
			<td>318806</td>
			<td>store at 4 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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 &#176;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&#176;)</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 
			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 &#176;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 &#181;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 &#176;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&#39;VIVO 15 Serum-free medium</td>
			<td>Lonza</td>
			<td>BEBP04-744Q</td>
			<td>1000 ml Culture medium, store at 4 &#176;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</...

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

Research

JoVE Journal

Immunology and Infection

5.6K 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

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...