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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.
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’s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient’s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these “educated” immune cells to the patient’s blood. Exosomes are nano-sized extracellular vesicles between 30‒150 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.
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’s peripheral blood mononuclear cells (PBMC) are collected and circulated through a cell separator and co-cult....
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 C.......
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
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.......
We are grateful to Mr. Poddar and Mr. Ludwig for generous funding support via Hackensack UMC Foundation. We appreciate Laura Zhao for English editing.
....Name | Company | Catalog Number | Comments |
1.5 ml Microcentrifuge tube | Fisher Scientific | 05-408-129 | |
15 ml conical tube | Falcon | 352196 | |
24-well plate | Falcon | 351147 | Non-tissue culture plate |
3,3'-Diostadecyloxacarbocyanine perchlorate(Dio) | Millipore sigma | D4292-20MG | Store at 4 °C |
300 Mesh Grids | Ted Pella | 1GC300 | |
50 mL conical tube | Falcon | 352070 | |
6-well plate | Falcon | 353046 | Tissue culture plate |
96-well plate | Falcon | 353072 | Tissue culture plate |
ACK Lysis Buffer | Lonza | 10-548E | 100 ml |
Amicon-15 10kDa Centrifuge Fliter Unit | Millipore sigma | UFC901024 | |
Anti-Human Alix | Biolegend | 634501 | store at 4 °C, RRID: AB_2268110 |
Anti-Human Calnexin | Biolegend | 699401 | store at 4 °C, RRID: AB_2728519 |
Anti-Human CD11c antibody, Pe-Cy7 | BD Bioscience | 561356 | store at 4 °C, RRID: AB_10611859 |
Anti-Human CD14 antibody, Karma Orange | Beckman Coulter | B36294 | store at 4 °C, RRID: AB_2728099 |
Anti-Human CD163 antibody, PE | BD Bioscience | 556018 | store at 4 °C, RRID: AB_396296 |
Anti-Human CD19 antibody, PC5 | Beckman Coulter | IM2643U | store at 4 °C, RRID: AB_131160 |
Anti-Human CD206 antibody, FITC | BD Bioscience | 551135 | store at 4 °C, RRID: AB_394065 |
Anti-Human CD209 antibody, Brilliant Violet 421 | BD Bioscience | 564127 | store at 4 °C, RRID: AB_2738610 |
Anti-Human CD270 antibody, PE | Biolegend | 318806 | store at 4 °C, RRID: AB_2203703 |
Anti-Human CD3 antibody, Pacfic Blue | Biolegend | 300431 | store at 4 °C, RRID: AB_1595437 |
Anti-Human CD34 antibody, APC | Beckman Coulter | IM2427U | store at 4 °C, RRID: N/A |
Anti-Human CD4 antibody, APC | BD Bioscience | 555349 | store at 4 °C, RRID: AB_398593 |
Anti-Human CD45 antibody, Pe-Cy7 | Beckman Coulter | IM3548U | store at 4 °C, RRID: AB_1575969 |
Anti-Human CD56 antibody, PE | Beckman Coulter | IM2073U | store at 4 °C, RRID: AB_131195 |
Anti-Human CD63 antibody,PE | BD Bioscience | 561925 | store at 4 °C, RRID: AB_10896821 |
Anti-Human CD8 antibody, APC-Alexa Fluor 750 | Beckman Coulter | A94686 | store at 4 °C, RRID: N/A |
Anti-Human CD80 antibody, APC | Beckman Coulter | B30642 | store at 4 °C, RRID: N/A |
Anti-Human CD81 antibody, FITC | BD Bioscience | 561956 | store at 4 °C, RRID: AB_394049 |
Anti-Human CD86 antibody, APC-Alexa Fluor 750 | Beckman Coulter | B30646 | store at 4 °C, RRID: N/A |
Anti-Human CD9 antibody, FITC | ThermoScientic | MA5-16860 | store at 4 °C, RRID: AB_2538339 |
Anti-Human Galectin 9 antibody, Brilliant Violet 421 | Biolegend | 348919 | store at 4 °C, RRID: AB_2716134 |
Anti-Human OCT3/4 antibody, eFluor660 | ThermoScientic | 50-5841-82 | store at 4 °C, RRID: AB_11218882 |
Anti-Human SOX2 antibody, Alexa Fluor 488 | ThermoScientic | 53-9811-82 | store at 4 °C, RRID: AB_2574479 |
BCA Protein Assay Kit | ThermoFisher Scientific | 23227 | |
Bovine Serum Albumin | Millipore Sigma | A1933 | |
Buffy coat | New York Blood Center | 40-60 ml/unit | |
Cell scraper | Falcon | 353085 | |
Disposable semi-micro cuvette | VWR | 97000590 | |
Dissociation buffer | Gibco | 131510014 | 100 ml |
Dual-Chanber cell counting slides | Bio-Rad | 1450015 | |
Exosome-Human CD63 Detection reagent | ThermoFisher Scientific | 10606D | store at 4 °C |
Ficoll-Paque PLUS density grandient media | GE Health | 17-1440-03 | 500 ml |
Fixed-Angle Rotor(25°) | Thermo Scientifc | 75003698 | Maxium 24,700 x g |
Gallios flow cytometer | Beckman Coulter | 3 lasers 10 Color Max | |
Human cord blood | Cryo-Cell International | 40-100 ml/unit | |
Human Fc Block | BD Bioscience | 564220 | store at 4 °C |
Immun-Blot PVDF membrane | Bio-Rad | 1620177 | |
Millex-GP Syringe Filter Unit, 0.22 µm | Millipore Sigma | SLGP033RS | |
Optima XE-90 Ultracentriguge | Beckman Coulter | ||
Orbital Shaker MP4 | BioExpress | S-3500-1 | |
PBS | ThermoFisher Scientific | 10010049 | 500 ml |
Propidium Iodide | BD Bioscience | 56-66211E | store at 4 °C |
Nikon Eclipse Ti2 microscope | Nikon instruments Inc | Eclipse Ti2 | NIS-Elements software version 5.11.02 |
Hochest 33342 | Thermo Scientifc | 62249 | 5 ml |
Revert microsopy | Fisher scientific | 12563518 | |
Rotor 41 Ti | Beckman Coulter | 331362 | Maxium 41,000 rpm |
Sorvall St 16R Centrifuge | Thermo Scientifc | 75004381 | |
Swinging Bucket Rotor | Thermo Scientifc | 75003655 | |
TC-20 cell counter | Bio-Rad | ||
ThermoScientific Forma 380 Steri Cycle CO2 Incubator | ThermoFisher Scientific | ||
Transmission electron microscopy | JEOL | JEM-2100 PLUS | |
Ultracentrifuge tube | Beckman Coulter | 331372 | |
X'VIVO 15 Serum-free medium | Lonza | BEBP04-744Q | 1000 ml Culture medium, store at 4 °C |
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