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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
1.5 ml Microcentrifuge tubeFisher Scientific05-408-129
15 ml conical tubeFalcon352196
24-well plateFalcon351147Non-tissue culture
plate
3,3'-Diostadecyloxacarbocyanine perchlorate(Dio)Millipore sigmaD4292-20MGStore at 4 °C
300 Mesh GridsTed Pella1GC300
50 mL conical tubeFalcon352070
6-well plateFalcon353046Tissue culture plate
96-well plateFalcon353072Tissue culture plate
ACK Lysis BufferLonza10-548E100 ml
Amicon-15 10kDa Centrifuge Fliter UnitMillipore sigmaUFC901024
Anti-Human AlixBiolegend634501store at 4 °C, RRID: AB_2268110
Anti-Human CalnexinBiolegend699401store at 4 °C, RRID: AB_2728519
Anti-Human CD11c antibody, Pe-Cy7BD Bioscience561356store at 4 °C, RRID: AB_10611859
Anti-Human CD14 antibody, Karma OrangeBeckman CoulterB36294store at 4 °C, RRID: AB_2728099
Anti-Human CD163 antibody, PEBD Bioscience556018store at 4 °C, RRID: AB_396296
Anti-Human CD19 antibody, PC5Beckman CoulterIM2643Ustore at 4 °C, RRID: AB_131160
Anti-Human CD206 antibody, FITCBD Bioscience551135store at 4 °C, RRID: AB_394065
Anti-Human CD209 antibody, Brilliant Violet 421BD Bioscience564127store at 4 °C, RRID: AB_2738610
Anti-Human CD270 antibody, PEBiolegend318806store at 4 °C, RRID: AB_2203703
Anti-Human CD3 antibody, Pacfic BlueBiolegend300431store at 4 °C, RRID: AB_1595437
Anti-Human CD34 antibody, APCBeckman CoulterIM2427Ustore at 4 °C, RRID: N/A
Anti-Human CD4 antibody, APCBD Bioscience555349store at 4 °C, RRID: AB_398593
Anti-Human CD45 antibody, Pe-Cy7Beckman CoulterIM3548Ustore at 4 °C, RRID: AB_1575969
Anti-Human CD56 antibody, PEBeckman CoulterIM2073Ustore at 4 °C, RRID: AB_131195
Anti-Human CD63 antibody,PEBD Bioscience561925store at 4 °C, RRID: AB_10896821
Anti-Human CD8 antibody, APC-Alexa Fluor 750Beckman CoulterA94686store at 4 °C, RRID: N/A
Anti-Human CD80 antibody, APCBeckman CoulterB30642store at 4 °C, RRID: N/A
Anti-Human CD81 antibody, FITCBD Bioscience561956store at 4 °C, RRID: AB_394049
Anti-Human CD86 antibody, APC-Alexa Fluor 750Beckman CoulterB30646store at 4 °C, RRID: N/A
Anti-Human CD9 antibody, FITCThermoScienticMA5-16860store at 4 °C, RRID: AB_2538339
Anti-Human Galectin 9 antibody, Brilliant Violet 421Biolegend348919store at 4 °C, RRID: AB_2716134
Anti-Human OCT3/4 antibody, eFluor660ThermoScientic50-5841-82store at 4 °C, RRID: AB_11218882
Anti-Human SOX2 antibody, Alexa Fluor 488ThermoScientic53-9811-82store at 4 °C, RRID: AB_2574479
BCA Protein Assay KitThermoFisher Scientific23227
Bovine Serum AlbuminMillipore SigmaA1933
Buffy coatNew York Blood Center40-60 ml/unit
Cell scraperFalcon353085
Disposable semi-micro cuvetteVWR97000590
Dissociation bufferGibco131510014100 ml
Dual-Chanber cell counting slidesBio-Rad1450015
Exosome-Human CD63 Detection reagentThermoFisher Scientific10606Dstore at 4 °C
Ficoll-Paque PLUS density grandient mediaGE Health17-1440-03500 ml
Fixed-Angle Rotor(25°)Thermo Scientifc75003698Maxium 24,700 x g
Gallios flow cytometerBeckman Coulter3 lasers
10 Color Max
Human cord bloodCryo-Cell International40-100 ml/unit
Human Fc BlockBD Bioscience564220store at 4 °C
Immun-Blot PVDF membraneBio-Rad1620177
Millex-GP Syringe Filter Unit, 0.22 µmMillipore SigmaSLGP033RS
Optima XE-90 UltracentrigugeBeckman Coulter
Orbital Shaker MP4BioExpressS-3500-1
PBSThermoFisher Scientific10010049500 ml
Propidium IodideBD Bioscience56-66211Estore at 4 °C
Nikon Eclipse Ti2 microscopeNikon instruments IncEclipse Ti2NIS-Elements software version 5.11.02
Hochest 33342Thermo Scientifc622495 ml
Revert microsopyFisher scientific12563518
Rotor 41 TiBeckman Coulter331362Maxium 41,000 rpm
Sorvall St 16R CentrifugeThermo Scientifc75004381
Swinging Bucket RotorThermo Scientifc75003655
TC-20 cell counterBio-Rad
ThermoScientific Forma 380 Steri Cycle CO2 IncubatorThermoFisher Scientific
Transmission electron microscopyJEOLJEM-2100 PLUS
Ultracentrifuge tubeBeckman Coulter331372
X'VIVO 15 Serum-free mediumLonzaBEBP04-744Q1000 ml Culture medium, store at 4 °C

References

  1. Zhao, Y. Stem cell educator therapy and induction of immune balance. Current Diabetes Reports. 12 (5), 517-523 (2012).
  2. Zhao, Y., et al. Reversal of type 1 diabe....

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