Differentiation and Characterization of Dendritic Cells from Human Embryonic Stem Cells

Kanishka Mohib1, Lisheng Wang2

1 Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada, 2 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.11
DOI:  10.1002/0471142735.im22f11s98
Online Posting Date:  August, 2012
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Abstract

Human embryonic stem cells (hESCs) offer great hope in regenerative medicine. Their ability to give rise to almost any type of cell present in the adult body makes them an invaluable tool in finding cures for a variety of diseases. While considerable protocols have been devised to efficiently differentiate hESCs into various cells types including cells of hematopoietic origin, this protocol will focus on the derivation of dendritic cells (DC), a potent antigen‐presenting cell. DCs are a highly important arm of the immune system, as they represent one of the few cells that bridge the innate and adaptive systems, leading to effective pathogen clearance. The study of DCs has led to potential applications in diverse fields, such as vaccine development, tumor immunology, and transplantation. In this protocol, we describe two different methods of differentiating hESCs into DCs. The first method uses OP9 bone marrow stromal supporting cells as a coculture system, while the second method utilizes the formation of embryoid body (EB, cellular aggregate) as an approach. To assure the successful outcome and subsequent assessment of the differentiated DCs, supporting protocols have been included in this chapter. Curr. Protoc. Immunol. 98:22F.11.1‐22F.11.22. © 2012 by John Wiley & Sons, Inc.

Keywords: Human embryonic stem cells; hematopoietic progenitor cells; dendritic cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of hESC into DCs Using OP9 Cells
  • Alternate Protocol 1: Differentiation of hESCs into HPC Through Embryoid Body Formation
  • Support Protocol 1: Determination of Dendritic Cell Markers by Flow Cytometry
  • Support Protocol 2: hESC‐DC Phagocytosis of Dextran Beads
  • Support Protocol 3: Allogeneic T Cell Stimulation Using hESC‐Derived DCs
  • Support Protocol 4: Maintenance of OP9 Bone Marrow Stromal Cells
  • Support Protocol 5: hESC Maintenance on MEF
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Differentiation of hESC into DCs Using OP9 Cells

  Materials
  • hESCs (see protocol 7) growing in 6‐well plates
  • Phosphate‐buffered saline (PBS, Mg2+/Ca2+‐free Invitrogen, cat. no. 14190)
  • 200 U/ml collagenase IV (see recipe)
  • HPC differentiation medium (see recipe)
  • OP9 cells overgrown in 10‐cm dish (see protocol 6)
  • 0.05% trypsin‐EDTA (Invitrogen)
  • HPC expansion medium (see recipe)
  • Complete RPMI medium (see recipe)
  • GM‐CSF (R&D Systems)
  • IL‐4 (R&D Systems)
  • TNF‐α (R&D Systems)
  • Low‐attachment 6‐well plates (Corning)
  • 10‐cm culture dish (Corning)
  • 75‐cm2 low‐attachment flask (Corning)
  • Centrifuge

Alternate Protocol 1: Differentiation of hESCs into HPC Through Embryoid Body Formation

  • HPC‐EB medium (see recipe)
  • SCF (R&D Systems)
  • FLT3‐L (R&D Systems)
  • GM‐CSF (R&D Systems)
  • BMP‐4 (R&D Systems)
  • 4 U/ml collagenase B (see recipe)
  • Phosphate‐buffered saline (PBS, Mg2+/Ca2+‐free Invitrogen, cat. no. 14190)
  • Cell dissociation buffer (Invitrogen)
  • FACS buffer (see recipe)
  • PE‐conjugated mouse anti‐human PECAM1/CD31 antibody (Beckman Coulter)
  • FITC‐conjugated mouse anti‐human CD45 antibody (Beckman Coulter)
  • Mouse IgG‐PE monoclonal antibody isotype control (Beckman Coulter or BD Pharmingen)
  • Mouse IgG‐FITC (or IgG‐APC) monoclonal antibody isotype control (Beckman Coulter or BD Pharmingen)
  • 7‐AAD (7‐amino‐actinomycin D ready‐to‐use solution, Beckman Coulter)
  • IMDM medium (see recipe) with and without 10% FBS
  • 1× BIT 9500 medium (see recipe)
  • IL‐3 (R&D Systems)
  • IL‐6 (R&D Systems)
  • 15‐ml conical polypropylene tubes
  • 70‐µm mesh filter
  • 40‐µm cell strainer
  • 24‐well plate (Corning) coated with fibronectin (see recipe)
  • Additional reagents and equipment for fluorescence activated cell sorting (Chapter 5)

Support Protocol 1: Determination of Dendritic Cell Markers by Flow Cytometry

  Materials
  • hESC‐derived DCs ( protocol 1 or protocol 2)
  • FACS buffer (see recipe)
  • Human AB blocking serum (Sigma) or Fc block reagent (Miltenyi Biotech)
  • Fluorochrome‐conjugated CD1a, CD80, CD86, CD83, CD40, HLA‐DR, and DC‐SIGN
  • FACS buffer (see recipe)
  • Centrifuge
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 2: hESC‐DC Phagocytosis of Dextran Beads

  Materials
  • Immature and mature hESC‐derived DCs (see protocol 1 or protocol 2)
  • FACS buffer (see recipe)
  • 10 mg/ml dextran‐FITC‐labeled beads (mol. wt. 40,000; Sigma)
  • Phosphate‐buffered saline (PBS, Mg2+/Ca2+‐free Invitrogen, cat. no. 14190)
  • FACS buffer (see recipe) with 0.1% (w/v) NaN 3
  • Centrifuge

Support Protocol 3: Allogeneic T Cell Stimulation Using hESC‐Derived DCs

  Materials
  • hESC‐derived DCs (see protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS, Mg2+/Ca2+‐free Invitrogen, cat. no. 14190)
  • Complete RPMI medium (see recipe)
  • Mitomycin C (Sigma)
  • Purified T cells (see relevant units in Chapter 3)
  • Tritiated thymidine (PerkinElmer)
  • 96‐well round bottom plates (Corning)
  • 96 well filter mats (Perkin Elmer)
  • TomTec cell harvester (TomTec; http://www.tomtec.com)
  • Plastic filter pouch (Perkin Elmer)
  • Scintillation fluid (Perkin Elmer)
  • Liquid scintillation counter (Wallac)
  • Vacuum plastic sealer
  • Centrifuge
  • Additional reagents and equipment for counting cells

Support Protocol 4: Maintenance of OP9 Bone Marrow Stromal Cells

  Materials
  • OP9 cells (ATCC cat. no. CRL‐2749)
  • OP9 medium (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • 0.05% trysin‐EDTA (Invitrogen)
  • Centrifuge
  • 10‐cm plates (Corning), gelatin‐coated (see recipe)

Support Protocol 5: hESC Maintenance on MEF

  Materials
  • MEF cells (ATCC, cat. no. SCRT1040, SCRT1040.1, or SCRT1040.2a (MEF can also be prepared from mouse embryo according to the protocol provided by WiCell: https://www.wicell.org/index.php?option=com_content&task=category&id=310&Itemid=156&sectionid=16)
  • MEF medium (see recipe)
  • Mitomycin C (Sigma); can be dissolved as 1 mg/ml stock in sterile H 2O, as described by manufacturer
  • hESC lines (obtained from WiCell: http://www.wicell.org/ Please also see NIH Stem Cell Data Management System: http://stemcells.nih.gov/research/nihresearch/scunit/
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • hESC medium (see recipe) with and without 4 ng/ml bFGF
  • 200 U/ml collagenase IV (see recipe)
  • Cell dissociation buffer (Invitrogen)
  • 6‐well plates (Corning), gelatin coated (see recipe)
  • Centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3A)
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Literature Cited

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