Differentiation of Mast Cells from Embryonic Stem Cells

Martina Kovarova1, Beverly Koller2

1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.10
DOI:  10.1002/0471142735.im22f10s97
Online Posting Date:  April, 2012
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Abstract

In this unit, we describe a simple coculture‐free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow–derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured, the difficulty in introducing specific genetic changes into these cells, and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cells from embryonic stem cells addresses these limitations. ES‐derived mast cells can be generated in numbers sufficient for studies of the pathways involved in mast cell effector functions. These ES cell–derived mast cells respond to antigens and other stimuli by releasing histamine, cytokines, lipids, and other bioactive mediators. The derivation of human mast cells from ES cells carrying mutations introduced by homologous recombination should provide a novel means of testing the function of genes in both the development and the effector functions of mast cells. Curr. Protoc. Immunol. 97:22F.10.1‐22F.10.16. © 2012 by John Wiley & Sons, Inc.

Keywords: mast cells; embryonic stem cells; differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation and Characterization of Mast Cells from Mouse Embryonic Stem Cells
  • Support Protocol 1: Determination of FCɛRI and c‐Kit‐Positive Cells in mES Derived Mast Cells Cultures
  • Basic Protocol 2: Differentiation of Human Mast Cells from Embryonic Stem Cells
  • Support Protocol 2: Immunofluorescence Staining of Human Mast Cells for Tryptase and Chymase
  • Support Protocol 3: Coating Plates with Matrigel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Differentiation and Characterization of Mast Cells from Mouse Embryonic Stem Cells

  Materials
  • Mouse ES cells in feeder cell‐free culture maintained in gelatinized dishes in DMEM/LIF medium (see recipe for medium)
  • IMDM/LIF medium (see recipe)
  • Phosphate‐buffered solution (PBS, Invitrogen)
  • Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 15% fetal bovine serum (FBS, Cellgro)
  • Trypsin‐EDTA (0.05% trypsin, 0.53 mM EDTA in HBSS; Cellgro)
  • Differentiation medium (see recipe)
  • Fetal bovine serum (FBS, Cellgro)
  • Mast cell medium for mouse (see recipe)
  • 10‐cm cell culture plates (Corning)
  • 50‐ml conical tubes (Becton Dickinson)
  • Centrifuge (Beckman Coulter Allegra‐6KR or equivalent)
  • 10‐cm polystyrene Petri dishes (Fisher)
  • 10‐ml syringes (Becton Dickinson)
  • 20‐G needles (Becton Dickinson)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)

Support Protocol 1: Determination of FCɛRI and c‐Kit‐Positive Cells in mES Derived Mast Cells Cultures

  Materials
  • Cells ( protocol 1)
  • Mouse IgE (clone SPE‐7, Sigma)
  • Staining buffer (see reagents and solution)
  • Blocking antibody (anti‐mouse FcγIII/II, Pharmingen, cat. no. 553142)
  • Specific antibodies to c‐Kit and IgE, fluorescently labeled (see Table 22.10.1)
  • Centrifuge
  • 5‐ml polypropylene tubes (Becton Dickinson)
  • Additional reagents and equipment for FACS (Chapter 5)
    Table 2.1.1   MaterialsAntibodies for Staining FcɛRI and c‐Kit on Mast Cells Derived from mES Cells

    Sample number Type of staining Antibody used (cat. no.)
    1 Isotype control for FITC and PE Rat IgG1‐FITC (Pharmingen 553924); Rat IgG2b‐PE (Pharmingen, 553989)
    2 FITC IgE‐specific + PE isotype Anti‐IgE‐FITC (Pharmingen, 553415); Rat IgG2b‐PE (Pharmingen, 553989);
    3 PE c‐Kit–specific + FITC isotype Anti c‐Kit‐PE (Pharmingen, 553869); Rat IgG1‐FITC (Pharmingen, 553924)
    4 FITC and PE–specific Anti‐IgE‐FITC (Pharmingen, 553415); Anti c‐Kit‐PE (Pharmingen, 553869)

Basic Protocol 2: Differentiation of Human Mast Cells from Embryonic Stem Cells

  Materials
  • Human embryonic stem cells (clones H1, H9, Wicell) grown in feeder‐free culture in mTeSR medium (StemCell Technologies)
  • 1 µg/ml Dispase in DMEM/F12 medium (Invitrogen)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Differentiation medium (see recipe)
  • Mast cell medium I for human (see recipe)
  • Mast cell medium II for human (see recipe)
  • Matrigel‐coated plates (see protocol 5)
  • 6‐well plates (Corning) coated with Matrigel ( protocol 5)
  • Cell scrapers (Corning)
  • 50‐ml conical tubes (Becton Dickinson)
  • 10‐cm ultra‐low‐attachment dishes (Corning)
  • Centrifuge (Beckman Coulter Allegra‐6KR or equivalent)
  • 70‐µm cell strainer (BD Bioscience)
  • 10‐cm cell culture plates (Corning)

Support Protocol 2: Immunofluorescence Staining of Human Mast Cells for Tryptase and Chymase

  Materials
  • 30% (v/v) sulfuric acid
  • 90% (v/v) ethanol
  • 0.01% poly‐L‐lysine solution: dilute 0.1% poly‐L‐lysine solution (Sigma) 1:10 in PBS
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Methanol, –20°C
  • Acetone, –20°C
  • Blocking buffer (see recipe)
  • TBS buffer (see recipe)
  • Mouse anti‐human mast cell tryptase (G3, Chemicon)
  • Mouse anti‐human mast cell chymase (B7, Chemicon)
  • Rhodamine‐conjugated anti–mouse IgG antibody (AP127R, Chemicon)
  • ProLong Gold antifade reagent with DAPI (Invitrogen)
  • Coverslips, number 1, 12‐mm circular (Fisher Scientific)
  • Glass beakers
  • 4‐well plates (Nunclon)
  • Whatman filter paper
  • 5‐ml polypropylene tubes (Becton Dickinson)
  • Centrifuge (Beckman Coulter Allegra‐6KR or equivalent) with plate adaptor
  • Microscope slides (Fisher Scientific)
  • Upright fluorescence microscope

Support Protocol 3: Coating Plates with Matrigel

  Materials:
  • BD Matrigel Basement Membrane Matrix (BD Biosciences)
  • DMEM/F12 medium (Invitrogen)
  • 1‐ml (P‐1000) pipet tips and repeating pipettor
  • 1.7‐ml microcentrifuge tubes
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Figures

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Literature Cited

Literature Cited
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