Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood Vessels

Nicholas C. Kappas1, Victoria L. Bautch1

1 The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.3
DOI:  10.1002/0471143030.cb2303s34
Online Posting Date:  March, 2007
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Abstract

Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the capacity to give rise to all cell types in the adult body. The ability of ES cells to do so has opened the door for novel experimental approaches in the field of developmental biology. Under appropriate culture conditions, ES cells will differentiate and form embryoid bodies (EBs). Upon attachment to a permissive surface, EBs continue a programmed differentiation, and many of the cells differentiated from the EBs reflect those found in the developing embryo and yolk sac, such as hematopoietic cells, endoderm, and endothelial cells. Endothelial cells that arise during ES cell differentiation have the potential to form primitive blood vessels, comparable to the vessels that first form in vivo. This unit describes protocols for maintaining ES cells and the subsequent differentiation of EBs. This unit also provides methods for analyzing vascular marker expression in differentiated ES cultures.

Keywords: embryonic stem cells; vascular development; in vitro differentiation; endothelial cells

     
 
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Table of Contents

  • Basic Protocol 1: Programmed in Vitro Differentiation of Mouse Embryonic Stem Cells
  • Support Protocol 1: Benzidine Staining
  • Support Protocol 2: Double Staining with Pecam and Mac‐1
  • Support Protocol 3: Immunolocalization
  • Support Protocol 4: β‐Galactosidase Staining
  • Support Protocol 5: Culturing Mouse Embryonic Stem Cells in the Absence of Feeder Cells
  • Support Protocol 6: Preparation of 5637 Cell–Conditioned Medium (CM)
  • Support Protocol 7: Lot Testing of FBS for Passage of ES Cells
  • Support Protocol 8: Lot Testing of Fetal Bovine Serum for in Vitro Differentiation of Mouse ES Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Programmed in Vitro Differentiation of Mouse Embryonic Stem Cells

  Materials
  • 5‐ to 6‐day‐old ES cell culture ( protocol 6)
  • 1× CMF‐PBS (Sigma)
  • 2.4 U/ml dispase, grade II stock (Boehringer‐Mannheim; see recipe)
  • Differentiation medium (see recipe)
  • 50‐ml centrifuge tubes (Sarstedt), sterile
  • 10‐cm bacteriological petri dishes (do not use TC‐treated plates)
  • 24‐well tissue culture plates (Costar)
  • Medidroppers (Fisher Scientific), autoclaved
NOTE: All volumes are given assuming that one 6‐cm dish of ES cell colonies is being used. Double all volumes if using 10‐cm dishes.

Support Protocol 1: Benzidine Staining

  Materials
  • 8‐day differentiated ES cell cultures ( protocol 1)
  • Benzidine solution (see recipe), 4°C
  • Differentiation medium (see recipe)
  • 1× CMF‐PBS (Sigma)

Support Protocol 2: Double Staining with Pecam and Mac‐1

  Materials
  • Day‐10 differentiated cultures (from protocol 1)
  • 1× CMF‐PBS (Sigma)
  • 4% (w/v) paraformaldehyde fixative (see recipe)
  • 1:500 trypsin solution (see recipe)
  • 100% heat‐inactivated fetal bovine serum
  • Staining medium (see recipe)
  • Primary and secondary antibodies (see recipe);
    • Rat anti‐mouse CD31 (PECAM‐1; Mec 13.3; BD Pharmingen)
    • FITC‐AffinPure F(ab′) 2 fragment of donkey anti‐rat IgG (Jackson
    • Immunoresearch)
    • Mac‐1‐BNHS (biotin coupled to Mac‐1 antibody); clone M1/70;
    • Rat anti‐mouse Mac‐1 (BD Pharmingen)
    • Streptavidin‐RPE (Southern Biotechnology Associates)
  • Rat serum staining medium (see recipe)

Support Protocol 3: Immunolocalization

  Materials
  • Differentiated ES cell culture (from protocol 1)
  • 1× CMF‐PBS (Sigma)
  • Methanol/actetone fixative (see recipe): 1:1 (v/v) methanol (Fisher Scientific)/acetone (Mallinckrodt) or 4% (w/v) paraformaldehyde (PFA; Polysciences; see recipe)
  • Staining medium (see recipe)
  • Primary and secondary antibodies (see recipe)
  • Fluorescent microscope, inverted (equipped with epifluorescence and camera)
NOTE: All volumes given are for staining cultures from a 24‐well plate. Gently add and remove all solutions from culture plates, as harsh removal and addition of solutions may result in detachment of cultures.

Support Protocol 4: β‐Galactosidase Staining

  Materials
  • Day‐8 differentiated cultures in 24‐well plate ( protocol 1)
  • 0.1 M phosphate buffer, pH 7.3 to 7.5 (see recipe)
  • Glutaraldehyde fixative solution (see recipe)
  • Xgal stain (see recipe)
  • Wash buffer (see recipe)
NOTE: All volumes given are for staining cultures from a 24‐well plate. Gently add and remove all solutions from culture dishes, as harsh removal and addition of solutions may result in detachment of cultures.

Support Protocol 5: Culturing Mouse Embryonic Stem Cells in the Absence of Feeder Cells

  Materials
  • 3‐ to 4‐day‐old ES cell dishes (unit 23.2), without feeder layer, passage 3 to 45
  • 1× CMF‐PBS (Sigma)
  • 0.25× trypsin/EDTA (see recipe)
  • Trypsin stop solution (see recipe)
  • ES cell culture medium (see recipe)
  • Gelatin‐coated dishes (see recipe)
  • 6‐cm tissue culture (TC)–treated culture dishes (Corning)
NOTE: All volumes are given assuming that one 6‐cm dish of ES cell colonies is being used. Double all volumes if using 10‐cm dishes. Prewarm all solutions to 37°C prior to use.

Support Protocol 6: Preparation of 5637 Cell–Conditioned Medium (CM)

  Materials
  • 5637 human bladder carcinoma cell line (ATCC #HTB9)
  • 5637 cell growth medium (recipe)
  • Collection medium (see recipe)
  • 6‐ and 15‐cm tissue culture (TC)–treated culture dishes (Corning)
  • 15‐ and 50‐ml centrifuge tubes (Sarstedt)
  • 200‐ml centrifuge tubes, if desired (NUNC)
  • 1‐, 3‐, 10, and 25‐ml disposable pipets
  • 0.22‐µm cellulose acetate filter units with bottle (Corning) or Nalgene SFCA 0.2‐µm bottle top filter with 33‐mm neck to fit standard 500‐ml glass bottles
  • 500‐ml bottles, autoclaved
NOTE: The following protocol describes the collection of 5637 medium from one 15‐cm plate, however, more plates are typically processed for collection. Adjust volumes as needed if more plates are to be processed for 5637 collection.

Support Protocol 7: Lot Testing of FBS for Passage of ES Cells

  Materials
  • ES cell culture medium (see recipe), prepared with different lots of FBS
  • 3‐ to 4‐day‐old ES cell culture dish ( protocol 6)
  • 6‐cm tissue culture–treated culture dishes (Corning)
  • Additional reagents and equipment on in vitro differentiation of ES cells ( protocol 1), passaging of ES cells ( protocol 7), and fixing and staining differentiated cultures ( protocol 4)

Support Protocol 8: Lot Testing of Fetal Bovine Serum for in Vitro Differentiation of Mouse ES Cells

  Materials
  • Differentiation medium (see recipe), prepared with different lots of FBS
  • 5‐day‐old to 6‐day‐old ES cell culture ( protocol 6)
  • Additional reagents and equipment for in vitro differentiation of ES cells ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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