Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

Michaela Rothová1, Jurriaan J. Hölzenspies1, Alessandra Livigni2, Santiago Nahuel Villegas3, Joshua M. Brickman2

1 Centre (DanStem), University of Copenhagen, Copenhagen, 2 MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh, 3 Instituto de Neurociencias, Consejo Superior de Investigaciones Cientificas, Universidad Miguel Hernandez de Elche, Alicante
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.3
DOI:  10.1002/9780470151808.sc01g03s36
Online Posting Date:  February, 2016
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Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive endoderm with pronounced anterior character. ADE‐containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum‐free adherent monolayer culture. ESC‐derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut derivatives. © 2016 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; differentiation; endoderm; anterior; activin; FGF; Hhex

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Table of Contents

  • Introduction
  • Basic Protocol 1: Monolayer Differentiation of mESCs to ADE
  • Alternate Protocol 1: Embryoid Body Differentiation of mESCs to ADE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Monolayer Differentiation of mESCs to ADE

  • N2B27 medium (see recipe)
  • 0.1% gelatin (see recipe)
  • mESC adherent culture in gelatin‐coated 25‐cm2 flask, ∼80% confluent, grown in mESC medium (see recipe) containing LIF
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (CMF‐PBS; Sigma, cat. no. D8537)
  • 0.025% trypsin (see recipe)
  • mESC medium without LIF
  • Recombinant cytokine stock solutions (see recipe):
    • 20 μg/ml activin A (1000×)
    • 10 μg/ml bone morphogenetic protein 4 (BMP4; 1000×)
    • 100 μg/ml epidermal growth factor (EGF; 5000×)
    • 50 μg/ml fibroblast growth factor 4 (FGF4; 5000×)
    • 2.5 mg/ml human recombinant insulin (5000×)
  • ADEM (see recipe)
  • 7.5% bovine serum albumin (BSA fraction V; Life Technologies, cat. no. 15260‐037; store in aliquots at –20°C; once thawed, store indefinitely at 4°C)
  • L‐Glutamine (Life Technologies, cat. no. 25030‐081)
  • 2‐Mercaptoethanol (Sigma, cat. no. M7522)
  • 6‐well tissue culture plates (Corning), 8‐well μ‐Slides (Ibidi, cat. no. 80821), or T25 or T75 tissue culture flasks
  • 15‐ and 50‐ml sterile conical tubes (or 30‐ml universal tubes)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.3; Michalska, 2007)

Alternate Protocol 1: Embryoid Body Differentiation of mESCs to ADE

  Additional materials (also see protocol 1Basic Protocol)
  • 75‐ or 150‐cm2 sterile culture flask
  • Bacteriological Petri dishes (Sterilin)
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Literature Cited

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