Deriving Functional Hepatocytes from Pluripotent Stem Cells

Dagmara Szkolnicka1, Sarah L. Farnworth1, Baltasar Lucendo‐Villarin1, David C. Hay1

1 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.5
DOI:  10.1002/9780470151808.sc01g05s30
Online Posting Date:  August, 2014
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Abstract

Despite major progress in the management of human liver disease, the only cure for a critically failing organ is liver transplantation. While a highly successful approach, the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore, the use of cell‐based therapies has been explored to provide support for the failing liver. In addition to developing new treatments, there is also an imperative to develop better human models ‘in a dish’. Such approaches will undoubtedly lead to a better understanding of the disease process, offering new treatment or preventative strategies. With both approaches in mind, we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly, our procedure is highly efficient (∼90%) and delivers active, drug‐inducible, and predictive human hepatocyte populations. Curr. Protoc. Stem Cell Biol. 30:1G.5.1‐1G.5.12. © 2014 by John Wiley & Sons, Inc.

Keywords: pluripotent stem cells; human embryonic stem cells; liver; hepatocyte; cell‐based therapies; models ‘in a dish’

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagating and Passaging hESCs
  • Support Protocol 1: Matrigel Stock Preparation
  • Support Protocol 2: Seeding the Cells at 2 × 105 Viable Cells per cm2
  • Basic Protocol 2: Human Embryonic Stem Cell Hepatocyte Differentiation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagating and Passaging hESCs

  Materials
  • Matrigel (BD Bioscience, Matrigel, cat. no. 354230)
  • mTeSR1 and supplement (Stem Cell Technologies, cat. no. 05850; see recipe)
  • Human embryonic stem cells (hESCs)
  • Gentle Cell Dissociation Reagent (Stem Cell Technologies, cat. no. 07174)
  • Y‐27632 (ROCK inhibitor; Stem Cell Technologies, cat. no. 07171; see recipe)
  • DMEM/F12 (Life Technologies, cat. no 11320‐033)
  • PBS (Life Technologies, cat. no. 14190‐094)
  • 0.4% Trypan blue solution (Life Technologies, cat. no. 15250‐061)
  • Multiwell culture plates
  • Class II Biological Safety Cabinet (Class II BSC)
  • Cell culture incubator set to 37°C with 5% CO 2
  • Hemacytometer
  • Centrifuge

Support Protocol 1: Matrigel Stock Preparation

  Materials
  • 10 ml Matrigel (BD Bioscience, Matrigel, cat. no. 354230)
  • Knockout DMEM (KO‐DMEM; Life Technologies, cat.no. 10829)
  • Sterile 30‐ml universal tubes

Support Protocol 2: Seeding the Cells at 2 × 105 Viable Cells per cm2

  Materials
  • Human embryonic stem cells (hESCs)
  • 0.4% Trypan blue solution (Life Technologies, cat. no. 15250‐061)
  • Hemacytometer

Basic Protocol 2: Human Embryonic Stem Cell Hepatocyte Differentiation

  Materials
  • Human embryonic stem cells (hESCs) in 6‐well Matrigel‐coated plates
  • RPMI/B27 differentiation medium (see recipe)
  • SR/DMSO differentiation medium (see recipe)
  • HepatoZYME (HZM) differentiation medium (see recipe)
  • Recombinant mouse wnt3a (R&D Systems, cat. no. 1324‐WN‐500/CF; see recipe)
  • Human activin A (AA; Peprotech, cat. no. 120‐14E; see recipe)
  • Human Hepatocyte Growth Factor (HGF; Peprotech, cat. no. 100‐39; see recipe)
  • Human Oncostatin M (OSM; Peprotech, cat. no. 300‐10; see recipe)
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Figures

Videos

Literature Cited

Literature Cited
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