Small‐Molecule‐Directed Hepatocyte‐Like Cell Differentiation of Human Pluripotent Stem Cells

Santosh Mathapati1, Richard Siller1, Agata A.R. Impellizzeri2, Max Lycke3, Karianne Vegheim4, Runar Almaas4, Gareth J. Sullivan5

1 Norwegian Center for Stem Cell Research, Oslo, 2 Department of Medical Genetics, Oslo University Hospital, Ullevål, Oslo, 3 Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 4 Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital, Oslo, 5 Institute of Immunology, Oslo University Hospital, Oslo
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.6
DOI:  10.1002/cpsc.13
Online Posting Date:  August, 2016
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Hepatocyte‐like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small‐molecule‐driven protocol for in vitro differentiation of hPSCs into HLCs without the use of growth factors. hPSCs are coaxed through a developmentally relevant route via the primitive streak to definitive endoderm (DE) using the small molecule CHIR99021 (a Wnt agonist), replacing the conventional growth factors Wnt3A and activin A. The small‐molecule‐derived DE is then differentiated to hepatoblast‐like cells in the presence of dimethyl sulfoxide. The resulting hepatoblasts are then differentiated to HLCs with N‐hexanoic‐Tyr, Ile‐6 aminohexanoic amide (Dihexa, a hepatocyte growth factor agonist) and dexamethasone. The protocol provides an efficient and reproducible procedure for differentiation of hPSCs into HLCs utilizing small molecules. © 2016 by John Wiley & Sons, Inc.

Keywords: definitive endoderm; hepatocyte; hESCs; hiPSCs; small molecules

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Table of Contents

  • Introduction
  • Basic Protocol 1: Small‐Molecule Differentiation of hPSCs Into HLCs Using Single‐Cell Seeding
  • Alternate Protocol 1: Small‐Molecule Differentiation of hPSCs into HLCs Using Standard Cell Passaging
  • Support Protocol 1: Preparation of Geltrex‐Coated Tissue Culture Dishes
  • Support Protocol 2: Feeder‐Independent Culture and Maintenance of hPSCs on Geltrex
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Small‐Molecule Differentiation of hPSCs Into HLCs Using Single‐Cell Seeding

  • Essential 8 Medium (see recipe), room temperature
  • 10 mM ROCK inhibitor Y‐27632 (1000×; see recipe)
  • Human pluripotent stem cells (hPSCs), maintained and expanded on Geltrex‐coated six‐well plates (see protocol 4)
  • Dulbecco's phosphate‐buffered saline, calcium and magnesium free (DPBS−/−; Life Technologies, cat. no. 14190)
  • Accutase (Life Technologies, A1115‐01)
  • 0.4% trypan blue solution (Life Technologies, 15250‐061)
  • 3 mM CHIR99021 (see recipe)
  • RPMI/B‐27 medium (see recipe)
  • SR‐DMSO differentiation medium (see recipe)
  • L‐15 differentiation medium (see recipe)
  • Sterile biosafety cabinet
  • 37°C, 5% CO 2 incubator
  • Phase‐contrast microscope
  • 40‐µm Corning cell strainer (Sigma‐Aldrich, CLS431750)
  • 50‐ml conical tubes (VWR, 734‐0453)
  • Geltrex‐coated six‐well plates (see protocol 3)
  • Additional reagents and equipment for determining viability by trypan blue exclusion

Alternate Protocol 1: Small‐Molecule Differentiation of hPSCs into HLCs Using Standard Cell Passaging

  • Geltrex stock solution (see recipe)
  • AdvDMEM/F12 medium: 1:1 Advanced Dulbecco's modified Eagle medium/Ham's F‐12 (Life Technologies, cat. no. 12634)
  • 50‐ml conical tube (VWR, 734‐0453)
  • Six‐well tissue culture plates (VWR, 7340991)

Support Protocol 1: Preparation of Geltrex‐Coated Tissue Culture Dishes

  Additional Materials (also see protocol 1Basic Protocol)
  • Geltrex‐coated six‐well plates (see protocol 3)
  • Frozen human pluripotent stem cells (hPSCs; in liquid nitrogen): e.g., hESC H1 cells (WiCell) or Sendai virus–derived hiPSC line AG05836B clone #27 (Coriell Institute, AG05836)
  • Dry ice
  • 70% (v/v) ethanol
  • 0.5 mM EDTA (see recipe)
  • Sterile 5‐ml serological pipets
  • 15‐ml conical tubes (VWR, 734‐0451)
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Literature Cited

Literature Cited
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