Differentiation of Hepatocytes from Pluripotent Stem Cells

Sunil K. Mallanna1, Stephen A. Duncan1

1 Medical College of Wisconsin, Milwaukee, Wisconsin
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.4
DOI:  10.1002/9780470151808.sc01g04s26
Online Posting Date:  September, 2013
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Abstract

ABSTRACT

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte‐like cells provides a platform to study the molecular basis of human hepatocyte differentiation, to develop cell culture models of liver disease, and to potentially provide hepatocytes for treatment of end‐stage liver disease. Additionally, hepatocyte‐like cells generated from human pluripotent stem cells could serve as platforms for drug discovery, determination of pharmaceutical‐induced hepatotoxicity, and evaluation of idiosyncratic drug‐drug interactions. Here, we describe a step‐wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte‐like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. Curr. Protoc. Stem Cell Biol. 26:1G.4.1‐1G.4.13. © 2013 by John Wiley & Sons, Inc.

Keywords: hepatocytes; liver development; definitive endoderm; iPSC; hESC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of Human Pluripotent Stem Cells to Hepatocyte‐Like Cells
  • Support Protocol 1: Culture of Human Pluripotent Stem Cells on an E‐Cad‐Fc Substrate
  • Support Protocol 2: Coating Tissue Culture Dishes with Matrigel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Differentiation of Human Pluripotent Stem Cells to Hepatocyte‐Like Cells

  Materials
  • Human pluripotent stem cells on either E‐cad‐FC or Matrigel‐coated 100‐mm tissue culture dishes (see protocol 2)
  • Pluripotent stem cell medium: mTeSR1 [STEMCELL Technologies, cat. no. 5850; alternatively, mTeSR can prepared in the laboratory as described previously (Ludwig et al., )] or MEF‐conditioned stem cell culture medium (see recipe)
  • DPBS (Ca++/Mg++‐free DPBS; Invitrogen, cat. no. 14190‐136)
  • DPBS with 0.02% EDTA , pH 7.4
  • Accutase (STEMCELL Technologies, cat. no. 7920), optional
  • RPMI cell differentiation medium (see recipe)
  • B27 without insulin (Invitrogen, cat. no. 0050129SA)
  • Activin A (R&D systems, cat. no. 338‐AC‐010)
  • Bone morphogenetic protein 4 (BMP4; R&D, cat. no. 314BP)
  • Fibroblast growth factor 2 (FGF2; Invitrogen, cat. no. PHG0023)
  • B27 with insulin (Invitrogen, cat. no. 17504044)
  • Hepatocyte growth factor (HGF; Peprotech, cat. no. 100‐39)
  • Clonetics hepatocyte culture medium (HCM Bullet kit; Lonza, cat. no. CC‐3198)
  • Oncostatin M (R&D Systems, cat. no. 295‐OM‐010)
  • 6‐well sterile tissue culture plates coated with Matrigel (see protocol 3)
  • 37°C incubator

Support Protocol 1: Culture of Human Pluripotent Stem Cells on an E‐Cad‐Fc Substrate

  Materials
  • StemAdhere (STEMCELL Technologies, cat. no. 7160) or E‐cad‐Fc (prepared as described in Nagaoka and Duncan, )
  • DPBS+ (Ca++/Mg++ containing DPBS; Invitrogen, cat. no. 14040‐117)
  • Pluripotent stem cell medium: mTeSR1[(STEMCELL Technologies, cat. no. 5850; mTeSR can be prepared in the laboratory as described previously (Ludwig et al., )], or MEF‐conditioned stem cell culture medium (see recipe)
  • DPBS (Ca++/Mg++‐free DPBS; Invitrogen, cat. no. 14190‐136)
  • DPBS/0.02% EDTA
  • Sterile nontreated polystyrene cell culture plates (100‐mm dishes, Corning, VWR, cat. no.25382‐456; 60‐mm dishes, Corning, VWR, cat. no. 25382‐452; 6‐well plates, CellStar BioExpress, T‐3026‐4; 24‐well plates, BD Falcon, VWR, cat. no. 15705‐060; 96‐well plates, Corning, VWR, cat. no. 25381‐056)
  • 27‐G needles
  • 37°C incubator

Support Protocol 2: Coating Tissue Culture Dishes with Matrigel

  Materials
  • Dulbecco's modified Eagle medium (DMEM):nutrient mixture Ham's F‐12 (1:1) (DMEM/F12; Invitrogen, cat. no. 11330)
  • Matrigel (Geltrex hESC‐qualified reduced growth factor basement membrane matrix; Invitrogen, cat. no. A1413301)—diluted to 2 mg/ml in DMEM/F12 medium
  • Ice
  • Sterile tissue culture dishes (Corning Bioexpress):
    • 100‐mm dishes (cat. no. T‐2877‐100)
    • 60‐mm dishes (cat. no. T‐2877‐60)
    • 6‐well plates (cat. no. T‐2989‐6)
    • 12‐well plates (cat. no. T‐2989‐12)
    • 24‐well plates (cat. no. T‐2989‐24)
    • 96‐well plates (T‐2989‐96)
    • 37°C incubator
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Figures

Videos

Literature Cited

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Key Reference
  Si‐Tayeb et al., 2010b. See above.
  The manuscript describes in detail the differentiation of human pluripotent stem cells into hepatocytes and formed the basis for the .
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