Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting

Ulf Diekmann1, Claudia Davenport1, Jasmin Kresse1, Ortwin Naujok1

1 Institute of Clinical Biochemistry, Hannover Medical School, Hannover
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.9
DOI:  10.1002/cpsc.22
Online Posting Date:  February, 2017
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Abstract

Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm‐committed cells in high numbers in a 4‐day treatment protocol. Second, a method for the purification of CXCR4‐positive endoderm cells by magnetic‐activated cell sorting (MACS) and fluorescence‐activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm‐derived somatic cells. © 2017 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cells; differentiation; definitive endoderm; MACS; FACS; purification; cell sorting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Feeder‐Free Cultivation of Human Pluripotent Stem Cells
  • Basic Protocol 2: Differentiation of Pluripotent Stem Cells into Definitive Endoderm
  • Support Protocol 1: Quantification of CXCR4‐Positive Definitive Endoderm Cells by Flow Cytometry
  • Support Protocol 2: Characterization and Quantitation Of PSC‐Derived Definitive Endoderm by Immunofluorescence
  • Basic Protocol 3: Sorting of CXCR4‐Positive Cells by Magnetic‐Activated Cell Sorting (MACS)
  • Basic Protocol 4: Sorting of CXCR4‐Positive Cells by Fluorescence‐Activated Cell Sorting (FACS)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Feeder‐Free Cultivation of Human Pluripotent Stem Cells

  Materials
  • Matrigel hESC‐Qualified Matrix (e.g., Corning, cat. no. 354277)
  • KnockOut DMEM/F‐12 (e.g, Thermo Fisher Scientific, cat. no. 12660012)
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (see Moore, )
  • Pluripotent stem cells (PSCs; e.g., WiCell HES−3, cat. no. ES03)
  • mTeSR1 (e.g., STEMCELL Technologies, cat. no. 05850) or StemMACS iPS‐Brew XF (e.g., Miltenyi Biotec, cat. no. 130‐104‐368) PSC culture medium
  • Non‐enzymatic passaging solution (see recipe)
  • 6‐well, plastic cell culture‐grade plates (e.g., Corning, cat. no. 353046)
  • 15‐ml and 50‐ml conical tubes
  • 37°C incubator

Basic Protocol 2: Differentiation of Pluripotent Stem Cells into Definitive Endoderm

  Materials
  • Matrigel hESC‐Qualified Matrix (e.g., Corning, cat. no. 354277)
  • KnockOut DMEM/F‐12 (e.g, Thermo Fisher Scientific, cat. no. 12660012) with and without 10% (v/v) fetal bovine serum (FBS)
  • Pluripotent stem cells (PSCs; e.g., WiCell HES−3, cat. no. ES03)
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (see Moore, )
  • 0.5% (w/v) trypsin/0.2% (w/v) EDTA (TE)
  • mTeSR1 (e.g., STEMCELL Technologies, cat. no. 05850) or StemMACS iPS‐Brew XF (e.g., Miltenyi Biotec, cat. no. 130‐104‐368) PSC culture medium
  • ROCK inhibitor (Y‐27632)
  • Endoderm induction medium I (see recipe)
  • Endoderm induction medium II (see recipe)
  • 6‐well, plastic cell culture‐grade plates (e.g., Corning, cat. no. 353046)
  • 37°C incubator
  • 15‐ml and 50‐ml conical tubes
  • Centrifuge
  • Hemocytometer

Support Protocol 1: Quantification of CXCR4‐Positive Definitive Endoderm Cells by Flow Cytometry

  Materials
  • Endoderm‐induced cells (see protocol 2)
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (see Moore, )
  • 0.5% (w/v) trypsin/0.2% (w/v) EDTA (TE)
  • KnockOut DMEM/F‐12 (e.g, Thermo Fisher Scientific, cat. no. 12660012) with 10% (v/v) fetal bovine serum (FBS)
  • PFE buffer (see recipe)
  • FcR blocking reagent
  • CD184 (CXCR4)‐APC, (e.g., Miltenyi Biotec, cat. no. 130‐098‐357)
  • EasySep APC Selection Kit (e.g., STEMCELL Technologies, cat. no. 18451) containing:
    • EasySep APC Selection Cocktail
    • EasySep Magnetic Nanoparticles
  • Endoderm induction medium II (see recipe)
  • ROCK inhibitor (Y‐27632)
  • 37°C incubator
  • 15‐ml conical tube
  • Centrifuge
  • 5‐ml round‐bottom tube with cell strainer (35 to 40 µM; e.g., Corning, cat. no. 352235)
  • Hemocytometer
  • 5‐ml round‐bottom tube (e.g., Corning, cat. no. 352058)
  • EasySep Magnet (e.g., STEMCELL Technologies, cat. no. 18000)
  • Flow cytometer

Support Protocol 2: Characterization and Quantitation Of PSC‐Derived Definitive Endoderm by Immunofluorescence

  Materials
  • Endoderm‐induced cells (see protocol 2)
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (see Moore, )
  • 0.5% (w/v) trypsin/0.2% (w/v) EDTA (TE)
  • KnockOut DMEM/F‐12 (e.g, Thermo Fisher Scientific, cat. no. 12660012) with 10% (v/v) fetal bovine serum (FBS)
  • FACS buffer (see recipe)
  • CD184 (CXCR4)‐APC, (e.g., Miltenyi Biotec, cat. no. 130‐098‐357)
  • 37°C incubator
  • 15‐ml conical tube
  • Centrifuge
  • 5‐ml round‐bottom tube with cell strainer (35 to 40 µM; e.g., Corning, cat. no. 352235)
  • Hemocytometer
  • Fluorescence‐activated cell sorter (FACS)
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Figures

Videos

Literature Cited

Literature Cited
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