Lentiviral Transduction and Clonal Selection of hESCs with Endothelial‐Specific Transgenic Reporters

Daylon James1, Qiansheng Zhan1, Christopher Kloss2, Nikica Zaninovic1, Zev Rosenwaks1, Shahin Rafii2

1 Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, New York, New York, 2 Department of Genetic Medicine, Weill Cornell Medical College, New York, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.12
DOI:  10.1002/9780470151808.sc01f12s17
Online Posting Date:  April, 2011
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Abstract

Generation of vascular endothelial cells (EC) from human embryonic stem cells (hESC) is a vital component of cell‐based strategies for treatment of cardiovascular disease. Before hESC‐derived ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce vascular differentiation. One approach to screening for culture conditions that support differentiation of hESCs to any cell type is their genetic modification with exogenous DNA sequence comprising a tissue‐specific gene promoter driving reporters such as fluorescent protein or antibiotic drug resistance. The protocols herein provide instructions for the generation of clonal hESC lines containing a reporter transgene that is specifically expressed in vascular endothelial derivatives. Additionally, they demonstrate the methodology employed to assess vascular differentiation from clonal lines. Together, these protocols provide a solid foundation for study of vascular differentiation, and may also be applied, in principle, to studies of other specialized cell types derived from hESCs. Curr. Protoc. Stem Cell Biol. 17:1F.12.1‐1F.12.14. © 2011 by John Wiley & Sons, Inc.

Keywords: vascular endothelium; hESC; differentiation; vasculogenesis; VE‐cadherin; lentivirus reporter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transduction of hESCs and Selection of Clones Containing Viral Integrations
  • Alternate Protocol 1: Selection of Virally Transduced hESCs Without FACs
  • Basic Protocol 2: Induction of Vascular Differentiation from Lentivirally Transduced hESCs and Selection of Clonal Lines with Specific and Robust Expression of Reporter Transgenes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Transduction of hESCs and Selection of Clones Containing Viral Integrations

  Materials
  • Concentrated lentivirus particles (Barde et al., ) containing a reporter transgene based on a 1.5‐kilobase fragment of the human VE‐cadherin gene promoter (James et al., ), which has been sub‐cloned into the lentiviral vector driving either mOrange or green fluorescent protein (GFP); available from the authors (djj2001@med.cornell.edu)
  • Human ES cells in standard feeder‐free culture (see Chapter 1), in 6‐well plates
  • hESC medium (see recipe) conditioned by growth of MEF feeder cells (Michalska, )
  • hESC medium (see recipe)
  • Y‐27632 ROCK inhibitor (Sigma)
  • Accutase cell detachment reagent (eBioscience; http://www.ebioscience.com/)
  • 0.5 M EDTA (Invitrogen)
  • 15‐ml conical polypropylene tubes (e.g., BD Falcon)
  • 70‐µm cell strainer
  • Fluorescence‐activated cell sorter (FACS)
  • Matrigel‐coated 6‐well plates (see recipe)
CAUTION: Following addition of lentivirus particles, all materials must be treated with appropriate biosafety precautions. The passage of hESC derivatives that have been transduced with lentivirus through cell sorting equipment must take into consideration biosafety risks, and must proceed at the discretion of the FACS operator and only following stringent washing of cellular input to eliminate lentivirus contamination.NOTE: All culture incubations are performed in a 37°C humidified 5% CO 2 incubator unless otherwise indicated.

Alternate Protocol 1: Selection of Virally Transduced hESCs Without FACs

  • 2 mg/ml Dispase (see recipe)
  • Serum‐free DMEM medium (e.g., Invitrogen)
  • 1000‐µl (P‐1000) pipet tip
  • 15‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Ultra‐low‐attachment culture dishes (6‐well format; Corning)
  • Matrigel coated 24‐well plates (see recipe)

Basic Protocol 2: Induction of Vascular Differentiation from Lentivirally Transduced hESCs and Selection of Clonal Lines with Specific and Robust Expression of Reporter Transgenes

  Materials
  • Candidate clonal lines of lentivirus‐transduced hESCs (in feeder‐free conditions; see protocol 1 or protocol 2)
  • hESC medium (see recipe) conditioned by growth of MEF feeder cells (Michalska, )
  • 2 mg/ml Dispase (see recipe)
  • Serum‐free DMEM medium (e.g., Invitrogen)
  • hESC medium (see recipe)
  • Differentiation medium: hESC medium (see recipe) supplemented with 5 ng/ml BMP4 (add from 10 µg/ml stock; see below)
  • 10 µg/ml stocks of recombinant human bone morphogenetic protein 4 (BMP4), recombinant human bone Activin A, and recombinant human vascular endothelial growth factor A isoform 165 (VEGF‐A) (all available from R&D Systems); reconstitute to 10 µg/ml in CMF‐DPBS (e.g., Invitrogen) and store up to 6 months at –80°C
  • 20 µg/ml stock of SB‐431542 (small molecule inhibitor of TGF‐β signaling; Tocris): reconstitute to 20 µg/ml in CMF‐DPBS (e.g., Invitrogen) and store up to 6 months at –80°C
  • Accutase cell detachment reagent (eBioscience; http://www.ebioscience.com/)
  • MEF medium (see recipe)
  • Phosphate‐buffered saline (PBS) containing 2% (v/v) donkey serum
  • Flow‐cytometry‐grade antibody to human CD31 (BD Biosciences) conjugated to Pacific Blue; directly conjugate a fluorophore that is amenable to combination with fluorescent protein reporter genes with CD31 antibody; use at a concentration of 10 µg/ml
  • Phosphate‐buffered saline (PBS)
  • 0.5 mM EDTA (Invitrogen)
  • Matrigel‐coated 10‐cm plates (see recipe)
  • 1000‐µl (P‐1000) pipet tip
  • 15‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Ultra‐low‐attachment culture dishes (6‐well format; Corning)
  • Gelatin‐coated (see recipe) 6‐well culture plates
  • Inverted fluorescence microscope (e.g., confocal microscope) with 488 excitation filter and bandpass 510/20 emission filter for eGFP and/or 535/561 excitation filter and bandpass 575/26 emission filter for mOrange
  • Flow cytometer or fluorescence activated cell sorter (FACS)
  • 70‐µm cell strainer
CAUTION: Following addition of lentivirus particles, all materials must be treated with appropriate biosafety precautions. The passage of hESC derivatives that have been transduced with lentivirus through cell sorting equipment must take into consideration biosafety risks, and must proceed at the discretion of the FACS operator and only following stringent washing of cellular input to eliminate lentivirus contamination.NOTE: All culture incubations are performed in a 37°C humidified 5% CO 2 incubator unless otherwise indicated.
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Figures

Videos

Literature Cited

Literature Cited
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