Isolation and Functional Characterization of Pluripotent Stem Cell–Derived Cardiac Progenitor Cells

Xiaojing Huang1, Sean M. Wu2

1 Cardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Boston, Massachusetts, 2 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.10
DOI:  10.1002/9780470151808.sc01f10s14
Online Posting Date:  September, 2010
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Abstract

The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here, we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and are differentiated using the hanging‐droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence‐activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters. Curr. Protoc. Stem Cell Biol. 14:1F.10.1‐1F.10.14. © 2010 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cells; induced pluripotent stem cells; cardiac progenitor cells; in vitro differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culturing and In Vitro Differentiation of Pluripotent Stem Cells
  • Alternate Protocol 1: In Vitro Differentiation of Pluripotent Stem Cells in a Two‐Dimensional High‐Throughput Format
  • Basic Protocol 2: Preparation of Differentiation Culture for Flow Cytometry Analysis or FACs
  • Basic Protocol 3: Flow Cytometric Analysis and FACs‐Based Purification of Cardiac Progenitor Cells
  • Basic Protocol 4: Culture and Functional Characterization of Cardiac Progenitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culturing and In Vitro Differentiation of Pluripotent Stem Cells

  Materials
  • Nkx2.5‐eGFP transgenic ES or iPS cells passaged fewer than 30× since original derivation, in standard culture in 6‐well plates on a feeder layer of growth‐arrested mouse embryonic fibroblasts (MEFs; unit 1.3); MEFs should be seeded at 400,000 cells/well
  • DMEM‐ES medium (see recipe)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • IMDM‐ES medium (see recipe)
  • 0.1% (w/v) gelatin solution (see recipe)
  • Differentiation medium (see recipe)
  • 10‐cm cell culture dish
  • 10‐ to 100‐µl multichannel pipettor
  • 150 × 15–mm slippable monoplate petri dishes (Fisher, cat. no. 08‐757‐14)
  • Additional reagents and equipment for counting cells (unit 1.3)

Alternate Protocol 1: In Vitro Differentiation of Pluripotent Stem Cells in a Two‐Dimensional High‐Throughput Format

  • ES or iPS cells with MEFs depleted (see protocol 1)
  • Collagenase/DNase solution (see recipe)
  • HEPES‐buffered saline (HBS; see recipe), optional
  • Flow cytometry buffer (see recipe)
  • Propidium iodide solution (1 mg/ml; Invitrogen, cat. no. P3566)
  • 96‐well cell culture plates, gelatin‐coated
  • Polypropylene cluster tubes for flow cytometry (Corning Costar, cat. no. 4411)

Basic Protocol 2: Preparation of Differentiation Culture for Flow Cytometry Analysis or FACs

  Materials
  • EB culture (see protocol 1)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • Collagenase/DNase solution (see recipe)
  • HEPES‐buffered saline (HBS; see recipe)
  • Flow cytometry buffer (see recipe)
  • Propidium iodide solution (1 mg/ml; Invitrogen, cat. no. P3566)
  • 10‐ml serological pipet or sterile cell scraper
  • 15‐ml or 50‐ml conical tubes
  • 1000‐µl pipettor
  • 37°C water bath
  • 40‐µm cell strainers

Basic Protocol 3: Flow Cytometric Analysis and FACs‐Based Purification of Cardiac Progenitor Cells

  Materials
  • EBs (dissociated according to protocol 3)
  • Differentiation medium (DM; see recipe)
  • TRIzol (Invitrogen, cat. no. 15596026) or other cell lysis solution for RNA isolation
  • Antibodies for cardiac troponin T (cTnT; e.g., clone 13‐11, Neomarkers), sarcomeric myosin heavy chain (sarcMHC; e.g., clone MF20, Developmental Studies Hybridoma Bank), sarcomeric actinin (sarcActinin; e.g., clone EA‐53, Sigma), smooth muscle actin‐α (SMAα; e.g., clone IA4, DakoCytomation), and smooth muscle myosin heavy chain (SM‐MHC; e.g., polyclonal, Biomedical Technologies)
  • FACS collection tubes (Becton Dickinson, cat. nos. 352054, 352063, or 352196)
  • Gelatin‐coated chamber slides or 6‐, 12‐, 24‐, or 96‐well plates
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Figures

Videos

Literature Cited

Literature Cited
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