Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells

Paul W. Burridge1, Alexandra Holmström1, Joseph C. Wu1

1 Department of Medicine (Division of Cardiology), Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.3
DOI:  10.1002/0471142905.hg2103s87
Online Posting Date:  October, 2015
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Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal‐derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost‐effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non‐enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection–based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell–derived cardiomyocytes, cryopreservation, and thawing. © 2015 by John Wiley & Sons, Inc.

Keywords: human induced pluripotent stem cells; differentiation; cardiac; cardiomyocyte; chemically defined; monolayer

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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture and Cardiomyocyte Differentiation of Human iPS Cells
  • Support Protocol 1: Characterization of Cardiomyocytes by Flow Cytometry
  • Support Protocol 2: Characterization by Immunofluorescent Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Culture and Cardiomyocyte Differentiation of Human iPS Cells

  • Human induced pluripotent stem cells (hiPS cells; see information in step 1)
  • E8‐Y medium (see recipe)
  • E8 medium (without Y27632; see recipe)
  • 0.5 mM EDTA (see recipe)
  • CDM3‐C (with CHIR99021; see recipe)
  • CDM3‐C59 (with Wnt‐C59; see recipe)
  • CDM3‐L (without D‐glucose, with L‐lactic acid; see recipe)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • TrypLE Express (Life Technologies, cat. no. 12605‐036)
  • Liberase TH, 260 U/50 mg (Roche, cat. no. 05401151001), resuspend in 10 ml WFI water (Corning, cat. no. 25‐055‐CV) and make 500‐μl aliquots; store at −20°C
  • DNase I, 277 U/μl (Life Technologies, cat. no 18047‐019)
  • CDM3 (see recipe)
  • Fetal bovine serum (FBS; Life Technologies, cat. no. 10082‐147)
  • Dimethylsulfoxide (DMSO; Fisher Scientific, cat. no. BP231‐1)
  • Liquid N 2
  • CDM3‐T (with T 3; see recipe)
  • 15‐ml (Corning Falcon, cat. no. 352097) and 50‐ml (Corning Falcon, cat. no. 352098) polystyrene conical tubes
  • Matrigel‐coated (see recipe) 6, 12, 24, 96, 384‐well cell culture plates (Greiner, cat. no. 657160, 665180, 662160, 655090, 781091, respectively)
  • Centrifuge (e.g., Thermo Sorvall ST8)
  • 100‐μm cell strainer (Corning Falcon, cat. no. 352360)
  • Luna Automated Cell Counter (Logos Biosystems, cat. no. L20001)
  • Cyrovials (Greiner, cat. no. 122261)
  • Coolcell LX (Biocision, cat. no. BCS‐405)
  • Additional reagents and equipment for generating hiPS cells [unit 4.1 (Park and Daley, ) and unit 4.2 (Ohnuki et al, )]
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are carried out in a humidified 37°C, 5% CO 2 incubator (Thermo Scientific Heracell VIOS) unless otherwise specified. We have found that 5% O 2 (hypoxic) incubators are not essential for the success of this protocol.NOTE: We do not place any media in a 37°C water bath before use due to concerns regarding the temperature stability of the FGF2 in the media (Chen et al., ). Bringing the media to room temperature is sufficient, and we have found no noticeable effects on cell growth by using 4°C media.

Support Protocol 1: Characterization of Cardiomyocytes by Flow Cytometry

  • Cardiomyocytes ( protocol 1Basic Protocol)
  • 1% (w/v) PFA in CMF‐DPBS (prepare from 20% PFA; Electron Microscopy Sciences, cat. no. 15713‐S)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • 90% (v/v) methanol (Fisher, cat. no. A412‐1)
  • 0.5% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS
  • 0.5% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS containing 0.1% (v/v) Triton X‐100 (Sigma Aldrich, cat. no. X100)
  • TNNT2 mouse monoclonal (13‐11) primary antibody (Thermo Scientific, cat. no. MS‐295‐P)
  • AlexaFluor 488–conjugated goat anti–mouse IgG 1 (Life Technologies, cat. no. A21121)
  • Flow cytometry tubes (Corning Falcon, cat. no. 352235)
  • Centrifuge accommodating TX‐750 rotor
  • 5/7 ml tube buckets with decanting aid for TX‐750 rotor (Thermo, cat. no. 75003732)
  • Flow cytometer capable for analyzing FITC and Texas Red such as Beckman Coulter CytoFLEX

Support Protocol 2: Characterization by Immunofluorescent Staining

  • Cardiomyocytes ( protocol 1Basic Protocol)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • 4% (w/v) PFA in CMF‐DPBS (prepare from 20% PFA; Electron Microscope Sciences, cat. no. 15713‐S)
  • 0.5% (v/v) Triton X‐100 (Sigma‐Aldrich, cat. no. X100) in CMF‐DPBS
  • 3% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS
  • TNNT2 (Troponin T) primary antibody, rabbit polyclonal IgG (Abcam, cat. no. ab45932)
  • ACTN2 (α‐actinin) primary antibody, mouse monoclonal IgG 1, clone EA‐53 (Sigma‐Aldrich, cat. no. A7811)
  • AlexaFluor 488–conjugated goat anti‐rabbit IgG (Life Technologies, cat. no. A11008)
  • AlexaFluor 594–conjugated goat anti‐mouse IgG1 (Life Technologies, cat. no. A21125)
  • Prolong Diamond with DAPI (Life Technologies, cat. no. P36962)
  • 8‐well Lab‐Tek II chamber slides (Thermo Nunc, cat .no. 154534)
  • 12‐well Matek glass No. 1.5 plates (Matek, cat no. P12G‐1.5‐14‐F)
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Literature Cited

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