Cardiac Differentiation of Human Embryonic Stem Cells and their Assembly into Engineered Heart Muscle

Poh Loong Soong1, Malte Tiburcy1, Wolfram‐Hubertus Zimmermann1

1 Department of Pharmacology, Heart Research Center Göttingen (HRCG), University Medical Center Göttingen, Göttingen, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.8
DOI:  10.1002/0471143030.cb2308s55
Online Posting Date:  June, 2012
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The advent of pluripotent human embryonic stem cells has created the unique opportunity for the development of a wide variety of humanized cellular tools for basic research, as well as industrial and clinical applications. It has, however, become apparent that embryonic stem cell derivatives in classical monolayer or embryoid body culture do not resemble bona fide tissues, mainly because of their limited organotypic organization and maturation in these culture formats. This shortcoming may be addressed by tissue engineering technologies aiming at the provision of a “natural” growth environment to facilitate organotypic tissue assembly. In this unit, we provide two harmonized basic protocols for (1) cardiac differentiation of human embryonic stem cells under serum‐free conditions and (2) the assembly of the stem cell–derived cardiomyocytes into engineered heart muscle. This protocol can be easily adapted to bioengineer heart muscle also from other stem cell–derived cardiomyocytes, including cardiomyocytes from human‐induced pluripotent stem cells. Curr. Protoc. Cell Biol. 55:23.8.1‐23.8.21. © 2012 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; pluripotent stem cells; cardiac differentiation; cardiomyocytes; tissue engineering; engineered heart muscle

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cardiac Differentiation in Human Embryonic Stem Cells Cultures
  • Basic Protocol 2: Bioengineering of Heart Muscle
  • Support Protocol 1: Preparation of Casting Molds for EHT Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cardiac Differentiation in Human Embryonic Stem Cells Cultures

  • hESCs (e.g., HES3 hESCs)
  • 100‐mm feeder cell‐coated culture plates (see protocol 3)
  • hESC maintenance medium (see recipe)
  • Dulbecco's phosphate‐buffered saline with Mg2+ and Ca2+ (DPBS+; Invitrogen, cat. no. 14040‐091)
  • Collagenase IV working solution (see recipe)
  • SF adaptation medium (see recipe)
  • bSFS medium (see recipe)
  • SB203580 (CalBiochem, cat. no. 559389‐5MG)
  • 100‐mm tissue culture dishes (Nunc, cat. no. 150679)
  • Stereomicroscope (Leica, cat. no. MZ8)
  • Glass Pasteur pipets
  • Stem Cell EZ passaging Tool (Invitrogen, cat. no. 23181‐010)
  • Cell scraper
  • Graded disposable serological pipets
  • 50‐ml centrifuge tubes (BD Falcon, cat. no. 352070), sterile
  • Centrifuge
  • 100‐mm ultra‐low petri dishes (Corning, cat. no. 3262)
  • 40‐µm cell strainers (BD Falcon, cat. no. 352340)
  • Tube rack

Basic Protocol 2: Bioengineering of Heart Muscle

  • Culture day 13 EBS in ultra‐low petri dishes (see protocol 1)
  • Collagenase I (see recipe)
  • DNase I (see recipe)
  • Iscove MEM F0465 (Biochrom)
  • 0.25%Trypsin‐EDTA (Invitrogen, cat. no. 24200‐056)
  • Fetal bovine serum (FBS; PAA, cat. no. A15102)
  • hEHT medium (see recipe)
  • Dulbecco's phosphate‐buffered saline without Mg2+ and Ca2+ (DPBS‐; Invitrogen, cat. no. 14190‐94)
  • 70% (v/v) ethanol
  • 15‐ and 50‐ml centrifuge tubes
  • 37°C shaking water bath
  • 10‐ml serological pipets
  • Centrifuge (Eppendorf, cat. no. 5804R)
  • Glass Pasteur pipet
  • Hemacytometer
  • Casting molds (see protocol 4; Figure A)
  • Silicone holders (Fig. B)
  • Sterile curved forceps
  • 100‐µl pipet tips, sterile

Support Protocol 1:

  • Human foreskin fibroblasts (HFF; ATCC# SCRC‐1041)
  • HFF maintenance medium (see recipe)
  • Human basic fibroblast growth factor (bFGF; Stemgent, cat. no. 03‐0002 or Miltenyi Biotech, cat. no. 130‐093‐841)
  • 37°C water bath
  • 15‐ml conical tubes
  • Hemacytometer
  • Standard inverted microscope
  • Irradiation source
  • 100‐mm tissue culture plates
  • 37°C, 5% CO 2 incubator

Support Protocol 2: Preparation of Casting Molds for EHT Culture

  • Sylgard silicone elastomer and curing agent 184 (Dow Corning, cat. no. 1673921)
  • Distilled water
  • Teflon spacer (Fig. A,i, custom made: 11‐mm diameter, 5‐mm height)
  • 60 × 20–mm glass petri dishes (Schott Duran, cat. no. 217554101)
  • 50‐ml centrifuge tubes
  • Forceps
  • Silicone core (Figure A,ii, 1‐mm diameter)
  • 55°C incubator
  • 5‐liter glass beakers
  • Silicone tubing (Figure A,iii, 4‐mm diameter; VWR, cat. no. 224‐0563)
  • Aluminum foil
  • Autoclave
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