Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures

Sherwin Ting1, Marti Lecina1, Shaul Reuveny2, Steve Oh3

1 Both of these authors have contributed the same amount of work to this paper, 2 Department of Biotechnology, Israel Institute for Biological Research, Ness‐Ziona, Israel, 3 Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Singapore
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.7
DOI:  10.1002/9780470151808.sc01d07s21
Online Posting Date:  May, 2012
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Abstract

We have developed an improved cardiomyocyte differentiation protocol where we stabilized embryoid bodies (EB) in serum‐ and insulin‐free medium (bSFS) supplemented with p38 MAP kinase inhibitor (SB203580) by addition of 10 µm laminin‐coated positively charged (protamine sulfate derivatized TSKgel Tresyl‐5PW) microcarriers. This protocol achieved a maximum 3‐fold cell expansion, differentiation efficiency of 20%, and an overall cardiomyocyte yield of 3 × 105 CM/ml in static conditions. In comparison, EB cultures achieved 1.5‐fold cell expansion, differentiation efficiency of 15%, and an overall cardiomyocyte yield of 1.1 × 105 CM/ml. The scalability of this platform was demonstrated in suspended spinner cultures, producing a maximum of 2.14 × 105 CM/ml in 50‐ml cultures. This yield is two‐fold higher than the control static EB‐based platform (1.1 × 105 CM/ml), and seven‐fold higher than yields reported in literature, 3.1–9 × 104 CM/ml. The robustness of this protocol was tested with HES‐3 and H1 cell lines. Curr. Protoc. Stem Cell Biol. 21:1D.7.1‐1D.7.14. © 2012 by John Wiley & Sons, Inc.

Keywords: human embryonic; stem cells; cardiomyocytes; SB203580; microcarriers; hESC; differentiation; scale up

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiating hESC to CM on Tosoh 10 Microcarriers in Ultra‐Low Attachment Plates under Static Conditions
  • Basic Protocol 2: Differentiating hESC to CM on Microcarriers in Spinner Flask
  • Support Protocol 1: Preparation of Tosoh 10 Microcarriers
  • Support Protocol 2: Laminin Coating of Microcarriers
  • Support Protocol 3: CM Harvesting for Flow Cytometry Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Differentiating hESC to CM on Tosoh 10 Microcarriers in Ultra‐Low Attachment Plates under Static Conditions

  Materials
  • hESC (HES‐3 cell line from ES International, and H1 from WiCell), expanded colonies cultured in 60 × 15–mm tissue culture dish on immortalized MEFs (see unit 1.3)
  • Phosphate‐buffered saline (PBS) with CaCl 2 and MgCl 2 (PBS+)
  • Differentiation medium (see recipe)
  • Laminin‐coated protamine‐derivatized Tosoh‐10 microcarriers ( protocol 4)
  • MAP kinase inhibitor SB203580 (Sigma‐Aldrich, cat. no. S8307) previously dissolved in dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. D2650)
  • 60 × 15–mm tissue culture dishes seeded with 1.5×106 MEF cells
  • 1‐ml, 200‐µl, and 10‐µl pipettor and sterile‐filtered tips
  • 5‐ml serological pipet, sterile
  • StemPro EZPassage disposable stem cell passaging tool (Invitrogen, cat. no. 23181‐00)
  • Stereomicroscope
  • Cell scraper, 24 cm (TPP, cat. no. 99002)
  • NucleoCounter (ChemoMetec A/S, NucleoCounter SCC‐100)
  • 12‐well ultra‐low‐attachment plates (Nunc, cat. no. 145385)
  • Orbital shaker (IKA Mixing Orbital Shaker KS260 Control, cat. no. 2980300)
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Differentiating hESC to CM on Microcarriers in Spinner Flask

  Materials
  • Sigmacote (Sigma‐Aldrich, cat. no. SL2)
  • Differentiation medium (see recipe)
  • Bovine serum albumin (BSA; Invitrogen, cat no. A10008‐01)
  • Laminin‐coated protamine‐derivatized Tosoh‐10 microcarriers (see protocol 4)
  • MAP kinase inhibitor SB203580 (Sigma‐Aldrich, cat. no. S8307), previously dissolved in DMSO at 5 mM (see recipe)
  • hESC (HES‐3 cell line from ES International, and H1 from WiCell), expanded colonies cultured in 100 × 15 mm tissue culture dish on immortalized MEFs
  • Differentiation medium for spinner flask (see recipe)
  • Pluronic F‐68 (10%) (Gibco, cat. no. 24040)
  • 100‐ml spinner flask (Bellco, cat. no. 1965‐00100)
  • Autoclave
  • 5‐, 10‐, 25‐, and 50‐ml serological pipets
  • 37°C humidified 5% CO 2 incubator
  • StemPro EZPassage disposable stem cell passaging tool (Invitrogen, cat. no. 23181‐00)
  • Stereomicroscope
  • Cell scraper, 24 cm (TPP, cat. no. 99002)
  • 1‐ml and 200‐µl pipettor with sterile‐filtered tips
  • NucleoCounter (ChemoMetec A/S, NucleoCounter SCC‐100)
  • 50‐ml tubes, sterile
  • Magnetic stirrer in 37°C humidified incubator with 5% CO 2
NOTE: Methods used for the preparation of MEFs are described in unit 1.3. 100 × 15‐mm tissue culture dishes were coated with 3 × 106 MEF cells. Two 100 × 15‐mm tissue culture dish should be able to generate at least 1 × 108 viable hESC in 7 days.

Support Protocol 1: Preparation of Tosoh 10 Microcarriers

  Materials
  • TSKgel Tresyl 5Pw (Tosoh, cat. no. 16208)
  • Coupling solution: 0.1 M carbonate buffer (NaHCO 2) containing 0.5 M NaCl (pH 8)
  • Protamine sulfate solution: Dissolve 32 mg protamine sulfate (Sigma, cat. no. P3369) in 4 ml coupling solution (concentration: 8 mg/ml)
  • Blocking solution: 0.1 M Tris⋅Cl buffer, pH 8
  • PBS (‐), without CaCl 2 and MgCl 2 (Invitrogen, cat. no. 14190‐144), pH 7.4
  • Weighing balance
  • 15‐ml centrifuge tube
  • 5‐ and 10‐ml serological pipets
  • Vortex mixer
  • Rotary agitator (e.g., Grant Instruments Ltd. 360° vertical multi‐function rotator PTR‐60)
  • Centrifuge
  • Hemacytometer
  • Controlled Cobalt‐60 source for γ‐irradiation

Support Protocol 2: Laminin Coating of Microcarriers

  Materials
  • 4 × 107 beads/ml of protamine‐derivatized TSKgel Tresyl 5Pw microcarriers ( protocol 3)
  • Natural mouse laminin (Invitrogen. cat. no. 23017015)
  • Differentiation medium (see recipe)
  • 50‐ml centrifuge tubes, sterile
  • 10‐ml serological pipets, sterile
  • 200‐µl sterile pipettor and tips
  • Laboratory platform rockers

Support Protocol 3: CM Harvesting for Flow Cytometry Analysis

  Materials
  • CM culture aggregates
  • PBS (‐), without CaCl 2 and MgCl 2 (pH 7.2; Invitrogen, cat. no. 14190‐144)
  • Collagenase solution (see recipe)
  • Trypsin/EDTA (Invitrogen, cat. no. 25200)
  • Differentiation medium (see recipe)
  • Fetal bovine serum (FBS; HyClone, cat. no. SV30160.03)
  • 15‐ and 50‐ml centrifuge tubes
  • 5‐ml serological pipets
  • Centrifuge
  • 1‐ml pipettor
  • 37°C incubator
  • 40‐µm cell strainer (BD Falcon, cat. no. 352340)
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Figures

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Literature Cited

Literature Cited
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