Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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 CaCl₂ and MgCl₂ (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% CO₂ 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% CO₂ 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% CO₂

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 (NaHCO₂ ) 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 CaCl₂ and MgCl₂ (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 CaCl₂ and MgCl₂ (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

Figure 1.D0.1 Microscopy of the microcarrier‐based HES‐3 cardiomyocyte differentiation process (static culture). (A ) hESC colony culture in 60 × 15–mm dishes after being sliced using a StemPro EZPassage disposable stem cell passaging tool (Invitrogen). (B ) Day 0 of differentiation: Seeding of hESC clumps (2 × 10⁶ cells/well) and Tosoh 10 microcarriers. Red arrow indicates one Tosoh 10 microcarrier. (C ) Day 1 of differentiation (aggregates have been broken up to prevent multi aggregation). (D ) Example of unwanted multi‐aggregation of the culture. Scale bars for (A) and (B) indicate 1 mm while scale bars in (C) and (D) indicate 100 µm.

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Figure 1.D0.2 Microscopy of HES‐3 cardiomyocytes generated in Tosoh 10 microcarrier cultures (A ) and embryoid bodies culture (B ). Day 16 in culture. Scale bar indicates 1 mm.

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Figure 1.D0.3 Comparison of cardiomyocyte (CM) yield obtained in static microcarrier culture (red) to EB culture (blue) using HES‐3. (A ) Cell expansion fold, (B ) % Positive cells (FACS, α‐actinin and myosin heavy chain). (C ) Cardiomyocyte yield (CM obtained per hESC seeded). Day 16 in culture. *** p value <0.01 n = 23.

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Literature Cited

Literature Cited
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Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited