Expanding Mouse Ventricular Cardiomyocytes Through GSK‐3 Inhibition

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Controlled proliferation of cardiomyocytes remains a major limitation in cell biology and one of the main underlying hurdles for true modern regenerative medicine. Here, a technique is described for robust expansion of early fetal?derived mouse ventricular cardiomyocytes on a platform usable for high?throughput molecular screening, tissue engineering and, potentially, in vivo translational experiments. This method provides a small?molecule approach to control proliferation or differentiation of early beating cardiomyocytes through modulation of the Wnt/??catenin signaling pathway. Moreover, isolation and expansion of fetal cardiomyocytes takes less than 3 weeks, yields a relatively pure (?70%) functional myogenic population, and is highly reproducible. Curr. Protoc. Cell Biol . 61:23.9.1?23.9.10. © 2013 by John Wiley & Sons, Inc.

Keywords: cardiomyocyte proliferation; differentiation; isolation; expansion; GSK?3 inhibitor; Wnt/??catenin signaling

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Introduction
Basic Protocol 1: Isolation of Fetal Ventricular Cardiomyocytes
Basic Protocol 2: Two‐Dimensional Culture of Fetal Ventricular Cardiomyocytes
Alternate Protocol 1: Three‐Dimensional Culture of Fetal Ventricular Cardiomyocytes
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Isolation of Fetal Ventricular Cardiomyocytes

Materials

Pregnant C57BL/6 or CD1 mouse, E11.5 to E14.5
1× phosphate‐buffered saline (PBS), sterile and ice cold
Collagenase digestion solution (see recipe )
0.25% trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
Fetal bovine serum (FBS; Gemini Bioproducts, cat. no. 100‐500)
Cardiomyocyte culture medium (see recipe )

Scissors
Forceps
10‐cm cell culture dish
Scalpel
15‐ml centrifuge tubes (BD, cat. no. 352097)
37°C water bath

Additional reagents and equipment for counting cells (unit )

Basic Protocol 2: Two‐Dimensional Culture of Fetal Ventricular Cardiomyocytes

Materials

0.1% (w/v) gelatin (Sigma, cat. no. G1890) in phosphate‐buffered saline (PBS)
Cardiomyocyte culture medium (see recipe )
10 mM 6‐bromoindirubin‐3′‐oxime (BIO, a GSK‐3 inhibitor; Sigma, cat. no. B1686) in DMSO (store up to 1 year at −80°C)
10 mM inhibitor of Wnt response‐1 (IWR, an Axin inhibitor; Sigma, cat. no. I0161) in DMSO (store up to 1 year at −80°C)
Dimethyl sulfoxide (DMSO)
4% paraformaldehyde (PFA) solution
Primary antibodies against:
- Cardiac troponin T (cTnT, mouse monoclonal, NeoMarkers, Ms‐295, 0.2 mg/ml, 1:250)
- Ki67 (rabbit monoclonal, Abcam, cat. no. 16667, 1 mg/ml, 1:300)
- α‐Sarcomeric actinin (α‐SA, mouse monoclonal, Sigma, cat. no. A7811, 1 mg/ml, 1:250)
- Connexin‐43 (Cx43, rabbit polyclonal, Sigma, cat. no. C6219, 1 mg/ml, 1:150)
Secondary antibodies:
- Donkey anti‐mouse, Alexa 488 (Invitrogen, cat. no. A‐21202, 1:400)
- Donkey anti‐rabbit, Alexa 594 (Invitrogen, cat. no. A‐21206, 1:400)
Saponin (Sigma, cat. no. 47036)
4′,6‐Diamidino‐2‐phenylindole, dihydrochloride (DAPI, Invitrogen, cat. no. D1306, 1:10,000)
TRIzol reagent (Invitrogen, cat. no. 15596‐026)
Qiagen RNeasy Mini Kit (Qiagen, cat. no. 74104)
iScript cDNA Synthesis Kit (Bio‐Rad, cat. no. 170‐8891)

24‐, 96‐, or 384‐well cell culture plate(s)
37°C/5% CO₂ culture incubator

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Figures

Figure 23.9.1 Isolation and plating of ventricular myocytes. (A ) Dissected mouse uterus containing multiple embryos. (B ) Mouse embryo at ∼E12.5. Dashed lines indicate where incisions should be made to yield the heart. (C ) Fetal mouse heart. Dashed lines indicate excision of atrial tissue. (D ) Pooling of ventricular tissue in 15‐ml tubes. Two to three tubes can be used for biological replicates. (E ) Representative image of ventricular cells stained for cardiac troponin T (cTnT; green), the proliferation marker Ki67 (red), and DAPI (blue). Scale bar, 50 µm. (F ) Quantification of the number of cTnT+ cells relative to total cells 1 day after isolation (E12.5+1). (G ) Quantification of cTnT+ cell number per well of a 384‐well plate. In F and G, error bars indicate standard deviation; n = 3 each in six technical replicates.

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Figure 23.9.2 Two‐dimensional expansion and differentiation of ventricular myocytes. Representative images of ventricular cells cultured in (A ) DMSO, (B ) BIO, or (C ) IWR, stained for cardiac troponin T (cTnT; green), Ki67 (red), and DAPI (blue). Scale bar, 50 µm. (D ) Quantification of cTnT+ cells at day 1 (E12.5+1, baseline) and after 3 and 6 additional days of culture (E12.5+4, E12.5+7) in the presence or absence of BIO (1 or 2 µM) or IWR (8 or 16 µM). Error bars indicate standard deviation; n = 3 each in six technical replicates for each time point.

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Figure 23.9.3 Three‐dimensional culture of ventricular myocytes. Representative bright‐field images of ventricular cells cultured in aggregates treated with (A ) DMSO, (B ) BIO, or (C ) IWR. Scale bar, 50 µm. (D ) Quantification of the diameter of ventricular tissue constructs after treatment with DMSO, BIO (1 or 2 µM), or IWR (8 or 16 µM). Error bars indicate standard deviation; n = 3 each in three technical replicates. (E ) qRT‐PCR analysis for structural cardiac genes after treatment with DMSO, BIO (2.5 µM), or IWR (16 µM). Error bars indicate standard deviation; n = 3.

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Literature Cited

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Internet Resources
	http://pga.mgh.harvard.edu/primerbank/
	PCR primer sequences are available online at PrimerBank.

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Expanding Mouse Ventricular Cardiomyocytes Through GSK‐3 Inhibition

Abstract

Table of Contents

Materials

Basic Protocol 1: Isolation of Fetal Ventricular Cardiomyocytes

Basic Protocol 2: Two‐Dimensional Culture of Fetal Ventricular Cardiomyocytes

Figures

Videos

Literature Cited