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. 2013 Dec 2:61:23.9.1-23.9.10.
doi: 10.1002/0471143030.cb2309s61.

Expanding mouse ventricular cardiomyocytes through GSK-3 inhibition

Jan W Buikema  1   2   3 , Peter-Paul M Zwetsloot  1   3 , Pieter A Doevendans  3 , Joost P G Sluijter  3 , Ibrahim J Domian  1   2   4
Affiliations

Expanding mouse ventricular cardiomyocytes through GSK-3 inhibition

Jan W Buikema et al. Curr Protoc Cell Biol. .

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/β-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.

Keywords: GSK-3 inhibitor; Wnt/β-catenin signaling; cardiomyocyte proliferation; differentiation; expansion; isolation.

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Figures

Figure 1
Figure 1. Isolation and plating of ventricular myocytes
(a) Image of 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 15mL tubes. 2–3 tubes can be used for biological replicates. (e) representative image of ventricular cells stained for cardiac troponin T (cTnT) (green), Ki67 (red) and DAPI (blue). (f) percentage of cTnT+ cells 1 day after isolation (E12.5+1). Scale bar represents 50μm. (g) quantification of cTnT+ cell number per well of a 384-well plate. Error bars indicate standard deviation. (n=3, each in 6 technical replicates).
Figure 2
Figure 2. 2-dimensional expansion of 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 represents 50μm. (d) Quantification of cTnT+ cells at day 1 (baseline) (E12.5+1), 3 (E12.5+4) and 6 (E12.5+7) additional days of culture in the presence or absence of BIO or IWR. Error bars indicate standard deviation. (n=3, each in 6 technical replicates for each time point).
Figure 3
Figure 3. 3-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 represents 50μm. (d) Quantification of the diameter of ventricular tissue constructs in μm. (n=3, each in 3 technical replicates). (e) qPCR analysis for structural cardiac genes on cells treated with BIO, DMSO or IWR. (n=3). Error bars indicate standard deviation.
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