Cozzarelli Prize Winner: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI 53706, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2012; 109(27):E1848-57. DOI: 10.1073/pnas.1200250109
Source: PubMed


Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

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Available from: Kexian Zhu, Oct 30, 2014
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    • "Thus, the approach is also amenable to scale-up. Progress in differentiation techniques has yielded multiple methods that produce pure cardiac cells by mimicking the embryonic developmental signals that control mesoderm induction using activin-Nodal, BMP, Wnt and FGF [6,17,36e38], and subsequent cardiac specification using inhibition of Wnt [6], BMP [36] and TGFb [36] [39] pathways. Unfortunately, these culture systems used expensive growth factors and yielded low purity of CMs. "
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    ABSTRACT: Pluripotent stem cell (PSC) usage in heart regenerative medicine requires producing enriched cardiomyocytes (CMs) with mature phenotypes in a defined medium. However, current methods are typically performed in 2D environments that produce immature CMs. Here we report a simple, growth factor-free 3D culture system to rapidly and efficiently generate 85.07 ± 1.8% of spontaneously contractile cardiac spheres (scCDSs) using 3D-cultured human and monkey PSC-spheres. Along with small molecule-based 3D induction, this protocol produces CDSs of up to 95.7% CMs at a yield of up to 237 CMs for every input pluripotent cell, is effective for human and monkey PSCs, and maintains 81.03 ± 12.43% of CDSs in spontaneous contractibility for over three months. These CDSs displayed CM ultrastructure, calcium transient, appropriate pharmacological responses and CM gene expression profiles specific for maturity. Furthermore, 3D-derived CMs displayed more mature phenotypes than those from a parallel 2D-culture. The system is compatible to large-scaly produce CMs for disease study, cell therapy and pharmaceutics screening. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 10/2015; 65. DOI:10.1016/j.biomaterials.2015.06.024 · 8.56 Impact Factor
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    • "DEVELOPMENT protocol for cardiac directed differentiation is based on studies from our laboratory and others showing that cardiac specification involves a biphasic modulation of Wnt/β-catenin signaling. Specifically, robust Wnt/β-catenin signaling activation is required to direct mesoderm, and specification into the cardiac lineage involves downregulation of Wnt/β-catenin signaling (Ueno et al., 2007; Paige et al., 2010; Lian et al., 2012; Palpant et al., 2013). The protocol used for directing cardiac differentiation is detailed in the supplementary Materials and Methods and Fig. S1. "
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    ABSTRACT: During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/β-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/β-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations. Cardiogenic mesoderm was generated under conditions specifying anterior-like mesoderm, whereas blood-forming endothelium was generated from posterior-like mesoderm, and vessel-forming CD31(+) endothelial cells were generated from all mesoderm origins. Surprisingly, inhibition of β-catenin signaling led to the highly efficient respecification of anterior-like endothelium into beating cardiomyocytes. Cardiac respecification was not observed in posterior-derived endothelial cells. Thus, activin/BMP gradients specify distinct mesodermal subpopulations that generate cell derivatives with unique angiogenic, hemogenic and cardiogenic properties that should be useful for understanding embryogenesis and developing therapeutics. © 2015. Published by The Company of Biologists Ltd.
    Development 07/2015; DOI:10.1242/dev.117010 · 6.46 Impact Factor
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    • "Human iPSC lines were maintained on Matrigel-coated plates (BD Biosciences) in Essential 8 Medium (GIBCO, Life Technology). Human iPSC-CMs were generated as described previously (Lian et al., 2012). Briefly, pluripotent stem cells were treated with 6 mM CHIR99021 ( "
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    ABSTRACT: β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell stem cell 06/2015; 17(1). DOI:10.1016/j.stem.2015.04.020 · 22.27 Impact Factor
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