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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    • "In this protocol, human iPS cells are differentiated as a monolayer, which eliminates embryoid body formation variability (Burridge et al., 2007) but requires careful control of pluripotent cell seeding density. Differentiation towards cardiomyocyte lineage is induced using small molecules to modulate the WNT signaling pathway, first with a GSK3B inhibitor to potentiate WNT signaling and then 2 days later with a WNT inhibitor to attenuate WNT signaling (Gonzalez et al., 2011; Lian et al., 2012; Burridge et al., 2014). The basic culture medium used throughout cardiac differentiation and cardiomyocyte maintenance is CDM3 (Burridge et al., 2014), a chemically defined medium consisting of RPMI 1640, rice-derived recombinant human albumin, and Lascorbic acid 2-phosphate. "
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    ABSTRACT: Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost-effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing. © 2015 by John Wiley & Sons, Inc.
    Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 10/2015; 87:21.3.1-21.3.15. DOI:10.1002/0471142905.hg2103s87
    • "Cells are then treated with 10 lM CHIR99021 supplemented E6 media—E8 media lacking the pluripotency factors FGF2 and TGF- b1—to simulate Wnt signaling during gastrulation. CHIR99021, a potent GSK3 inhibitor, has been extensively used to drive mesoderm specification in cardiac and other mesoderm-lineage differentiation protocols [1] [3] [24]. There is a notably high level of cell death—approximately 25% of total cells—24 h after the initial CHIR99021 treatment (Fig. 1B), which we observe to correlate with more efficient mesoderm induction. "
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    ABSTRACT: Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2 weeks using collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies.
    Methods 09/2015; DOI:10.1016/j.ymeth.2015.09.019 · 3.65 Impact Factor
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