Article

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: 9.81). 05/2012; 109(27):E1848-57. DOI: 10.1073/pnas.1200250109
Source: PubMed

ABSTRACT 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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    • "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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    • " by TGF - β to signal initiation and subsequent mesoderm formation ( Gadue et al . 2006 ; Bakre et al . 2007 ; Maretto et al . 2003 ) , before being repressed at later stages for cardiac lineage specification ( Ueno et al . 2007 ) . This property has been exploited for efficient in vitro generation of embryonic stem cell - derived cardiomyocytes ( Lian et al . 2012 ) . Due to their EndMT properties , most multipotent mesoderm progenitors are common to SMCs and endothelial cells . However , the separation between SMC and endothelial cell commitment and the generation of SMC - specific HAND+ progenitors is directed by Notch signalling and the mediation of Wnt and bone morphogenetic protein expressio"
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