Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Abstract

BackgroundWnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.Methodology/Principal FindingsWe tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.Conclusions/SignificanceOur data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.

Chemical Biology of Pluripotent Stem Cells: Focus on Cardiomyogenesis

“…But, after the mesoderm formation, Wnt signaling prevents differentiation of committed cells into cardiomyocytes [53,56]. Similar time-dependent biphasic effects of Wnt signaling on cardiomyogenesis are also noted in human ES cells [57]. Given the importance of Wnt// β-catenin signaling in cardiomyogenesis, it is not surprising then that selective modulators of this pathway have a strong impact on in vitro cardiomyogenesis.…”

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…The Activin/BMP4 protocol usually results in > 30 % of beating cardiomyocytes within 2 weeks [14]. Some researchers, however, have reported significant variance in the yields obtained with cell lines and poor reproducibility of different experiments with the same cell line [14,68,121].…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…The canonical Wnt pathway is also involved at the first stage of cardiac induction. b-catenin was showed to induce EMT in ESCs and expression of mesodermal markers [221,265,266].…”

“…Electrophysiological studies revealed that iPSC-CM display heterogeneous culture including atrial-, nodal-, and ventricular-like action potential (AP) [299,300] that also show variable AP characteristics between studies and within studies with different cell lines [214], differentiation methods [266], and time in culture [301].…”