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.
Highlights
Heart failure is a leading cause of death among all patient populations, in large part due to the heart’s limited ability for selfrepair
Using the standard activin A/BMP4 directed differentiation protocol, we found over a 300-fold increase in b-myosin heavy chain (b-MHC) relative to cultures treated with medium alone without cytokines (p,0.01) (Figure 1A)
While studies in frog and chick embryos suggested that Wnt/b-catenin signaling suppressed cardiac differentiation, evidence from mouse P19CL6 teratocarcinoma cells indicated a positive role for Wnt/b-catenin signaling in cardiogenesis [10,11,12]
Summary
Heart failure is a leading cause of death among all patient populations, in large part due to the heart’s limited ability for selfrepair. Our group has demonstrated that sequential treatment with activin A and BMP4 results in enhanced generation of mesoderm followed by induction toward a cardiac fate, with 10–50% of cells differentiating into definitive cardiomyocytes [8,9]. Before these cells can be used for therapy in humans, it will be necessary to understand the signaling pathways that affect the differentiation and maturation of ES cells and their progeny. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4
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