Abstract 14065: Inhibition of TGF-beta/activin Signaling just before Differentiation or in the Early Phase of Differentiation Enhances Cardiogenesis Induced by Canonical Wnt Signaling in Mouse ES Cells
Introduction: Cardiac myocytes derived from embryonic stem (ES) cells are promising cell source for cardiac repair, but only a small percentage of ES cells spontaneously differentiate into cardiac myocytes. Previously we showed that Wnt-3a induced cardiac mesoderm cells resulted in remarkable enhancement of cardiac differentiation. However, the efficacy of cardiogenesis by Wnt-3a is still not enough for clinical application.
Hypothesis: Based on the developmental studies, we hypothesized that TGF-beta/activin signaling combined with Wnt-3a could improve cardiogenesis in ES cells.
Methods and Results: R1 mouse ES cells were differentiated in suspension for 7 days to form embryoid bodies (EBs) and grown under adherent conditions for another 7 days. EBs received 100 ng/ml of mouse recombinant Wnt-3a from day 2 to 6 to induce cardiac mesoderm cells. EBs were additionally stimulated with 100 ng/ml of human recombinant Activin A from day 0 to 2. The proportion of beating EBs (% beating EBs) was examined under microscope at day 9, 11 and 14. The alpha-myosin heavy chain (MHC) expression normalized for GAPDH expression was examined by quantitative RT-PCR at day 14. At day 14, addition of Activin A did not increase % beating EBs and alpha-MHC expression. Next, we examined the inhibitory effect of TGF-beta/activin signaling. EBs were additionally inhibited with 10 μmol/l of SB431542 (SB), a specific inhibitor of TGF-beta/activin signaling from day -2 to 0 or day 0 to 2. At day 14, addition of SB from day -2 to 0 or day 0 to 2 significantly enhanced % beating EBs (SB (day -2 to 0) + Wnt-3a: 60.4 ± 11.7%, SB (day 0 to 2) + Wnt-3a: 71.7 ± 5.6%, Wnt-3a only (control): 22.1 ± 3.9%, p < 0.01, n = 3) compared to Wnt-3a alone stimulation. Moreover the alpha-MHC expression was significantly increased by inhibition of TGF-beta/activin signaling (SB (day -2 to 0) + Wnt-3a: 4.9 ± 0.5-fold, SB (day 0 to 2) + Wnt-3a: 5.5 ± 0.8-fold vs. Wnt-3a only, p < 0.01, n = 3).
Conclusions: The transient inhibition of TGF-beta/activin signaling just before cardiac differentiation or in the beginning of cardiac differentiation increases cardiogenesis induced by canonical Wnt signaling. Inhibition of TGF-beta/activin signaling at the onset of cardiac differentiation plays a critical role in early cardiogenesis.
- © 2010 by American Heart Association, Inc.