Parallel development of cardiomyocytes and neurons in embryonic stem cell culture
Section snippets
Materials and methods
Cell culture. Mouse D3 ES cells (American Type Culture Collection, Manassas, VA) were cultured for 4 days in differentiation medium IMDM with 15% FBS (Gibco, Invitrogen, Carlsbad, CA) in 100 mm non-adhesive Petri dishes to allow the cells to aggregate and form embryoid bodies as described previously [7]. At day 5, fibronectin (5 μg/ml) or the combination of all-trans retinoic acid (Sigma, St. Louis, MO) (2.5 × 10−7 M) with fibronectin was added to the culture. These cells were cultured for another 4
Results
To investigate whether neuronal and cardiomyogenic specification may occur in parallel in differentiating ES cell culture, we examined the effects of retinoic acid, and molecules of extracellular matrix laminin and fibronectin, applied alone or in combination, on ES cell differentiation (for schematic representation of ES cell culture treatments please see Fig. 1). After 15 days of culturing, we analyzed generated cell phenotypes. Examination of live ES cultures under inverted microscope
Discussion
It was established that retinoic acid blocks the expression of the mesodermal genes Brachyury, cardiac actin, and ζ-globin acting both pro-neuronal and anti-mesodermal on mouse ES cells in culture [8]. Moreover, a recent observation has demonstrated that retinoic acid signaling restricts cardiac specification in the zebrafish embryo [9]. Reduction of retinoic acid signaling caused the formation of cardiomyocytes, via fate transformations that increased cardiac progenitor’s density. The authors
Acknowledgments
This work was supported in parts by grants from North Carolina Biotechnology Center #2004-MRG-1104 (A.K.M.) and NIH R01 #HL-60047 (L.C.K.).
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