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Embryonic Stem Cells |
1 Department of Molecular Physiology and Biological Physics, The Robert M. Berne Cardiovascular Research Center, The University of Virginia, Charlottesville, Virginia
2 Biomedical Engineering, The University of Virginia, Charlottesville, Virginia
3 Veternary Biomedical Sciences, University of Missouri, Columbia, Missouri
* To whom correspondence should be addressed. E-mail: gko{at}virginia.edu.
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Abstract |
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The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin resistance gene under the control of either a smooth muscle (SM) 
-actin or SM-myosin heavy chain (SMMHC) promoter. Negative selection, either overnight or for three days, was then used to purify
SMCs from EBs. Purified SMCs expressed multiple SMC markers by immunofluorescence,
immunoblotting, quantitative RT-PCR and FACS and were designated APSC (SM -Actin-Puromycin-Selected Cells) or MPSC (SM-MHC-PSC) respectively. Both SMC lines displayed agonist induced Ca2+ transients, expressed functional Ca2+ channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as
endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for three days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESC which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC derived cell lines, even after prolonged differentiation; highlighting the critical requirement
for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
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