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EMBRYONIC STEM CELLS

Assessment of Contractility of Purified Smooth Muscle Cells Derived from Embryonic Stem Cells

^aDepartment of Molecular Physiology and Biological Physics,
^bThe Robert M. Berne Cardiovascular Research Center,
^cBiomedical Engineering, The University of Virginia, Charlottesville, Virginia, USA;
^dVeterinary Biomedical Sciences, University of Missouri, Columbia, Missouri, USA

Key Words. Embryonic stem cell • Smooth muscle • Embryoid body • Teratoma • Contraction

Correspondence: Gary K. Owens, Ph.D.,Department of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800736, Charlottesville, Virginia 22908-0736, USA Telephone: 434-924-2652; Fax: 434-982-0055; email: gko{at}virginia.edu

Received on January 2, 2006; accepted for publication on March 28, 2006.

First published online in STEM CELLS EXPRESS  April 6, 2006.

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 -actin (SMA) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMA-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), 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 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs 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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