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ToxSci Advance Access published online on December 15, 2005
Toxicological Sciences, doi:10.1093/toxsci/kfj078
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© The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received September 17, 2005
Accepted November 23, 2005

In Vitro Toxicology

Embryonic Stem Cells in Predictive Cardiotoxicity: Laser Capture Microscopy Enables Assay Development

Khuram W. Chaudhary 1, Nestor X. Barrezueta 2, Mary B. Bauchmann 2, Anthony J. Milici 2, Gretchen Beckius 2, Donald B. Stedman 2, John E. Hambor 2, William L. Blake 2, John D. McNeish 2, Anthony Bahinski 3, and Gabriela Gebrin Cezar 4 *

1 Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017 U.S.A.; Current address: Lexicon Genetics Inc., 8800 Technology Forest Place, The Woodlands, Texas 77381 U.S.A.
2 Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340 U.S.A.
3 Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017 U.S.A.
4 Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340 U.S.A.; Current address: University of Wisconsin-Madison, Department of Animal Sciences, 1675 Observatory Drive, Madison, Wisconsin, 53706 U.S.A.

* To whom correspondence should be addressed.
Gabriela Gebrin Cezar, E-mail: ggcezar{at}wisc.edu


  Abstract

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using Laser Microdissection and Pressure Catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 µM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.


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