Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development.

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 microM 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.

[1]  B. Thiers,et al.  The Gene Expression Signatures of Melanoma Progression , 2006 .

[2]  Ling Lin,et al.  Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. , 2005, Human molecular genetics.

[3]  F. Eusebi,et al.  Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes. , 2005, Cardiovascular research.

[4]  M. LeWinter,et al.  Embryonic stem cells: differentiation into cardiomyocytes and potential for heart repair and regeneration , 2005, Coronary artery disease.

[5]  T. Nakahata,et al.  Identification of cardiac stem cells with FLK1, CD31, and VE‐cadherin expression during embryonic stem cell differentiation , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  E. Petricoin,et al.  Pathology of the Future: Molecular Profiling for Targeted Therapy , 2005, Cancer investigation.

[7]  湯浅 慎介 Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells , 2005 .

[8]  Linzhao Cheng,et al.  Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro , 2004, The Lancet.

[9]  Toshio Miki,et al.  Use and application of stem cells in toxicology. , 2004, Toxicological sciences : an official journal of the Society of Toxicology.

[10]  A. Moorman,et al.  Regional expression of L‐type calcium channel subunits during cardiac development , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[11]  M. Rubin,et al.  Molecular genetics of human prostate cancer , 2004, Modern Pathology.

[12]  George Q. Daley,et al.  Derivation of embryonic germ cells and male gametes from embryonic stem cells , 2004, Nature.

[13]  A. Moorman,et al.  Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. , 2003, Journal of molecular and cellular cardiology.

[14]  D. Torella,et al.  Adult Cardiac Stem Cells Are Multipotent and Support Myocardial Regeneration , 2003, Cell.

[15]  P. Zandstra,et al.  Scalable production of embryonic stem cell-derived cardiomyocytes. , 2003, Tissue engineering.

[16]  James A Thomson,et al.  Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization , 2003, Circulation research.

[17]  H. Jongsma,et al.  Expression of the Electrophysiological System During Murine Embryonic Stem Cell Cardiac Differentiation , 2003, Cellular Physiology and Biochemistry.

[18]  S. Nishikawa,et al.  Chamber‐specific differentiation of Nkx2.5‐positive cardiac precursor cells from murine embryonic stem cells , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  R. W. Hansen,et al.  The price of innovation: new estimates of drug development costs. , 2003, Journal of health economics.

[20]  A. Vogel,et al.  Mechanisms of pulsed laser ablation of biological tissues. , 2003, Chemical reviews.

[21]  Gabriel Acevedo-Bolton,et al.  Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis , 2003, Nature.

[22]  R. Burgemeister,et al.  Live cell catapulting and recultivation. , 2003, Pathology, research and practice.

[23]  K. Boheler ES cell differentiation to the cardiac lineage. , 2003, Methods in enzymology.

[24]  Alon Spira,et al.  High-Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-Derived Cardiomyocytes: A Novel In Vitro Model for the Study of Conduction , 2002, Circulation research.

[25]  J. Sambrook,et al.  DNA Microarrays: A Molecular Cloning Manual , 2002 .

[26]  E. Lakatta,et al.  The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  D. Bers Cardiac excitation–contraction coupling , 2002, Nature.

[28]  L. Leinwand,et al.  Myosin heavy chain isoform expression in the failing and nonfailing human heart. , 2000, Circulation research.

[29]  B. Herman,et al.  Measurement of intracellular calcium. , 1999, Physiological reviews.

[30]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[31]  E. Marbán,et al.  Calcium cycling and contractile activation in intact mouse cardiac muscle , 1998, The Journal of physiology.

[32]  B. Fleischmann,et al.  Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. , 1997, Cardiovascular research.

[33]  H. Rackwitz,et al.  Specific immunohistochemical detection of cardiac/fetal alpha-actin in human cardiomyocytes and regenerating skeletal muscle cells. , 1996, Differentiation; research in biological diversity.

[34]  G. Koh,et al.  Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. , 1996, The Journal of clinical investigation.

[35]  B. Koller,et al.  A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human inflammation. , 1995, Experimental cell research.

[36]  L. Samuelson,et al.  Myosin heavy chain expression in contracting myocytes isolated during embryonic stem cell cardiogenesis. , 1995, Circulation research.

[37]  A M Wobus,et al.  Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. , 1994, Circulation research.

[38]  E. Jaimovich,et al.  Calcium fluxes, ion currents and dihydropyridine receptors in a new immortal cell line from rat heart muscle. , 1993, Journal of molecular and cellular cardiology.

[39]  L. Brunton,et al.  Excitation-contraction coupling and cardiac contractile force , 1992 .

[40]  W. Friedman,et al.  Developmental changes in cardiac myocyte calcium regulation. , 1990, Circulation research.