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.
Chaudhary
Khuram W
KW
Pfizer Global Research and Development, Chesterfield, Missouri 63017, USA.
Barrezueta
Nestor X
NX
Bauchmann
Mary B
MB
Milici
Anthony J
AJ
Beckius
Gretchen
G
Stedman
Donald B
DB
Hambor
John E
JE
Blake
William L
WL
McNeish
John D
JD
Bahinski
Anthony
A
Cezar
Gabriela Gebrin
GG
D000818:Animals; D001681:Biological Assay / Q000379:methods; D015415:Biomarkers / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005318:Fetal Heart / Q000166:cytology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006331:Heart Diseases / Q000139:chemically induced* / Q000473:pathology; D007834:Lasers; D051379:Mice; D008811:Mice, Inbred DBA; D042282:Microdissection / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009543:Nifedipine / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015262:Xenobiotics / Q000633:toxicity*
D015415:Biomarkers; D015262:Xenobiotics; D009543:Nifedipine; D002118:Calcium