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Published on: August 4, 2022
Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development
Khuram W Chaudhary1, Nestor X Barrezueta, Mary B Bauchmann
1Pfizer Global Research and Development, Chesterfield, Missouri 63017, USA.
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 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.
Insights
Laser microdissection and pressure catapulting (LMPC) isolates pure embryonic stem (ES) cell-derived cardiomyocytes. This method enables reliable in vitro drug discovery and cardiotoxicity testing for safer novel drugs.
Area of Science:
- Stem cell biology
- Cardiovascular research
- Drug discovery
Background:
- Embryonic stem (ES) cells are valuable for in vitro drug discovery and safety testing.
- ES cell-derived cardiomyocytes can predict drug cardiotoxicity.
- Cellular heterogeneity in differentiated ES cells hinders reliable in vitro model development.
Purpose of the Study:
- To develop a robust method for isolating homogeneous ES cell-derived cardiomyocytes.
- To enable reliable in vitro pharmacology assays for drug screening and cardiotoxicity assessment.
Main Methods:
- Utilized laser microdissection and pressure catapulting (LMPC) to isolate ES-derived cardiomyocytes.
- Applied isolated cells to 96-well format in vitro pharmacology assays.
- Confirmed expression of key cardiac developmental and functional markers (Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, phospholamban).
Main Results:
- LMPC successfully isolated homogeneous ES-derived cardiomyocytes.
- Isolated cells expressed essential cardiac markers.
- Functional assays demonstrated expected cardiac responses to calcium and nifedipine.
Conclusions:
- Laser microdissection and pressure catapulting (LMPC) is a robust technology for isolating homogeneous ES-derived cell types.
- This method is applicable to developing reliable in vitro assays for drug discovery and cardiotoxicity testing.
