Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Matthew Waas, Ranjuna Weerasekera, Erin M. Kropp, Marisol Romero-Tejeda, Ellen Poon, Kenneth R. Boheler, Paul W. Burridge, Rebekah L. Gundry*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Modern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fit-for-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Aug 9 2018

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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