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 N. Poon, Kenneth R. Boheler, Paul W. Burridge, Rebekah L. Gundry

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity 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 has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation.

Original languageEnglish (US)
Pages (from-to)395-410
Number of pages16
JournalStem Cell Reports
Volume12
Issue number2
DOIs
StatePublished - Feb 12 2019

Keywords

  • cardiomyocytes
  • flow cytometry
  • mass spectrometry
  • quality control
  • standard operating protocol
  • troponin

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Developmental Biology
  • Cell Biology

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