Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry

• Published in: Stem cell reports Vol. 12; no. 2; pp. 395 - 410
• Main Authors: Waas, Matthew, Weerasekera, Ranjuna, Kropp, Erin M., Romero-Tejeda, Marisol, Poon, Ellen N., Boheler, Kenneth R., Burridge, Paul W., Gundry, Rebekah L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.02.2019; Elsevier
• Subjects: Amino Acid Sequence; cardiomyocytes; Cell Differentiation - physiology; Cell Line; flow cytometry; Flow Cytometry - methods; Humans; mass spectrometry; Myocytes, Cardiac - cytology; Pluripotent Stem Cells - cytology; quality control; Resource; standard operating protocol; troponin; cardiomyocytes; mass spectrometry; flow cytometry; quality control; troponin; standard operating protocol
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2018.12.016

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. [Display omitted] •TNNI3 and TNNT2 proteins are present in day 25 hPSC-CMs•Commonly used reagents can lead to non-specific binding of anti-troponin antibodies•A fit-for-purpose workflow describes how to develop a flow cytometry protocol•A robust protocol for routine quality control testing was validated for hPSC-CMs Waas and colleagues demonstrate pitfalls with popular antibodies and sample preparation conditions commonly used for the assessment of cardiomyocyte identity within differentiation cultures. By using a rigorous fit-for-purpose workflow, the authors developed and validated a comprehensive protocol to accurately assess cardiomyocyte identity within hPSC-CM cultures. The new protocol includes stepwise instructions to facilitate its implementation by experts and novices alike.