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

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.