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

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.

ORGANISM(S): Homo Sapiens

SUBMITTER: Matthew Waas  

PROVIDER: PXD014971 | panorama | Mon Aug 12 00:00:00 BST 2019

REPOSITORIES: PanoramaPublic