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Stem Cell Reports. 2019 Feb 12;12(2):395-410. doi: 10.1016/j.stemcr.2018.12.016. Epub 2019 Jan 24.

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

Author information

1
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
2
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
3
School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
4
School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21205, USA.
5
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA; Center for Biomedical Mass Spectrometry Research, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: rgundry@mcw.edu.

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.

KEYWORDS:

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

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