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

doi:10.1016/j.stemcr.2018.12.016

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^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

10 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 language	English (US)
Pages (from-to)	395-410
Number of pages	16
Journal	Stem cell reports
Volume	12
Issue number	2
DOIs	https://doi.org/10.1016/j.stemcr.2018.12.016
State	Published - Feb 12 2019

Funding

This work was supported by the National Institutes of Health ( R01-HL126785 and R01-HL134010 to R.L.G.; F31-HL140914 to M.W.; R00-HL121177 and R01-CA220002 to P.W.B.); American Heart Association ( 15GRNT24980002 to R.L.G. and 18TPA34230105 to P.W.B.), and Entopsis Asia, Ltd, to K.R.B. E.M.K. is a member of the MCW-MSTP which is partially supported by a T32 grant from NIGMS, GM080202 . Funding sources were not involved in study design, data collection, interpretation, analysis, or publication. We thank Dr. Paul Goldspink and Linda Berg Luecke (MCW) for insightful discussions and careful review of the manuscript. Mass spectrometry analyses were performed using instrumentation in the Center for Biomedical Mass Spectrometry Research at the Medical College of Wisconsin. Flow cytometry analyses were performed using instrumentation in the Blood Center of the Wisconsin Flow Cytometry Core.

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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10.1016/j.stemcr.2018.12.016

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title = "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.",

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AU - Romero-Tejeda, Marisol

AU - Poon, Ellen N.

AU - Boheler, Kenneth R.

AU - Burridge, Paul W.

AU - Gundry, Rebekah L.

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AB - 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.

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

Abstract

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Keywords

ASJC Scopus subject areas

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