| Autor: |
Matthew Waas, Ranjuna Weerasekera, Erin M. Kropp, Marisol Romero-Tejeda, Ellen N. Poon, Kenneth R. Boheler, Paul W. Burridge, Rebekah L. Gundry |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
|
| Zdroj: |
Stem Cell Reports, Vol 12, Iss 2, Pp 395-410 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
2213-6711 |
| DOI: |
10.1016/j.stemcr.2018.12.016 |
| Popis: |
Summary: 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. : 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. Keywords: quality control, flow cytometry, troponin, cardiomyocytes, mass spectrometry, standard operating protocol |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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