Why new standards for stem cell derivatives?

Human pluripotent stem cells are a viable source for in vitro generation of cardiac cells useful for the study of human development, heart disease modeling, drug testing, and regenerative medicine. Cellular heterogeneity, variability among lines, and immaturity pose challenges to their use. Thus, the ability to accurately and precisely assess cell identity in differentiation cultures remains paramount to well-defined studies that can be replicated among laboratories.

Our laboratory works to address recent calls for added rigor in biomedical research by developing protocols, reagents, and knowledge that will facilitate reproducible generation, characterization, and selection of well-defined human pluripotent stem cell derivatives. By establishing rigorous standards for quality control evaluation, we believe our efforts will enhance the use of stem cell derivatives in a broad range of research and clinical applications, especially by enabling more accurate comparisons of results among studies.

Cell surface markers for stem cell derivatives: cell type, chamber specificity, maturation stage

We have used mass spectrometry to identify >1100 cell surface proteins on stem cell derived cardiomyocytes and cardiac fibroblasts and primary adult cardiomyocytes and cardiac fibroblasts. From these data, we are uncovering new markers for cell type, chamber, and maturation stage identity.

Poon EN, et al., The Cell Surface Marker CD36 Selectively Identifies Matured, Mitochondria-Rich hPSC-cardiomyocytes, Cell Research, 39&7): 626-6292020, PMID: 32157205.

Berg Luecke et al., Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts, 2023.

Flow Cytometry Based Assessment of Troponin Positivity

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The question of how to confidently and accurately measure the percentage of cardiomyocytes within an hPSC-CM culture may seem straightforward. However, while flow cytometry is well-suited for this purpose, there is currently no consensus regarding which marker, antibody, or protocol is best suited to enable comparisons of hPSC-CM culture heterogeneity among experiments or laboratories. This poses serious challenges to evaluating outcomes generated among laboratories and studies. In our recent study, we demonstrate previously undocumented pitfalls with popular antibodies and sample preparation conditions commonly used for the assessment of cardiomyocyte identity within differentiation cultures. To solve these problems, we used a rigorous fit-for-purpose workflow, to develop and validate a comprehensive protocol to accurately assess cardiomyocyte identity within hPSC-CM cultures.

Waas M, et al., Stem Cell Reports. 2019 Feb 12;12(2):395-410.

Berg Leucke, Waas M, and Gundry RL, Curr Protoc Stem Cell Biol. 2019 Sep;50(1):e94.

Waas M and Gundry RL, A call to adopt a "fit for purpose" approach to antibody validation for flow cytometry analyses of stem cell models and beyond.Am J Physiol Heart Circ Physiol. 2019 Nov 1;317(5):H954-H957.

Podcast interview with Dr. Gundry about this issue is here:
https://www.podbean.com/eu/pb-58645-c82268.