Why New Standards for hPSC-CM?
Human pluripotent stem cells are a viable source for in vitro generation of cardiomyocytes useful for the study of human development, heart disease modeling, drug testing, and regenerative medicine. As the number of laboratories and studies that utilize pluripotent stem cell-derived cardiomyocytes (hPSC-CM) has grown steadily over the past decade, 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 hPSC-CM. By establishing rigorous standards for quality control evaluation of hPSC-CM, we believe our efforts will enhance the use of hPSC-CM in a broad range of research and clinical applications, especially by enabling more accurate comparisons of results among studies.
Flow Cytometry Based Assessment of Troponin Positivity
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.
Looking for reliable methods for assessing intracellular proteins in hPSC-derivatives by flow cytometry? Check out our detailed methods and SOP here:
Our protocol for generating stem cell derived cardiomyocytes:
Cell Surface Markers for Stem Cell Derived Cardiomyocytes: Cell type, chamber specificity, maturation stage
Our long-term goals include the development of cell surface marker "barcodes" that enable identification and isolation of stage- and subtype-specific cardiomyocytes which can then be tested for their suitability for a wide variety of research and clinical applications. We have already used mass spectrometry to identify >800 cell surface proteins on hPSC-CM, including several new putative markers for cell type, chamber, and maturation stage identity. Ongoing efforts to validate the novel monoclonal antibodies that recognize these molecules are actively underway (which takes years…). Check this space again soon to learn about these novel reagents and methods when they are validated and published.