Abstract 346: A Standard Flow Cytometry Protocol for Assessing Human Pluripotent Stem Cell-derived Cardiomyocyte Identity by Troponin Positivity

Directed differentiation of human pluripotent stem cells (hPSC) into cardiomyocytes (hPSC-CM) offers an inexhaustible supply of cells for basic science research and translational applications. However, despite significant advancements in defining the factors most critical for differentiation, the resulting cultures remain a heterogeneous mixture of cells. Ultimately, as heterogeneity can pose challenges to interpreting functional data, the ability to accurately and precisely assess cell identity in differentiation cultures is paramount to well-defined and reproducible studies. To date, a standardized flow cytometry protocol that is broadly accepted among laboratories has not been established for assessing cell type heterogeneity within hPSC-CM cultures, posing challenges to evaluating outcomes generated among laboratories and studies. A survey of studies published over the past seven years (1/2010-10/2017) reveals the wide range of antibodies and experimental conditions reported for flow cytometry-based assessment of hPSC-CM. Although our literature survey revealed that a preponderance of studies relied on TNNT2 as a marker of cardiomyocyte identity, TNNI3 is more specific to cardiomyocytes than TNNT2 throughout development. We applied targeted mass spectrometry to confirm the presence of TNNI3 in our hPSC-CM. We then investigated five commercially available anti-TNNI3 and three sample preparation techniques for use in the assessment of heterogeneity of hPSC-CM cultures by flow cytometry. Results demonstrate two of the five anti-TNNI3 clones appear suitable for marking hPSC-CM and reveal differential susceptibility of antibody clones to sample preparation conditions. To further test the rigor and utility of our flow cytometry protocol, it was shared with two collaborating laboratories and applied to heterogeneous mixtures of positive and negative cell types. Results from these two laboratories demonstrate the protocol successfully distinguishes positive and negative cell types similarly, despite using different cell lines and differentiation protocols. We propose a workflow for establishing the fit-for-purpose use of antibodies and a standard protocol for assessing hPSC-CM identity by troponin positivity.