Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2158; p. 223
• Main Authors: Galdos, Francisco X, Darsha, Adrija K, Paige, Sharon L, Wu, Sean M
• Format: Journal Article
• Language: English
• Published: United States 2021
• Subjects: Cell Differentiation; Cell Separation - methods; CRISPR-Cas Systems; Fluorescence; Gene Editing; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Regeneration; Flow cytometry; Genome editing; CRISPR/Cas9; Fluorescent reporters; hiPSC; Cardiac differentiation
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-0668-1_17

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.