title: High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.
abstract: There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin.
authors.forename: Subarna
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Stanford Cardiovascular Institute, Stanford University School of Medicine.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Burridge
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Kropp
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Chuppa
authors.affiliation: Department of Anesthesiology, Medical College of Wisconsin.
authors.forename: Wai-Meng
authors.initials: WM
authors.lastname: Kwok
authors.affiliation: Stanford Cardiovascular Institute, Stanford University School of Medicine.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University; Division of Cardiology, Johns Hopkins University School of Medicine.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Boheler
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin; Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin; rgundry@mcw.edu.
authors.forename: Rebekah L
authors.initials: RL
authors.lastname: Gundry
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D053595:Embryonic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D018398:Homeodomain Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; C485533:IRX4 protein, human