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. Video LinkThe video component of this article can be found at

Inhibition of an NAD+ Salvage Pathway Provides Efficient and Selective Toxicity to Human Pluripotent Stem Cells

“…For all experiments, a 24‐hour pulse treatment with 5 μM STF‐31 was performed on day 10 hiPSC‐derived cardiomyocytes (DF6‐9‐9T). Flow cytometry was performed 72 hours after initiation of STF‐31 treatment for TNNI3 (troponin I type 3) and IRX4 (Iroquois class homeodomain protein) as previously described [22]. Quantitative real‐time polymerase chain reaction (qPCR) for TNNI3 , TNNT2 (troponin T2), and NKX2‐5 (homeobox protein Nkx‐2.5) was performed using TaqMan assays (Life Technologies) 24–72 hours after initiation of STF‐31 treatment as previously described [19, 22].…”

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

“…The immature phenotype, low levels of extracellular matrix proteins and the membrane structure of hPSC‐CMs make them considerably more tolerant of enzymatic dissociation for FACS analyses or automated cell manipulation than adult CMs (Bhattacharya et al , 2014; Scheel et al , 2014). Ion currents and AP can be measured with a sufficient level of precision with these systems that the chances of generating large‐scale, standardized methods for measuring cardiotoxicity are increased (Scheel et al , 2014).…”

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

“…To obtain cardiac myocytes, a slightly modified version of the Boheler and Lian differentiation methods is used 30,31 . It is imperative that the differentiation starts during the log-phase of cell growth, but also that the starting population is sufficiently confluent to obtain a useable number of cells after sorting (approximately 75% is optimal).…”