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Updated: Dec 26, 2025
Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization
Published on: January 17, 2020
Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques
Cassady E Rupert1, Chinedu Irofuala1, Kareen L K Coulombe1
1Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI, United States of America.
Insights
Optimizing human induced pluripotent stem cell (hiPSC) differentiation for cardiac therapies requires specific conditions. This study identifies optimal CHIR99027 concentration and seeding density for high-purity cardiomyocyte generation.
Area of Science:
- Cardiovascular Biology
- Stem Cell Biology
- Biotechnology
Background:
- Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are crucial for cardiac research and therapy development.
- Large-scale, high-purity generation of these cells remains a significant challenge for widespread application.
Purpose of the Study:
- To optimize cardiac differentiation protocols for hiPSCs using design of experiments (DOE).
- To develop and apply novel image analysis for assessing cell seeding and differentiation.
- To evaluate the impact of metabolic selection on cardiomyocyte bioenergetics and tissue function.
Main Methods:
- Design of experiments (DOE) to vary CHIR99027 concentration and cell seeding density.
- Novel image analysis for quantifying plate coverage at differentiation initiation.
- Metabolic selection using lactate to purify cardiomyocyte populations.
- Comparison of bioenergetic phenotypes and engineered tissue mechanics.
Main Results:
- Optimal differentiation conditions (3 μM CHIR99027, 72 x 103 cells/cm2 seeding density) achieved 50-90% cardiac purity across three hiPSC lines.
- Metabolic selection with lactate shifted cardiomyocyte metabolism towards oxidative phosphorylation.
- Enhanced metabolic maturity did not directly translate to improved contractile function in engineered tissues after one week.
Conclusions:
- Identified widely adaptable methods and parameters for refining hiPSC-cardiomyocyte differentiation.
- Demonstrated the utility of metabolic selection for purifying cardiomyocytes.
- Highlighted that metabolic maturation alone does not guarantee functional maturation in engineered cardiac tissues within one week.
Abstract:
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the cardiac differentiation space of three hiPSC lines when varying CHIR99027 concentration and cell seeding density, and a novel image analysis is developed to evaluate plate coverage when initiating differentiation. Metabolic selection via lactate purifies hiPSC-cardiomyocyte populations, and the bioenergetic phenotype and engineered tissue mechanics of purified and unpurified hiPSC-cardiomyocytes are compared. Findings demonstrate that when initiating differentiation one day after hiPSC plating, low (3 μM) Chiron and 72 x 103 cells/cm2 seeding density result in peak cardiac purity (50-90%) for all three hiPSC lines. Our results confirm that metabolic selection with lactate shifts hiPSC-cardiomyocyte metabolism towards oxidative phosphorylation, but this more "mature" metabolic phenotype does not by itself result in a more mature contractile phenotype in engineered cardiac tissues at one week of culture in 3D tissues. This study provides widely adaptable methods including novel image analysis code and parameters for refining hiPSC-cardiomyocyte differentiation and describes the practical implications of metabolic selection of cardiomyocytes for downstream tissue engineering applications.
