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Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity
Published on: July 10, 2019
Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via
Xiao-Xu Qiu1, Yang Liu1, Yi-Fan Zhang1
1Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine and Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, Shanghai, China.
Background:
Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation.
Methods And Results:
We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production.
Conclusions:
We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.
Insights
Combining rapamycin and CHIR99021 significantly improves cardiomyocyte differentiation from human pluripotent stem cells. This optimized method enhances efficiency, yield, and consistency for disease modeling and drug screening.
Area of Science:
- Stem cell biology
- Cardiovascular research
- Molecular medicine
Background:
- Human pluripotent stem cells offer a renewable source for cardiac disease modeling.
- Current small molecule-driven cardiomyocyte differentiation methods suffer from low efficiency and line-to-line variability.
- Challenges include low endogenous growth factor expression and high apoptosis rates in pluripotent stem cells.
Purpose of the Study:
- To identify small molecules that enhance cardiomyocyte differentiation efficiency and reliability.
- To develop a robust, growth factor-free protocol for generating cardiomyocytes from various human pluripotent stem cell lines.
- To elucidate the role of mammalian target of rapamycin (mTOR) in cardiac differentiation.
Main Methods:
- Screening of small molecules using an H9-human cardiac troponin T-eGFP reporter cell line.
- Utilizing a monolayer-based, growth factor-free differentiation model.
- Investigating the effects of combined rapamycin and CHIR99021 treatment on human pluripotent stem cells.
Main Results:
- Co-treatment with rapamycin and CHIR99021 is essential for efficient and consistent cardiomyocyte differentiation across multiple cell lines (e.g., H7, H9, UQ1).
- This combination increased cardiomyocyte yield (1:24) and reduced medium consumption by 50%.
- Inhibition of mTOR via rapamycin blunts p53 translation and mitochondrial reactive oxygen species, overcoming apoptosis during differentiation.
Conclusions:
- Mammalian target of rapamycin (mTOR) plays a stage-specific, regulatory role in cardiac differentiation.
- An optimized protocol using rapamycin and CHIR99021 enables large-scale generation of functional cardiomyocytes.
- This approach facilitates robust in vitro disease modeling and drug screening applications.
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