Rapamycin and CHIR99021 coordinate robust cardiomyocyte differentiation from human pluripotent stem cells via reducing p53-dependent apoptosis

X. X. Xiao-Xu, Yang Liu, Yi Fan Zhang, Ya Na Guan, Qian Qian Jia, Chen Wang, He Liang, Yong Qin Li, Huang Tian Yang, Yong Wen Qin, Shuang Huang, Xian Xian Zhao, Qing Jing

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 linespecific 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.

Original languageEnglish (US)
Article numbere005295
JournalJournal of the American Heart Association
Volume6
Issue number10
DOIs
StatePublished - Oct 1 2017

Fingerprint

Pluripotent Stem Cells
Sirolimus
Cardiac Myocytes
Apoptosis
Induced Pluripotent Stem Cells
Growth Differentiation Factors
Cell Line
Troponin T
Preclinical Drug Evaluations
Chir 99021
Heart Diseases
Reactive Oxygen Species
Intercellular Signaling Peptides and Proteins

Keywords

  • Apoptosis
  • Cardiac differentiation
  • Human embryonic stem cells
  • Mammalian target of rapamycin

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Rapamycin and CHIR99021 coordinate robust cardiomyocyte differentiation from human pluripotent stem cells via reducing p53-dependent apoptosis. / Xiao-Xu, X. X.; Liu, Yang; Zhang, Yi Fan; Guan, Ya Na; Jia, Qian Qian; Wang, Chen; Liang, He; Li, Yong Qin; Yang, Huang Tian; Qin, Yong Wen; Huang, Shuang; Zhao, Xian Xian; Jing, Qing.

In: Journal of the American Heart Association, Vol. 6, No. 10, e005295, 01.10.2017.

Research output: Contribution to journalArticle

Xiao-Xu, XX, Liu, Y, Zhang, YF, Guan, YN, Jia, QQ, Wang, C, Liang, H, Li, YQ, Yang, HT, Qin, YW, Huang, S, Zhao, XX & Jing, Q 2017, 'Rapamycin and CHIR99021 coordinate robust cardiomyocyte differentiation from human pluripotent stem cells via reducing p53-dependent apoptosis', Journal of the American Heart Association, vol. 6, no. 10, e005295. https://doi.org/10.1161/JAHA.116.005295
Xiao-Xu, X. X. ; Liu, Yang ; Zhang, Yi Fan ; Guan, Ya Na ; Jia, Qian Qian ; Wang, Chen ; Liang, He ; Li, Yong Qin ; Yang, Huang Tian ; Qin, Yong Wen ; Huang, Shuang ; Zhao, Xian Xian ; Jing, Qing. / Rapamycin and CHIR99021 coordinate robust cardiomyocyte differentiation from human pluripotent stem cells via reducing p53-dependent apoptosis. In: Journal of the American Heart Association. 2017 ; Vol. 6, No. 10.
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abstract = "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 linespecific 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.",
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AU - Xiao-Xu, X. X.

AU - Liu, Yang

AU - Zhang, Yi Fan

AU - Guan, Ya Na

AU - Jia, Qian Qian

AU - Wang, Chen

AU - Liang, He

AU - Li, Yong Qin

AU - Yang, Huang Tian

AU - Qin, Yong Wen

AU - Huang, Shuang

AU - Zhao, Xian Xian

AU - Jing, Qing

PY - 2017/10/1

Y1 - 2017/10/1

N2 - 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 linespecific 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.

AB - 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 linespecific 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.

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