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A simple and efficient method for purification of human pluripotent stem cell-derived cardiomyocytes via activating p53

Acta Pharmacologica Sinica volume 46, pages 2583–2585 (2025)Cite this article

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To address this, we developed a purification method for hPSC-CMs based on the different sensitivity of cells to death-regulatory signaling. p53 is robustly activated during CM maturation [5], indicating the apoptotic-resistance of CMs after p53 activation. Indeed, no additional cell death was observed in H9 human embryonic stem cell (hESC)-derived CMs (H9-CMs) after treatment with 20 μmol/L of nutlin-3a, a p53 activator, for 4 days (Fig. 1b). We thus tested whether activating p53 can selectively purify hPSC-CMs by killing non-CM cells. Various concentrations of nutlin-3a were tested for purification of H9-CMs from differentiation day 21 for a 2-day treatment followed by a 4-day recovery. As shown in Fig. S1, flow cytometry analysis showed that the CMs represented by CTNT positive cells were 60.84% ± 2.35%, 65.4% ± 0.19%, 80.9% ± 1.21%, 90.74% ± 0.56%, 93.46% ± 0.80%, and 96.12% ± 0.55% in the cells treated with nutlin-3a at 0, 2.5, 5, 10, 20, and 40 μmol/L, respectively. No statistical significance in the purity of CMs was observed between the H9-CMs treated with 20 and 40 μmol/L of nutlin-3a (Fig. S1b).

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Fig. 1: A simple and efficient method for the purification of hPSC-CMs by activating p53.

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Acknowledgements

We would like to thank Xiang Miao, Yu-jia Zhai, and Yi-fan Bu from the Institutional Center for Shared Technologies and Facilities of SINH, CAS for technical assistance. The authors also thank WiCell Research Institute for providing the H1, H7 and H9 hESCs. The work was supported by grants from National Key R&D Program of China (2022YFA1105100 and 2022YFA1104500 to HTY), National Natural Science Foundation of China (82100319 to PZ), Youth Innovation Promotion Association CAS (No. 2022276 to PZ), “LingYan” Research and Development Project (No. 2024C03155 to PL), the National Natural Science Foundation of China (No. 82370354 to PL).

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Author notes

  1. These authors contributed equally: Peng Zhang, Sen-le Rao

Authors and Affiliations

  1. Laboratory of Molecular Cardiology, CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences (CAS), Shanghai, 200031, China

    Peng Zhang, Sen-le Rao & Huang-tian Yang

  2. Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, China

    Xiao-chen Wang & Ping Liang

  3. National Stem Cell Translational Resource Center, Shanghai East Hospital (East Hospital Affiliated to Tongji University), Tongji University School of Life Sciences and Technology, Shanghai, 200120, China

    Ji-zhen Lu & Wen-wen Jia

Authors
  1. Peng Zhang
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  2. Sen-le Rao
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  3. Xiao-chen Wang
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  5. Ji-zhen Lu
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  7. Huang-tian Yang
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Contributions

PZ and HTY conceived the study. PZ and HTY designed experiments; PZ, SLR, XCW, PL, JZL, and WWJ performed the experiments. PZ and HTY analyzed the data and wrote the manuscript.

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Correspondence to Peng Zhang or Huang-tian Yang.

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Competing interests

The authors have filed a patent (202410535584.6) for the application of purification of hPSC-CMs via activating p53.

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The animals were cared for in accordance with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011). All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Nutrition and Health (SIBS-2022-YHT-1).

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Zhang, P., Rao, Sl., Wang, Xc. et al. A simple and efficient method for purification of human pluripotent stem cell-derived cardiomyocytes via activating p53. Acta Pharmacol Sin 46, 2583–2585 (2025). https://doi.org/10.1038/s41401-025-01543-5

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