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Original Research

Open Access Special Issue

WNT-targeted compound and phytoestrogen promoted cardiogenic differentiation of human induced pluripotent stem cells (hiPSCs) in vitro

  • Javad Kazemi1
  • Hosein Shahsavarani2,3
  • Parviz Pakzad1
  • Mohammadali Shokrgozar2,4

1Department of Cellular and Molecular Biology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad University, 1651153311 Tehran, Iran

2Department of Cell and Molecular Sciences, Faculty of Life Science and Biotechnology, Shahid Beheshti University, 1983963113 Tehran, Iran

3Laboratory of Regenerative Medicine and Biomedical Innovation, Pasteur Institute of Iran, 1316943551 Tehran, Iran

4Department of National Cell Bank, Pasteur Institute of Iran, 1316943551 Tehran, Iran

DOI: 10.31083/jomh.2021.097

Submitted: 14 April 2021 Accepted: 02 July 2021

Online publish date: 23 August 2021

*Corresponding Author(s): Hosein Shahsavarani E-mail:
*Corresponding Author(s): Mohammadali Shokrgozar E-mail:

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Background and objectives: Despite the advances made in the prevention and treatment of cardiovascular diseases (CVD) in the last decade, they are still the leading cause of death in males at the rate of 50% worldwide. Considering the protective role of estrogen to decrease CVD rates in young females, it was suggested that using hormone therapy can be considered to improve heart regeneration. Using in vitro induced pluripotent stem cells (iPSCs) has become one of the most significant tools in CVD treatment in both genders. We design a novel optimal protocol for the differentiation of iPSCs to cardiomyocytes which may be valuable for CVD treatment in men.

Methods: Human iPSCs were initially cultivated on mouse embryonic fibroblasts and then, transferred to a specific culture medium for differentiation process. In vitro differentiation of iPSCs into cardiomyocytes was induced at three phases on RPMI-1640 medium including CHIR99021 (5 µM) on days 0–3, BMP4 (20 ng/mL), and bFGF (100 ng/mL) on days 3–5, 10 µM of XAV939 on 6–8, and phytoestrogen + ascorbic acid on days 8–13. Scanning electron microscopy and Real-time PCR using specific primers were applied to confirm produced cardiomyocytes.

Results: We found that the simultaneous use of small chemical molecules such as CHIR99021 and XAV 939, growth factors, such as BMP4, bFGF, and herbal-derived phytoestrogen from red clover could efficiently differentiate hiPSCs from the mesoderm and cardiomyocytes after 13 days. Using phytoestrogen increased the induction of cardiac markers including cTnT and GATA-4 in a shorter time; consequently, the proposed formulation has the potential to be used in developing a novel approach for cardiac repair or regeneration.

Conclusion: Presented data indicated that the serial use of XAV939 and phytoestrogen at different times and stages can successfully induce cardiogenesis from hiPSCs. Thus, the proposed approach can be used for improved translational strategies for cardiac regeneration with fewer side effects.


Cardiac regeneration; hiPSCs; Phytoestrogen; Small molecules

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Javad Kazemi,Hosein Shahsavarani,Parviz Pakzad,Mohammadali Shokrgozar. WNT-targeted compound and phytoestrogen promoted cardiogenic differentiation of human induced pluripotent stem cells (hiPSCs) in vitro. Journal of Men's Health. 2022.doi:10.31083/jomh.2021.097.


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