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

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

[1] Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380: 2095–2128.

[2] Sarrafzadegan N, Mohammmadifard NJAoIm. Cardiovascular disease in Iran in the last 40 years: prevalence, mortality, morbidity, challenges and strategies for cardiovascular prevention. Archives of Iranian Medicine. 2019; 22: 204–210.

[3] Wang H, Naghavi M, Allen C, Barber RM, Bhutta ZA, Carter A, et al. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388: 1459–1544.

[4] Sawa Y. Surgical Regeneration Therapy Using Myoblast Sheets for Severe Heart Failure. Japanese Journal of Thoracic Surgery. 2017; 70: 9–13. (In Japanese)

[5] Murry CE, Reinecke H, Pabon LM. Regeneration gaps: observations on stem cells and cardiac repair. Journal of the American College of Cardiology. 2006; 47: 1777–1785.

[6] de Simone G, Devereux RB, Daniels SR, Meyer RA. Gender differences in left ventricular growth. Hypertension. 1995; 26: 979–983.

[7] Wake R, Yoshiyama M. Gender Differences in Ischemic Heart Disease. Recent Patents on Cardiovascular Drug Discovery. 2009; 4: 234–240.

[8] Kararigas G, Dworatzek E, Petrov G, Summer H, Schulze TM, Baczko I, et al. Sex-dependent regulation of fibrosis and inflammation in human left ventricular remodelling under pressure overload. European Journal of Heart Failure. 2014; 16: 1160–1167.

[9] Naftolin F, Friedenthal J, Nachtigall R, Nachtigall LJF. Cardiovascular health and the menopausal woman: the role of estrogen and when to begin and end hormone treatment. F1000Research. 2019; 8: F1000 Faculty Rev-1576.

[10] Johansen N, Tonstad S, Liavaag AH, Selmer RM, Tanbo TG, Michelsen TM. Risk of cardiovascular disease after preventive salpingo-oophorectomy. International Journal of Gynecologic Cancer. 2020; 30: 575–582.

[11] Oka S-i, Sabry AD, Cawley KM, Warren JSJFicm. Multiple levels of PGC-1α dysregulation in heart failure. Frontiers in Cardiovascular Medicine. 2020; 7: 2.

[12] Balafkan N, Mostafavi S, Schubert M, Siller R, Liang KX, Sullivan G, et al. A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates. Scientific Reports. 2020; 10: 18498.

[13] Burridge PW, Keller G, Gold JD, Wu JC. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 2012; 10: 16–28.

[14] Guan X, Xu W, Zhang H, Wang Q, Yu J, Zhang R, et al. Transplanta-tion of human induced pluripotent stem cell-derived cardiomyocytes improves myocardial function and reverses ventricular remodeling in infarcted rat hearts. Stem Cell Research & Therapy. 2020; 11: 73.

[15] Marzouni ET, Dorcheh SP, Nejad-Moghaddam A, Ghanei M, Goodarzi H, Hosseini SE, et al. Adipose-derived mesenchymal stem cells ameliorate lung epithelial injury through mitigating of oxidative stress in mustard lung. Regenerative Medicine. 2020; 15: 1861–1876.

[16] Ito M, Hara H, Takeda N, Naito AT, Nomura S, Kondo M, et al. Characterization of a small molecule that promotes cell cycle activation of human induced pluripotent stem cell-derived cardiomyocytes. Journal of Molecular and Cellular Cardiology. 2019; 128: 90–95.

[17] Sharma A, Li G, Rajarajan K, Hamaguchi R, Burridge PW, Wu SM. Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation. Journal of Visualized Experiments. 2015; 52628.

[18] Singh VP, Pinnamaneni JP, Pugazenthi A, Sanagasetti D, Mathison M, Wang K, et al. Enhanced Generation of Induced Cardiomyocytes Using a Small-Molecule Cocktail to Overcome Barriers to Cardiac Cellular Reprogramming. Journal of the American Heart Association. 2020; 9: e015686.

[19] Liu Y, Chen B, Yang X, Fugate JA, Kalucki FA, Futakuchi-Tsuchida A, et al. Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nature Biotech-nology. 2018; 36: 597–605.

[20] Hirschi KK, Li S, Roy K. Induced pluripotent stem cells for regener-ative medicine. Annual Review of Biomedical Engineering. 2014; 16: 277–294.

[21] Sepac A, Si-Tayeb K, Sedlic F, Barrett S, Canfield S, Duncan SA, et al. Comparison of cardiomyogenic potential among human ESC and iPSC lines. Cell Transplantation. 2012; 21: 2523–2530.

[22] Hwang GH, Park SM, Han HJ, Kim JS, Yun SP, Ryu JM, et al. Purification of small molecule-induced cardiomyocytes from human induced pluripotent stem cells using a reporter system. Journal of Cellular Physiology. 2017; 232: 3384–3395.

[23] Ueno S, Weidinger G, Osugi T, Kohn AD, Golob JL, Pabon L, et al. Biphasic role for Wnt/β-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104: 9685–9690.

[24] Desmarais JA, Unger C, Damjanov I, Meuth M, Andrews P. Apoptosis and failure of checkpoint kinase 1 activation in human induced pluripotent stem cells under replication stress. Stem Cell Research & Therapy. 2016; 7: 17.

[25] An WF, Germain AR, Bishop JA, Nag PP, Metkar S, Ketterman J, et al. Discovery of potent and highly selective inhibitors of GSK3b. In Probe Reports from the NIH Molecular Libraries Program. National Center for Biotechnology Information (US). 2014.

[26] Huang T, Li L, Moalim-Nour L, Jia D, Bai J, Yao Z, et al. A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells in Response to Wnt Signaling. Stem Cells. 2015; 33: 1419–1433.

[27] Tan JY, Sriram G, Rufaihah AJ, Neoh KG, Cao T. Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embry-onic stem cells through GSKi-mediated differentiation. Stem Cells and Development. 2013; 22: 1893–1906.

[28] Naujok O, Lentes J, Diekmann U, Davenport C, Lenzen S. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Research Notes. 2014; 7: 273.

[29] Yoshino Y, Ishioka C. Inhibition of glycogen synthase kinase-3 beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells. Scientific Reports. 2015; 5: 13249.

[30] Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109: E1848–E1857.

[31] Laco F, Woo TL, Zhong Q, Szmyd R, Ting S, Khan FJ, et al. Unraveling the Inconsistencies of Cardiac Differentiation Efficiency Induced by the GSK3β Inhibitor CHIR99021 in Human Pluripotent Stem Cells. Stem Cell Reports. 2018; 10: 1851–1866.

[32] Zhao M, Tang Y, Zhou Y, Zhang J. Deciphering Role of Wnt Signalling in Cardiac Mesoderm and Cardiomyocyte Differentiation from Human iPSCs: Four-dimensional control of Wnt pathway for hiPSC-CMs differentiation. Scientific Reports. 2019; 9: 19389.

[33] Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell. 2011; 8: 228–240.

[34] Andersen P, Tampakakis E, Jimenez DV, Kannan S, Miyamoto M, Shin HK, et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nature Communications. 2018; 9: 3140.

[35] Ulmer BM, Stoehr A, Schulze ML, Patel S, Gucek M, Mannhardt I, et al. Contractile Work Contributes to Maturation of Energy Metabolism in hiPSC-Derived Cardiomyocytes. Stem Cell Reports. 2018; 10: 834–847.

[36] Correia C, Koshkin A, Duarte P, Hu D, Carido M, Sebastião MJ, et al. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnology and Bioengineering. 2018; 115: 630–644.

[37] Lemoine MD, Mannhardt I, Breckwoldt K, Prondzynski M, Flenner F, Ulmer B, et al. Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density. Scientific Reports. 2017; 7: 5464.

[38] Machiraju P, Greenway SC. Current methods for the maturation of induced pluripotent stem cell-derived cardiomyocytes. World Journal of Stem Cells. 2020; 11: 33–43.

[39] Ahmed RE, Anzai T, Chanthra N, Uosaki HJFiC, Biology D. A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes. Frontiers in Cell and Developmental Biology. 2020; 8: 178.

[40] Iorga A, Cunningham CM, Moazeni S, Ruffenach G, Umar S, Eghbali M. The protective role of estrogen and estrogen receptors in cardiovascular disease and the controversial use of estrogen therapy. Biology of Sex Differences. 2017; 8: 33.

[41] Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, et al. Interaction of phytoestrogens with estrogen receptors alpha and beta. Biological & Pharmaceutical Bulletin. 2001; 24: 351–356.

[42] Azadian Z, Shafiei M, Hosseini S, Kazemi J, Alipour A, Shahsavarani H. EfficientIn VitroDifferentiation of Adipose Tissue-Derived Mes-enchymal Stem Cells into the Cardiomyocyte Using Plant-Derived Natural Compounds. Proceedings of the Singapore National Academy of Science. 2019; 13: 47–63.

[43] Parikh A, Wu J, Blanton RM, Tzanakakis ES. Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation. Tissue Engineering Part B, Reviews. 2015; 21: 377–392.

[44] Liu Q, Van Bortle K, Zhang Y, Zhao M, Zhang JZ, Geller BS, et al. Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid. Scientific Reports. 2018; 8: 12960.

[45] Ozhan G, Weidinger G. Wnt/β-catenin signaling in heart regenera-tion. Cell Regeneration. 2015; 4: 3.

[46] Hatami L, Valojerdi MR, Mowla SJ. Effects of oxytocin on cardiomy-ocyte differentiation from mouse embryonic stem cells. International Journal of Cardiology. 2007; 117: 80–89.

[47] Morselli E, Santos RS, Criollo A, Nelson MD, Palmer BF, Clegg DJ. The effects of oestrogens and their receptors on cardiometabolic health. Nature Reviews. Endocrinology. 2017; 13: 352–364.

[48] Tava A, Pecio Ł, Lo Scalzo R, Stochmal A, Pecetti L. Phenolic Content and Antioxidant Activity in Trifolium Germplasm from Different Environments. Molecules. 2019; 24: 298.

[49] Minami I, Yamada K, Otsuji TG, Yamamoto T, Shen Y, Otsuka S, et al. A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Reports. 2012; 2: 1448–1460.

[50] Shi Y, Zhang X, Hu Z, Zhang C, Liao D, Huang H, et al. Genistein Protects H9c2 Cardiomyocytes against Chemical Hypoxia-Induced Injury via Inhibition of Apoptosis. Pharmacology. 2019; 103: 282–290.

[51] Dixit P, Katare R. Challenges in identifying the best source of stem cells for cardiac regeneration therapy. Stem Cell Research & Therapy. 2015; 6: 26.

[52] Abraham CS, Prasana JC, Muthu S, Rizwana B F, Raja M. Quantum computational studies, spectroscopic (FT-IR, FT-Raman and UV–Vis) profiling, natural hybrid orbital and molecular docking analysis on 2,4 Dibromoaniline. Journal of Molecular Structure. 2018; 1160: 393–405.

[53] Anagnostis P, Paschou SA, Katsiki N, Krikidis D, Lambrinoudaki I, Goulis DG. Menopausal Hormone Therapy and Cardiovascular Risk: where are we now? Current Vascular Pharmacology. 2019; 17: 564–572.

[54] Pu D, Tan R, Yu Q, Wu J. Metabolic syndrome in menopause and associated factors: a meta-analysis. Climacteric : the Journal of the International Menopause Society. 2017; 20: 583–591.

[55] Wolters M, Dejanovic GM, Asllanaj E, Günther K, Pohlabeln H, Bramer WM, et al. Effects of phytoestrogen supplementation on in-termediate cardiovascular disease risk factors among postmenopausal women: a meta-analysis of randomized controlled trials. Menopause. 2020; 27: 1081–1092.

[56] Sansai K, Na Takuathung M, Khatsri R, Teekachunhatean S, Hanprasertpong N, Koonrungsesomboon N. Effects of isoflavone interventions on bone mineral density in postmenopausal women: a systematic review and meta-analysis of randomized controlled trials. Osteoporosis International. 2020; 31: 1853–1864.

[57] Liu A, Zhang D, Yang X, Song Y. Estrogen receptor alpha activates MAPK signaling pathway to promote the development of endometrial cancer. Journal of Cellular Biochemistry. 2019; 120: 17593–17601.

[58] Hosoda K, Furuta T, Yokokawa A, Ishii KJA, chemistry b. Iden-tification and quantification of daidzein-7-glucuronide-4′-sulfate, genistein-7-glucuronide-4′-sulfate and genistein-4′, 7-diglucuronide as major metabolites in human plasma after administration of kinako. Analytical and Bioanalytical Chemistry. 2010; 397: 1563–1572.