Salvianolic acid B–vitamin C synergy in cardiac differentiation from embryonic stem cells Sunny Sun-Kin Chan a , Jyh-Hong Chen b , Shiaw-Min Hwang c , I-Ju Wang a , Hui-Jing Li a , Richard T. Lee d , Patrick C.H. Hsieh a,e,f,g, * a Institute of Clinical Medicine and Research Center for Clinical Medicine, National Cheng Kung University & Hospital, Tainan, Taiwan, ROC b Division of Cardiology, Department of Medicine, National Cheng Kung University & Hospital, Tainan, Taiwan, ROC c Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC d Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, USA e Department of Surgery, National Cheng Kung University & Hospital, Tainan, Taiwan, ROC f Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan, ROC g Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC article info Article history: Received 15 July 2009 Available online 28 July 2009 Keywords: Embryonic stem cells Cardiomyocyte differentiation Natural products abstract Inefficient cardiomyocyte differentiation limits the therapeutic use of embryonic stem (ES) cell-derived cardiomyocytes. While large collections of proprietary chemicals had been screened to improve ES cell differentiation into cardiomyocytes, the natural product library remained unexplored. Using a mouse ES cell line transfected with a cardiomyocyte-specific a-myosin heavy chain promoter-driven enhanced green fluorescent protein (EGFP) reporter, we screened 24 natural products with known cardioprotective actions. Salvianolic acid B (saB), while produced minimal effect on its own, concentration-dependently synergized with vitamin C in inducing cardiomyocyte differentiation, as demonstrated by an increase in EGFP + cells, beating area in embryoid bodies, and expression of cardiomyocyte maturity markers. This synergy is specific to cardiomyocyte differentiation, and is involved with collagen synthesis. The present study demonstrates the saB–vitamin C synergy in inducing ES cell differentiation into matured and func- tional cardiomyocytes, and this may lead to a practicable cocktail approach to generate ES cell-derived cardiomyocytes for cardiac stem cell therapy. Ó 2009 Elsevier Inc. All rights reserved. Introduction One of the major obstacles hindering the clinical use of embry- onic stem (ES) cells-derived cardiomyocytes for cardiac regenera- tive therapy is inefficient cardiac differentiation leading to insufficient amount of cardiomyocytes [1]. The most common approach to solve this problem is to apply knowledge of develop- mental biology in employing cardiogenesis-related endogenous factors for inducing stem cells cardiomyocyte differentiation [2–4]. Some researchers, on the other hand, have screened large libraries of proprietary chemicals to identify the most potent cardiac differentiation inducer [5,6]. However, most of these com- pounds are not readily and commonly available due to intellec- tual property issues and material transfer agreement concerns, resulting in under-evaluated for reproducibility and unnecessary delays for clinical assessment. Natural products, on the other hand, not only pose no such problems, they have diverse biolog- ical properties and putative synergistic beneficial effects. How- ever, their cardiomyocyte differentiation potentials remain largely unknown. Due to the heterogeneous nature of ES cells and variations in the stem cell differentiation process, a sensitive screening system with excellent accuracy and precision is essential for the identifica- tion of a target inducer [7]. To achieve this goal, we used an ES cell line stably transfected with an enhanced green fluorescent protein (EGFP) reporter driven by the promoter of a cardiomyocyte-spe- cific marker, a-myosin heavy chain (aMHC), such that EGFP will only express upon cardiomyocyte differentiation [8]. Here we report screening of selected natural products with known cardio- protective activities in promoting ES cell differentiation into ma- tured and functional cardiomyocytes. Materials and methods Cell culture of mouse ES-paMHC-EGFP cells. Mouse ES-paMHC- EGFP cells, obtained by transfecting the aMHC promoter-driven EGFP reporter into the CGR8 ES cell line, were used to screen 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.07.122 * Corresponding author. Address: Institute of Clinical Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan City 70428, Taiwan, ROC. Fax: +886 6 3028049. E-mail address: phsieh@mail.ncku.edu.tw (P.C.H. Hsieh). Biochemical and Biophysical Research Communications 387 (2009) 723–728 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc