Bone morphogenetic protein-4 enhances cardiomyocyte differentiation of cynomolgus monkey ES cells in Knockout Serum Replacement medium

¹ Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
² International Center for Young Scientist (ICYS), National Institute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan
³ Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts
⁴ Biomaterials Center, National Institute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan
⁵ Biomaterials Center, National Institute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
⁶ Cardiovascular Institute, Mount Sinai Medical School, New York, New York

^* To whom correspondence should be addressed. E-mail: hossein.hosseinkhani{at}nims.go.jp.

	Abstract

Despite extensive research in the differentiation of rodent embryonic stem (ES) cells into cardiomyocytes, there have been few studies of this process in primates. In this study we examined the role of bone morphogenic protein-4 (BMP-4) to induce cardiomyocyte differentiation of cynomolgus monkey ES cells. To study the role of BMP-4, embryonic bodies (EBs) were formed and cultured in Knockout Serum Replacement (KSR) medium containing BMP-4 for 8 days and subsequently seeded in gelatin-coated dishes for 20 days. It was found that ES cells differentiated into cardiomyocytes upon stimulation with BMP-4 in KSR medium, which resulted in a large fraction of beating EBs (16%) and the up-regulation of cardiac-specific proteins in a dose and time-dependent manner. In contrast, the addition of BMP-4 in FBS containing medium resulted in a lower fraction of beating EBs (6%). BMP-4 acted principally between mesendodermal and mesoderm progenitors and subsequently enhanced their expression. Ultrastructural observation revealed that beating EBs contained mature cardiomyocytes with sarcomeric structures. In addition, immunostaining, RT-PCR and western blotting for cardiac markers confirmed the increased differentiation of cardiomyocytes in these cultures. Moreover, electrophysiological studies demonstrated that the differentiated cardiomyocytes were electrically activated. These findings may be useful in developing effective culture conditions to differentiate cynomolgus monkey ES cells into cardiomyocytes for studying developmental biology and for regenerative medicine.

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