Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

• Published in: Journal of cellular biochemistry Vol. 112; no. 12; pp. 3555 - 3562
• Main Authors: Ou, Dong-Bo, He, Yong, Chen, Rui, Teng, Ji-Wei, Wang, Hong-Tao, Zeng, Di, Liu, Xiong-Tao, Ding, Lu, Huang, Jin-Yan, Zheng, Qiang-Sun
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.12.2011
• Subjects: Animals; Base Sequence; Cardiac fibroblasts; Cardiomyocytes; Cell culture; Cell Differentiation; Coculture Techniques; Collagen; Collagen (type I); Differentiation; DNA Primers; Embryo cells; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - ultrastructure; Fibroblasts; Heart; Immunocytochemistry; Immunohistochemistry; Mice; Microscopy, Electron, Transmission; Myocardium - cytology; Polymerase chain reaction; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Stem cells; Transmission electron microscopy
• ISSN: 0730-2312; 1097-4644; 1097-4644
• DOI: 10.1002/jcb.23283

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two‐dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three‐dimensional (3D) culture combined with indirect cardiac fibroblasts co‐culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co‐cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC‐derived CMs (ESCMs) were analyzed by real time RT‐PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co‐culture was significantly higher than that without co‐culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late‐stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late‐stage development. The combined effects of 3D and co‐culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber‐like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co‐culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells. J. Cell. Biochem. 112: 3555–3562, 2011. © 2011 Wiley Periodicals, Inc.