title: Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.
abstract: 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.
authors.affiliation: Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
authors.forename: Dong-Bo
authors.initials: DB
authors.lastname: Ou
authors.forename: Yong
authors.initials: Y
authors.lastname: He
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Ji-Wei
authors.initials: JW
authors.lastname: Teng
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Wang
authors.forename: Di
authors.initials: D
authors.lastname: Zeng
authors.forename: Xiong-Tao
authors.initials: XT
authors.lastname: Liu
authors.forename: Lu
authors.initials: L
authors.lastname: Ding
authors.forename: Jin-Yan
authors.initials: JY
authors.lastname: Huang
authors.forename: Qiang-Sun
authors.initials: QS
authors.lastname: Zheng
mesh_terms: D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D018920:Coculture Techniques; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009206:Myocardium / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers