e0230 The effect of co-culturing with native cardiomyocytes on ascorbic acid-induced cardiomyogenic differentiation in embryonic stem cells

Objective Ascorbic acid has been reported to promote the differentiation of embryonic stem cells (ESCs) into cardiomyocytes (CMs). However, appropriate culture protocols are needed to improve the differentiation efficiency and produce adequate numbers of CMs for therapeutic cell transplantation. This study investigated the effect of co-culturing with native CMs on ascorbic acid-induced cardiomyogenic differentiation in embryonic stem cells, to develop a novel protocol for generating functional CMs from ESCs. Methods Native CMs were isolated from the hearts of 1-day-old Sprague-Dawley rats. Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with or without 0.1 mM of ascorbic acid (Sigma) for cardiomyogenic differentiation. They were divided into four groups: ascorbic acid & co-culture group, co-culture group, ascorbic acid group, and control group. In the co-culture system, EBs were co-cultured with native CMs by the hanging cell culture inserts (PET 1 μm) (Millicell; Millipore, Bedford, MA, USA). The native CMs were purposely placed on culture plate inserts to prevent direct contact with subnatant EBs. Both the ESCs and native CMs grew in the same medium but they were easy to separate. The structural and functional properties of ESC-derived CMs (ESCM) were evaluated by microscopic observation, immunocytochemistry, RT-PCR, and transmission electron microscopy. Results The average percentages of EBs exhibited rhythmic contractions in co-culture and ascorbic acid group, co-culture group, ascorbic acid group, and control group were 86.6±9.52%, 65.60±10.77%, 29.6±6.03%, and 17.76±5.99%, respectively. The percentage of beating EBs in co-culture & ascorbic acid group was much higher and the homogeneity of EBs were significantly improved over that seen in other groups (p<0.01), simultaneously, the automaticity of beating also maintained for more time. The majority (>90%) of cells in EBs were ESCM that acquired almost the same structural and functional properties as typical CMs. Conclusions The present study demonstrates the cardiomyogenic differentiation of ESCs can be efficiently controlled by co-cultured with native CMs, and this may lead to a practicable cocktail approach to generate ESC-derived CMs for stem cell-based regenerative medicine.