Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.

Various types of embryoid body (EB) that were formed from mouse embryonic stem (ES) cells under various culture conditions were characterized in terms of gene expression pattern to estimate the differentiation status of the bodies. The gene expression of typical markers (i.e., GATA-4, GATA-6, transthyretin [TTR], alpha-fetoprotein [AFP], Nkx2.5, and alpha-myosin heavy chain [alpha-MHC]) was quantitatively analyzed in various types of EB, and the gene expression pattern of those marker genes was graphically shown for each EB. The gene expression pattern accurately represented the differentiation status of the EBs. The gene expression pattern indicated that the Nkx2.5 and alpha-MHC genes were highly expressed in the EBs formed from 1000 ES cells in a low-adherence 96-well plate. By transferring the EBs into an attachment culture, cardiomyocytes were more efficiently generated in the outgrowth of the EBs. When we increased the seeding cell number from 1000 to 4000 ES cells, the gene expression pattern changed, that is, the expression levels of the TTR and AFP genes increased, whereas those of the Nkx2.5 and alpha-MHC genes decreased, and the trend of differentiation changed from cardiomyogenesis to visceral yolk-sac-like structure formation.

A Round-bottom 96-well Polystyrene Plate Coated with 2-methacryloyloxyethyl Phosphorylcholine as an Effective Tool for Embryoid Body Formation.

In this study, we proposed a culture method for forming embryoid bodies (EBs) from mouse embryonic stem (ES) cells using a round-bottom 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine (MPC plate). MPC is a phospholipid biocompatible polymer and prevents cells from adhering to the culture surface. The ES cells were seeded at 1000 cells per well in the MPC plate with 200 mul of medium. After 5 days of static incubation, a spherical cell aggregate termed EB was formed in a well. The size (diameter) of resulting EB was approximately 550 mum and it contained approx. 22,000 cells. It seems that the non-adhesiveness and the roundness of the well are important factors to form a good EB. Transferring the EBs to the attached differentiation culture, the EBs spread out and flattened, and the beating cells (cardiomyocytes) were effectively generated in the outgrowth of EBs. The round-bottom 96-well polystyrene plate coated with MPC is an effective tool for EB formation.

A simple method for forming embryoid body from mouse embryonic stem cells.

We proposed a simple method for forming an embryoid body (EB) from mouse embryonic stem (ES) cells using a polypropylene 1.5-ml conical tube with a screw cap. An ES cell suspension containing 2 x 10(4) cells was incubated in a conical tube. After 5 d of incubation, a single EB of 440 microm average diameter was formed in the conical tube. The formation efficiency of EB, which is the ratio of the number of tubes showing EB formation to the number of tubes seeded with ES cells, was greater than 99% in the conical tube, while it was approximately 60% in a hanging drop culture. The 5-day-old EB formed by the conical tube method had a sufficient differentiation ability. The beating of the cardiac muscle was microscopically observed in the populations derived from the 5-day-old EB.