Severe inhibition of in vitro cardiomyogenesis in mouse embryonic stem cells ectopically expressing EGAM1C homeoprotein.

Embryoid bodies were prepared from mouse embryonic stem cells expressing exogenous EGAM1C to analyze their ability to differentiate toward terminally differentiated cell types. The generation of cardiomyocytes was severely suppressed in Egam1c transfectants without upregulation of Nkx2-5, a crucial gene for cardiomyogenesis. These results indicate that EGAM1C is capable of affecting terminal differentiation in mouse embryonic stem cells.

Enzymatic conversion of atmospheric aldehydes into alcohol in a phospholipid polymer film.

We developed a unique method for converting atmospheric aldehyde into alcohol using formaldehyde dehydrogenase from Pseudomonas putida (PFDH) doped in a polymer film. A film of poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate) (PMB), which has a chemical structure similar to that of a biological membrane, was employed for its biocompatibility. A water-incorporated polymer film entrapping PFDH and its cofactor NAD(+) was obtained by drying a buffered solution of PMB, PFDH, and NAD(+). The aldehydes in the air were absorbed into the polymer film and then enzymatically oxidized by PFDH doped in the PMB film. Interestingly, alcohol and carboxylic acid were produced by the enzymatic reaction, indicating that PFDH catalyzes dismutation of aldehyde in the PMB film. Importantly, a PFDH-PMB film catalyzes aldehyde degradation without consuming the nucleotide cofactor, thereby allowing repeated use of the film. The activity of PFDH in the PMB film was higher than that in other common water-soluble polymers, suggesting that the hydrational state in a phospholipid polymer matrix is suitable for enzymatic activity.

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.

Suppression of the inflammatory response from adherent cells on phospholipid polymers.

The expression of interleukin-1beta (IL-1beta) messenger RNA (mRNA) in macrophage-like cells cultured on phospholipid polymers was evaluated to determine the extent of the inflammatory response. As phospholipid polymers, poly(2-methacryloyloxyethyl phosphorylcholine(MPC)-co-n-butyl methacrylate(BMA)s (PMBs) were synthesized. Poly(ethylene terephthalate) (PET), poly(2-hydroxyethyl methacrylate) (PHEMA), and segmented poly(ether urethane) (Tecoflex 60) were used as reference biomedical polymers. The protein adsorption onto the polymer surfaces from a cell culture medium was determined. The amount of the total protein adsorbed onto the PMBs was lower than that adsorbed onto the reference polymers, and the amount of adsorbed protein decreased with an increase in the MPC units in the PMBs. Human premyelocytic leukemia cell line (HL-60) was used, and the expression of IL-1beta mRNA was investigated with the reverse transcription polymerase chain reaction (RT-PCR) method. When HL-60 cells were cultured on PMBs, the expression of IL-1beta mRNA in the cells was much less than that on the reference polymers. In particular, the expression of IL-1beta mRNA in HL-60 cells cultured on the PMBs containing more than 10 mol % MPC units was not detected. This corresponded to the reduced amount of adsorbed proteins on the PMB surfaces. These results suggest that the PMBs effectively suppressed the activation and inflammatory response of adherent macrophagelike cells.