Effect of chemopreventive agents on differentiation of mouse embryonic stem cells.

Chemopreventive agents are derived from edible plants and from ancient time is a part of daily intake for many humans and animals. There are several lines of compelling evidence from epidemiological, clinical and laboratory studies that these dietary constituents are associated in reducing cancer risks. However, developmental toxicity of these natural compounds cannot be excluded. In the present study, we examined the effect of chemopreventive agents on the differentiation of mouse embryonic stem cells (ESCs) as an in vitro embryotoxicity model. We assumed that inhibition of developmentally regulated genes in vitro might predict developmental toxicity also under in vivo conditions. We found that epigallocatechin gallate (EGCG) (20 microM) induced the expression of mesodermal and cardiomyocyte genes and a significant increase in the number and the percentage of cardiomyocytes. The increase of the subpopulation correlated with higher numbers of beating foci and beating frequencies. Curcumin on the other hand at 0.4 mM was seen to enhance expression of ectodermal transcripts. Quercetin (2.5 microM) was found to inhibit several developmentally regulated genes.

Isolation and functional characterization of alpha-smooth muscle actin expressing cardiomyocytes from embryonic stem cells.

Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter. The onset of Acta2 expression occurred in parallel with the appearance of beating areas, indicating the formation of cardiomyocytes. Antibiotic selection resulted in a high yield of cardiomyocytes and smooth muscle cells. The green fluorescent beating areas stained positively for multiple cardiomyocyte markers. Comparative electrophysiological analysis including fetal and alpha-MHC-expressing ES cell-derived cardiomyocyte controls showed that Acta2-positive cardiomyocytes contained pacemaker-, atrial- and ventricular-like phenotypes. Interestingly, the proportion of ventricular-like cells was much higher in the Acta2-positive cardiomyocytes population than in control alpha-MHC-expressing cardiomyocytes (75 % and 12 %, respectively). The findings of the present study provide a novel approach for the identification and enrichment of Acta2-positive cardiomyocytes, especially of the ventricular phenotype under in vitro conditions.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated. Acta2(+) SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca(2+)-activated potassium channel. Culturing of Acta2(+) SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell-derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases.

The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.