Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (enhanced green fluorescent protein [EGFP]) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the adenoassociated virus type 2 (AAV2) target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, stage-specific embryonic antigen-3 (SSEA3), and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection, with 90% ؎ 4% of cells expressing EGFP compared with 57% ؎ 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% ؎ 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% ؎ 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with ϳ30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application. STEM CELLS 2008;26: 496 -504 Disclosure of potential conflicts of interest is found at the end of this article.

Our study shows that LGT is a significant evolutionary mechanism among diplomonads in particular and protists in general. These findings provide insights into the evolution of biochemical pathways in early eukaryote evolution and have important implications for studies of eukaryotic genome evolution and organismal relationships. Furthermore, "fusion" hypotheses for the origin of eukaryotes need to be rigorously reexamined in the light of these results.

Abstract. Stem cells are a powerful resource for cellbased transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and noncanonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis.