A glutamine-rich factor affects stem cell genesis in leech.

Leech embryogenesis is a model for investigating cellular and molecular processes of development. Due to the unusually large size of embryonic stem cells (teloblasts; 50 - 300 µm) in the glossiphoniid leech, Theromyzon tessulatum, and the presence of identifiable stem cell precursors (proteloblasts), we previously isolated a group of genes up-regulated upon stem cell birth. In the current study, we show that one of these genes, designated Tpr (Theromyzon proliferation), is required for normal stem cell genesis; specifically, transient Tpr knockdown experiments conducted with antisense oligonucleotides and monitored by semi-quantitative RT-PCR, caused abnormal proteloblast proliferation leading to embryonic death, but did not overtly affect neuroectodermal or mesodermal stem cell development once these cells were born. Tpr encodes a large, glutamine-rich (~34%) domain that shares compositional similarity with strong transcriptional enhancers, many of which have been linked with trinucleotide repeat disorders (e.g., Huntingtons).

Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells.

Human embryonic stem (hES) cells, derived from blastocysts, are capable of unlimited self-renewal and differentiation into all cell lineages of the body. Because of their pluripotent nature, hES cells are valuable tools for understanding human development and advancing the field of regenerative medicine. However, one key to harnessing the therapeutic power of hES cells for biomedical applications begins with determining how these cells maintain their pluripotent and undifferentiated state. Studies in mice have implicated three factors in regulating pluripotency in embryonic stem cells, Oct4, Nanog, and Sox2. However, significant differences in growth regulation between mouse embryonic stem and hES cells have been identified, suggesting a need to determine when and how factors work in hES cells. To date, the transcription factors Oct4 and Nanog have been identified as critical regulators of stem cell fate by functional studies in hES cells. To determine the role of Sox2 in maintaining hES cell pluripotency and self-renewal, we used RNA interference to specifically knock down Sox2 gene expression. Reduction of Sox2 expression in hES cells results in loss of the undifferentiated stem cell state, as indicated by a change in cell morphology, altered stem cell marker expression, and increased expression of trophectoderm markers. In addition, knockdown of Sox2 results in reduced expression of several key stem cell factors, including Oct4 and Nanog, linking these three factors together in a pluripotent regulatory network. Disclosure of potential conflicts of interest is found at the end of this article.

Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.

Changes in gene expression at the precursor --> stem cell transition in leech.

The glossiphoniid leech, Theromyzon trizonare, displays particularly large and accessible embryonic precursor/stem cells during its early embryonic cleavages. We dissected populations of both cell types from staged embryos and examined gene expression profiles by differential display polymerase chain reaction methodology. Among the approximately 10,000 displayed cDNA fragments, 56 (approximately 0.5%) were differentially expressed at the precursor --> stem cell transition; 29 were turned off (degraded, precursor-specific); and 27 were turned on (transcribed, stem cell-specific). Several putative differentially expressed cDNAs from each category were confirmed by Northern blot analysis on staged embryos. DNA sequencing revealed that 19 of the cDNAs were related to a spectrum of genes including the CCR4 antiproliferation gene, Rad family members, and several transcriptional regulators, while the remainder encoded hypothetical (10) or novel (27) sequences. Collectively, these results identify dynamic changes in gene expression during stem cell formation in leech and provide a platform for examining the molecular aspects of stem cell genesis in a simple invertebrate organism.

Molecular adaptation in the ice worm, Mesenchytraeus solifugus: divergence of energetic-associated genes.

The ice worm, Mesenchytraeus solifugus, is the largest glacially obligate metazoan and among only a few metazoan species that complete their life cycle at 0 degrees C. We conducted a large-scale sequencing analysis of cDNAs isolated from ice worm anterior segments. Sequence comparisons among an available group of ice worm, arthropod, chordate, and nematode homologues suggest that ice worms encode proteins that are less bulky, are less polar, and contain fewer charged residues. Also, subunits of the catalytic F1 ATP synthase complex appear to have diverged more rapidly than other ice worm genes examined, suggesting a role in cold-temperature adaptation. Modeling of F1 ATP synthase beta and gamma subunits identified nonconservative, ice worm-specific amino acid substitutions at subunit contact sites and at sites proximal to the catalytic site.