Chromatin meets its organizers.

Chromatin organization and gene-gene interactions are critical components of carrying out developmental programs. Phillips-Cremins et al. identify a series of unexpected architectural proteins that work in a combinatorial manner to functionally organize chromatin in a cell-type-specific manner at the submegabase-length scale.

Propagation and maintenance of undifferentiated human embryonic stem cells.

Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clinical uses have not yet been realized from the study of hES cells. Anticipation that these cells would be immediately useful for creating models of human disease has not yet been fulfilled. However, because of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for many areas of research and medicine. A major problem complicating developments in hES cell research is the difficulty of propagating and maintaining these cells in vitro without differentiation. This review addresses this problem and potential solutions in detail. In addition, the current state of research regarding the growth and maintenance of hES cells is summarized, along with basic protocols utilized by our laboratory for the successful propagation, characterization, and investigation of hES cells.

Nuclear neighborhoods and gene expression.

The eukaryotic nucleus is a highly compartmentalized and dynamic environment. Chromosome territories are arranged nonrandomly within the nucleus and numerous studies have indicated that a gene's position in the nucleus can impact its transcriptional activity. Here, we focus on recent advances in our understanding of the influence of specific nuclear neighborhoods on gene expression or repression. Nuclear neighborhoods associated with transcriptional repression include the inner nuclear membrane/nuclear lamina and perinucleolar chromatin, whereas neighborhoods surrounding the nuclear pore complex, PML nuclear bodies, and nuclear speckles seem to be transcriptionally permissive. While nuclear position appears to play an important role in gene expression, it is likely to be only one piece of a flexible puzzle that incorporates numerous parameters. We are still at a very early, yet exciting stage in our journey toward deciphering the mechanism(s) that govern(s) the permissiveness of gene expression/repression within different nuclear neighborhoods.

Unique gene expression signatures of independently-derived human embryonic stem cell lines.

Human embryonic stem cells (hESCs) have the potential to differentiate to diverse cell types. This ability endows hESCs with promise for the development of novel therapeutics, as well as promise for the development of a rigorous genetic system to probe human gene function. However, in spite of the impending utility of hESCs for clinical and basic applications, little is known about their fundamental properties. Recent reports have documented transcriptional profiles of mouse embryonic stem cells (mESCs), adult stem cells and a single hESC line, H9. To date, however, the transcriptional profiles of independently-derived hESC lines have not been compared. In order to examine the similarities and differences in multiple hESC lines, we compared gene expression profiles of the HSF-1, HSF-6 and H9 lines. We found that the majority of genes examined were expressed in all three cell lines. However, we also observed that each line possessed a unique expression signature; the expression of many genes was limited to just one or two hESC lines. We suggest that the observed differences in gene expression between independently-derived hESC lines may reflect inherent differences in the initial culture of each line and/or the underlying genetics of the embryos from which the lines were derived.

Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma.

Genes required to maintain pluripotency in human embryonic stem (hES) cells are largely unknown, with the exception of OCT-4, a homolog of mouse Oct-4, which is critical for the establishment of the embryonic inner cell mass and the generation of totipotent mouse embryonic stem (mES) cell lines. In the current study, we identified two genes with expression similar to OCT-4, in that they are largely restricted to pluripotent hES cells, premeiotic germ lineage cells, and testicular germ cell tumor cells. Furthermore, we determined that upon hES cell differentiation, their expression is downregulated. The genes we identified in the current study include the human stella-related (STELLAR) gene, which encodes a highly divergent protein (with just 32.1% identity to mouse stella over the 159 amino acid sequence) that maps to human chromosome 12p13. Notably, human STELLAR is located distal to a previously uncharacterized homeobox gene, which is the human homolog of the recently identified murine gene, Nanog, and proximal to the GDF3 locus, whose transcription is restricted to germ cell tumor cells. Our characterization of STELLAR, NANOG, and GDF3 suggests that they may play a similar role in humans as in mice, in spite of their remarkable evolutionary divergence.

Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

Little is known of molecular requirements for specification of human germ cells. However, it is likely that they are specified through the action of sequentially expressed genes just as in model organisms. We sought to determine whether human embryonic stem (ES) cell lines, like those of mice, might be capable of forming germ cells in vitro. We compared transcriptional profiles of three pluripotent human ES cells to those of isolated inner cell mass (ICM) cells. We found that ICM cells expressed NANOS1, STELLAR and OCT4, whereas undifferentiated human ES cells expressed these genes along with the germ cell-specific gene, DAZL. Upon ES cell differentiation into embryoid bodies (EBs), we observed a shift in expression from RNA and protein markers of immature germ cells to those indicative of mature germ cells, including expression of VASA, BOL, SCP1, SCP3, GDF9 and TEKT1. Although ability to test the function of these putative VASA positive germ cells is limited, these results demonstrate that differentiation of human ES cells into EBs in vitro results in formation of cells that express markers specific to gonocytes.