Association of telomere length with authentic pluripotency of ES/iPS cells

Telomerase and telomeres are important for indefinite replication of stem cells. Recently, telomeres of somatic cells were found to be reprogrammed to elongate in induced pluripotent stem cells (iPSCs). The role of telomeres in developmental pluripotency in vivo of embryonic stem cells (ESCs) or iPSCs, however, has not been directly addressed. We show that ESCs with long telomeres exhibit authentic developmental pluripotency, as evidenced by generation of complete ESC pups as well as germline-competent chimeras, the most stringent tests available in rodents. ESCs with short telomeres show reduced teratoma formation and chimera production, and fail to generate complete ESC pups. Telomere lengths are highly correlated (r > 0.8) with the developmental pluripotency of ESCs. Short telomeres decrease the proliferative rate or capacity of ESCs, alter the expression of genes related to telomere epigenetics, down-regulate genes important for embryogenesis and disrupt germ cell differentiation. Moreover, iPSCs with longer telomeres generate chimeras with higher efficiency than those with short telomeres. Our data show that functional telomeres are essential for the developmental pluripotency of ESCs/iPSCs and suggest that telomere length may provide a valuable marker to evaluate stem cell pluripotency, particularly when the stringent tests are not feasible.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.     

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.     

Histone h1 depletion impairs embryonic stem cell differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.     

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency‐associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real‐time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct‐4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct‐4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.