Understanding the molecular mechanism by which pluripotency is maintained in human embryonic stem cells (hESC) is important for the development of improved methods to derive, culture and differentiate these into cells of potential therapeutic use. Large-scale transcriptional comparison of the hES-NCL1 line derived from a day 8 embryo with H1 line derived from a day 5 embryo (WiCell Inc.) showed that only 0.52% of the transcripts analysed varied significantly between the two cell lines. This is within the variability range that has been reported when hESC derived from days 5-6 embryos have been compared with each other. This implies that transcriptional differences between the cell lines are likely to reflect their genetic profile rather than the embryonic stage from which they were derived. Bioinformatic analysis of expression changes observed when these cells were induced to differentiate as embryoid bodies suggested that quite a few of the downregulated genes were components of signal transduction networks. Subsequent analysis using western blotting, flow cytometry and antibody arrays implicated components of the PI3K/AKT kinase, MAPK/ERK and NFkappabeta pathways and confirmed that these components are decreased upon differentiation. Disruption of these pathways in isolation using specific inhibitors resulted in loss of pluripotency and/or loss of viability suggesting the importance of such signalling pathways in embryonic stem cell maintenance.

Evolutionary theory predicts that cellular maintenance, stress defense, and DNA repair mechanisms should be most active in germ line cells, including embryonic stem cells that can differentiate into germ line cells, whereas it would be energetically unfavorable to keep these up in mortal somatic cells. We tested this hypothesis by examining telomere maintenance, oxidative stress generation, and genes involved in antioxidant defense and DNA repair during spontaneous differentiation of two human embryonic stem cell lines. Telomerase activity was quickly downregulated during differentiation, probably due to deacetylation of histones H3 and H4 at the hTERT promoter and deacetylation of histone H3 at hTR promoter. Telomere length decreased accordingly. Mitochondrial superoxide production and cellular levels of reactive oxygen species increased as result of increased mitochondrial biogenesis. The expression of major antioxidant genes was downregulated despite this increased oxidative stress. DNA damage levels increased during differentiation, whereas expression of genes involved in different types of DNA repair decreased. These results confirm earlier data obtained during mouse embryonic stem cell differentiation and are in accordance with evolutionary predictions. STEM CELLS 2008;26:455-464 Disclosure of potential conflicts of interest is found at the end of this article.

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so-called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial-like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial-like cells. Further differentiation results in the formation of terminally differentiated epithelial-like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC-derived and adult limbal epithelial-like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss.

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses.We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESCderived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs. Stem Cells 2005;23:306-314

The primary emphasis of tissue engineering is the design and fabrication of constructs for the replacement of nonfunctional tissue. Because tissue represents a highly organized interplay of cells and extracellular matrix, the fabrication of replacement tissue should mimic this spatial organization. This report details studies evaluating the use of a three-dimensional, direct-write cell deposition system to construct spatially organized viable structures. A direct-write bioassembly system was designed and fabricated to permit layer-by-layer placement of cells and extracellular matrix on a variety of material substrates. Human fibroblasts suspended in polyoxyethylene/polyoxypropylene were coextruded through a positive displacement pen delivery onto a polystyrene slide. After deposition, approximately 60% of the fibroblasts remained viable. Bovine aortic endothelial cells (BAECs) suspended in soluble collagen type I were coextruded via microdispense pen delivery onto the hydrophilic side of flat sheets of polyethylene terephthalate. After deposition with a 25-gauge tip, approximately 86% of the BAECs were viable. When maintained in culture for up to 35 days, the constructs remained viable and maintained their original spatial organization. These results indicate the potential for utilizing a direct-write, three-dimensional bioassembly tool to create viable, patterned tissue-engineered constructs.

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Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

Embryonic stem cells (ESC) are a unique cell population with the ability to self-renew and differentiate into all three germ layers. Human ESC express the telomerase reverse transcriptase (TERT) gene and the telomerase RNA (TR) and show telomerase activity, but TERT, TR, and telomerase are all downregulated during the differentiation process. To examine the role of telomerase in human ESC self-renewal and differentiation, we modulated the expression of TERT. Upregulation of TERT and increased telomerase activity enhanced the proliferation and colony-forming ability of human ESC, as well as increasing the S phase of the cell cycle at the expense of a reduced G1 phase. Upregulation of TERT expression was associated with increases in CYCLIN D1 and CDC6 expression, as well as hyperphosphorylation of RB. The differentiated progeny of control ESC showed shortening of telomeric DNA as a result of loss of telomerase activity. In contrast, the differentiated cells from TERToverexpressing ESC maintained high telomerase activity and accumulated lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Although the TERT-overexpressing human ESC are able to form teratoma composed of three germ layers in vivo, their in vitro differentiation to all primitive and embryonic lineages was suppressed. In contrast, downregulation of TERT resulted in reduced ESC proliferation, increased G1, and reduced S phase. Most importantly, downregulation of TERT caused loss of pluripotency and human ESC differentiation to extraembryonic and embryonic lineages. Our results indicate for the first time an important role for TERT in the maintenance of human ESC pluripotency, cell cycle regulation, and in vitro differentiation capacity.