Rebecca Stewart

An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells

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.

Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells

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.

Differentiation of Human Embryonic Stem Cells into Corneal Epithelial‐Like Cells by In Vitro Replication of the Corneal Epithelial Stem Cell Niche

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.

A key role for telomerase reverse transcriptase unit in modulating human embryonic stem cell proliferation, cell cycle dynamics, and in vitro differentiation.

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 TERT‐overexpressing 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.

Epigenetic Marking Prepares the Human HOXA Cluster for Activation During Differentiation of Pluripotent Cells

Activation of Hox gene clusters is an early event in embryonic development since individual members play important roles in patterning of the body axis. Their functions require precise control of spatiotemporal expression to provide positional information for the cells of the developing embryo, and the manner by which this control is achieved has generated considerable interest. The situation is different in pluripotent cells, where HOX genes are not expressed but are held in potentio as bivalent chromatin domains, which are resolved upon differentiation to permit HOX cluster activation. In this study we have used differentiation of the pluripotent embryonal carcinoma cell line NTera2SP12 and the human embryonic stem cell line H9 to examine epigenetic changes that accompany activation of the HOXA cluster and show that specific genomic loci are marked by lysine methylation of histone H3 (H3K4 tri‐ and dimethyl, H3K9 trimethyl) and acetylation of histone H4 even in the undifferentiated cells. The precise locations of such modified histones may be involved in controlling the colinear expression of genes from the cluster.

Silencing of the expression of pluripotent driven-reporter genes stably transfected into human pluripotent cells

AIMS & METHODS Marking of human embryonic stem (ES) and embryonal carcinoma (EC) cells with pluripotent promoter-driven reporter gene cassettes provides an important tool for studies related to maintenance of pluripotency, cell differentiation and cell selection. OCT4, TERF1 and telomerase reverse transcriptase component (TERT) are considered as pluripotent marker genes since they are expressed in both human ES and EC cells and significantly downregulated during the differentiation process. Our aim was to use core promoter regions from such pluripotent genes to drive expression of reporter genes that would be suitable for human ES cell selection amongst differentiated cells. RESULTS Human ES and EC cells were stably transfected with a number of TERT, OCT4 and TERF1 promoter-driven EGFP or NTR gene cassettes. Gradual loss of reporter gene expression was observed from 24 h post-transfection during transient transfection studies, while almost complete loss of reporter expression was observed upon stable transfections. The loss of reporter gene expression was partly reversed by addition of a histone deacetylase inhibitor and a demethylating agent, suggesting that in vitro methylation of these exogenous constructs and the epigenetic architecture around the site of integration are likely to play a major role in their transcriptional activity. Inclusion of gene-regulatory elements in addition to the core promoters has been shown to minimize such effects and should be considered as an important strategy in such studies. CONCLUSIONS Together our data suggest that human ES and EC cells are able to silence pluripotent promoter-driven reporter genes with high efficiency. Whether differentiated cells derived from human ES and EC cells retain this activity is unknown and need to be investigated before large-scale comparative reporter-based transfection studies can be used as a tool in human embryonic stem cell biology.

A Critical Appraisal of Factors Affecting the Accuracy of Results Obtained when Using Flow Cytometry in Stem Cell Investigations: Where Do You Put Your Gates?

Flow cytometry is used extensively in stem cell investigations but there is wide variation in the methods used for data analysis between laboratories. Data analysis can be challenging in stem cell biology as there is often no clear distinction between positive and negative populations. We have undertaken a critical appraisal of factors that affect the accuracy of results in stem cell applications. We used mouse embryonic stem cells and determined the expression of three common antigens in stem cell investigations, namely CD15, CD184, and c‐kit. We have compared different cell preparation methods and gating strategies and also evaluated the use of isotype controls and unstained cells as controls for the identification of positive populations. The use of a “doublet discriminator” using a side scatter area signal versus side scatter height signal dot plot to identify single cells for analysis increases the accuracy of results regardless of the method used to dissociate cells. Isotype controls can be helpful in mimicking cellular nonspecific binding of the experimental antibody reaction. Isotype controls behave differently on stem cells at different stages of differentiation. Analysis of a viable single cell population with careful selection of control cell populations increases the accuracy of results.