Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367.

Artificial transcription factors are powerful tools for regulating gene expression. Here we report results with engineered zinc-finger transcription factors (ZF-TFs) targeting four protein-coding genes, OCT4, SOX2, KLF4 and c-MYC, and one noncoding ribonucleic acid (RNA) gene, the microRNA (miRNA) miR302/367 cluster. We designed over 300 ZF-TFs whose targets lie within 1 kb of the transcriptional start sites (TSSs), screened them for increased messenger RNA or miRNA levels in transfected cells, and identified potent ZF-TF activators for each gene. Furthermore, we demonstrate that selected ZF-TFs function with alternative activation domains and in multiple cell lines. For OCT4, we expanded the target range to -2.5 kb and +500 bp relative to the TSS and identified additional active ZF-TFs, including three highly active ZF-TFs targeting distal enhancer, proximal enhancer and downstream from the proximal promoter. Chromatin immunoprecipitation (FLAG-ChIP) results indicate that several inactive ZF-TFs targeting within the same regulatory region bind as well as the most active ZF-TFs, suggesting that efficient binding within one of these regulatory regions may be necessary but not sufficient for activation. These results further our understanding of ZF-TF design principles and corroborate the use of ZF-TFs targeting enhancers and downstream from the TSS for transcriptional activation.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Water-Soluble Nystatin and Derivative.

Fungal infections are increasingly causing more morbidity and mortality, especially for immunocompromised people. In recent years, there is growing evidence that new medicine-resistant fungal strains are posing added challenges in the clinic. Nystatin is a known antifungal from the polyene family. Due to Nystatin limited solubility and high toxicity, it is used mainly to treat oral and dermal fungal infections. In search for new Nystatin derivatives and formulations, we obtained amide derivatives and a deoxycholate formulation that were not described previously for this compound. Furthermore, we tested the potency of the derivatives and formulation by the USP(81) method and minimum inhibitory concentration of Candida albicans and Aspergillus niger. Additionally, the in vitro toxicity and stability were tested, and it was found that the ethanol amide derivative of Nystatin was fully water-soluble (up to 100 mg/mL) with the same potency of Nystatin but with 13.5 times lower toxicity. The ethanol amide derivative of Nystatin is a promising candidate for future drug development.

Human embryonic stem cells as a model for early human development.

Although, the study of human development--from fertilized egg to mature embryo--is extremely important, the early differentiation of human tissues remains an enigma. Moreover, the relatively high percentage of unexplained pregnancy loss--a major concern of human embryologists and gynaecologists--emphasizes the need for an appropriate model for studying early human development. The availability of human pluripotent stem cells might allow us to study previously inaccessible basic processes that occur during human embryogenesis, such as gastrulation and organogenesis.

Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells.

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs.

Epigenetic regulation of X-inactivation in human embryonic stem cells.

X chromosome inactivation (XCI) allows dosage compensation of the expression from sex chromosome in mammalian female cells. Although this mechanism is extensively studied in the mouse model organism, the corresponding mechanism during human development is largely unknown. The generation of human embryonic stem cells (hESCs) provides an invaluable tool to address early embryogenesis in humans. Even though hESCs were supposed to shed light on the XCI process in early human embryogenesis, previous studies largely indicated inconsistency in the status of XCI in these cells. Recently, new data suggested that in vitro culture might affect epigenetic mechanisms such as XCI. In this review we will present the existing data regarding XCI variations in hESC as compared to data from the mouse embryo and embryonic stem cells. We will also suggest possible explanations for the conflicting observations in the literature regarding XCI in hESCs.

Molecular analysis of LEFTY-expressing cells in early human embryoid bodies.

Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

Human embryonic stem cells as a powerful tool for studying human embryogenesis.

Human embryonic stem cells (HESC) are pluripotent stem cell lines derived from the inner cell mass (ICM) of human blastocyst-stage embryos. They are characterized by their unlimited capacity to self-renew in culture. In addition, they have a broad developmental potential, as demonstrated by their ability to form practically any cell type in vivo and in vitro. These two features have made HESC extremely important in basic and applied research. In addition, they may serve as a powerful tool for studying human development. HESC can recapitulate embryogenesis by expressing developmentally regulated genes and by activating molecular pathways as they occur in vivo. Moreover, they can be used to analyze the effect of specific mutations on particular developmental events and may enable us to identify critical factors that play a role in the processes of cell commitment, differentiation, and adult cell reprogramming. Thus, modeling human embryogenesis by the use of HESC may allow new insights into developmental processes, which would otherwise be inaccessible for research.

Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies.

BACKGROUND: The aim of this study was to characterize human embryonic stem (ES) cells at the molecular level by performing large-scale complementary DNA (cDNA) analysis using DNA micro-arrays. METHODS: The transcription profile of human ES cells was determined by comparing it to 2, 10 and 30-day old embryoid bodies (EBs) using Affymetrix Genechip human micro-arrays (U133). RESULTS: According to this analysis we demonstrate that two human ES cell lines are more close to each other than to their differentiated derivatives. We also show the spectrum of cytokine receptors that they express, and demonstrate the presence of five genes that are highly specific to human ES cells and to germ cells. Moreover, by profiling different stages in the differentiation of human embryoid bodies, we illustrate the clustering of five sets of temporally expressed genes, which could be related to the sequential stages of embryonic development. Among them are known genes that are involved in early pattern formation. CONCLUSIONS: The present study provides a molecular basis for the identity of human ES cells and demonstrates that during their in vitro differentiation they express embryonic specific genes in a stage specific manner.