Early Events in Xenograft Development from the Human Embryonic Stem Cell Line HS181 - Resemblance with an Initial Multiple Epiblast Formation

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart

Modulation of cellular and viral functions in Epstein-Barr virus infected cells

The aim of the study was to investigate the interplay of cellular and viral genes in Epstein- Barr virus (EBV) infected cells. Two aspects of this interaction have been investigated. The first aspect is related to the expression of major histocompatibility complex (MHC) class I molecules in virus infected malignant cells. MW class I expression plays a key role in the regulation of immune responses and is frequently altered in human tumors. We analyzed the mechanisms of this MW class 1 downregulation in Burkitt's lymphomas (BLs) that arise in HLA All positive individuals by comparing five pairs of BL lines and Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The cell lines were compared for the presence of HLA A11 gene and A11- specific mRNA, the reactivity to IFN-alpha treatment, the restoration of HLA All expression by transfection or hybridization, the activity of HLA All promoter driven chloramphenicol acetyl transferase reporter gene (pA l 1 CAT). Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down- regulation of HLA AI 1 in BL cells. Furthermore we analysed the involvement of EBV LMP1 in the process. The BL phenotype-dependent transcriptional defect observed for the HLA AI 1 promoter was shown to involve other WC class I promoters including HLA A2. Since the virus LMP1 was shown to be mainly responsible for changing the phenotype of BL cells towards LCL-like characteristics, we have tested how the class I promoter activity is affected by expression of LMP1. We can conclude, that expression of LMP1 correlates with activation of the HLA All promoter and up-regulation of several components of the transcription factor family NF-kappa-B/Rel. The second aspect of this thesis deals with viral strategies that promote escape from immune recognition. EBNA1 is expressed in all EBV associated malignancies and is the only viral protein expressed in BL. Endogenously expressed EBNA1 is not recognized by MHC class P restricted CTLs. This phenomenon is correlated with the presence of an internal glycine- alanine repeat (GAr) in the structure of the protein. The GAr generates a cis-acting inhibitory signal that interferes with antigen processing and MW class I restricted presentation by inhibiting the generation of antigenic peptides by ubiquitin-proteasome/dependent proteolysis. The mechanism of action of the GAr was investigated using as model a known target of the ubiquitin-poteasome system, the NF-kappa-B inhibitor I-kappa-B-alpha. Insertion of a minimal GA repeat of eight amino acids in different positions of I-kappa-B-alpha was sufficient to prevent tumor necrosis factor (TNF-alpha) induced ubiquitin-proteasome dependent degradation, and decrease its basal turnover in vivo. The chimeras are phosphorylated and ubiquitinated in response to TNF-alpha but than released and failed to associate with the proteasome. This explains how functionally competent I-kappa-N-alpha is protected from proteasomal disruption and identifies the GAr as a regulator of proteolysis. The analysis of the impact of length and amino acid composition on the capacity of various repeat sequences to inhibit the TNF-alpha-induced and physiological turnover of I-kappa-B-alpha demonstrated that inhibition is achieved by insertion of octamer peptides containing 3 hydrophobic amino acids, interspersed by no more then three consecutive glycines. The inhibitory activity was abolished by increasing the length of the spacer to four glycines, by elimination of the spacers, or by substitution of a single hydrophobic residue with a polar or charged residue. These findings suggest a model where inhibition of proteolysis requires the interaction of at least dime alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pockets of a putative receptor. The multifunctional EBNA1 protein is involved also in the replication of the EBV genome as an extra chromosomal element, and plays important role in the maintenance of the viral episome and is the key transcriptional regulator of latent viral gene expression including its own transcriptional regulation. We have investigated whether presence of the GAr modulates the functions of EBNA1. The GAr containing EBNA1 protein has an extremely slow turnover in virus infected cells while a GAr deleted EBNA1 is degraded by the ubiquitin-proteasome system. To assess the contribution of protein stability on EBNA l functions, destabilized variants were generated and studied for their capacity to promote the maintenance of oriP containing plasmids and regulate the viral Qp promoter. Both functions were strongly influenced by EBNA1 stability suggesting the GAr plays an important role in modulating the function of EBNA1 in virus infected cells

Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development

Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected