Marta P. Imreh

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.

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.

PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

Trisomy 12 in HESC leads to no selective in vivo growth advantage in teratomas, but induces an increased abundance of renal development

The aim of this investigation was to examine the impact of chromosome 12 amplification (tri‐12 cells) in human embryonic stem cells (HESC), following in vivo engraftment to an immunodeficient xeno‐model. For this we used sublines from the HESC line HS181, spontaneously exhibiting either low or high frequencies of tri‐12 cells. Fluorescent in situ hybridization (FISH) analysis revealed a random distribution of tri‐12 cells in the HS181 colonies in vitro. Similarly, the contribution of tri‐12 cells to the development of various tissues in teratomas in vivo seemed to be fully random with no particular preference regarding in vivo differentiation pathway of tri‐12 HS181 cells compared to HS181 cells with disomy 12 (di‐12 cells). On the other hand, following in vivo transplantation the ratio of tri‐12/di‐12 cells was significantly reduced (P < 0.001), indicating a negative selection for this trisomy in vivo. Moreover, injection of HS181 cultures containing tri‐12 cells resulted in a significantly increased abundance of areas compatible with renal formation (P < 0.001), relative teratomas derived from injection of di‐12 HS181 cells. However, such areas included no increased relative frequency of tri‐12 cells, suggesting indirect mechanism(s) for the increased abundance of renal development. The reasons for such developmental bias are unknown and warrant further investigation. J. Cell. Biochem. 100: 1518–1525, 2007.

Consistent downregulation of human lactoferrin gene, in the common eliminated region 1 on 3p21.3, following tumor growth in severe combined immunodeficient (SCID) mice

Lactoferrin (LF) is one of 19 active genes in the common eliminated region 1 at 3p21.3 identified by us. LF was transfected into mouse fibrosarcoma A9. Fourteen severe combined immunodeficient (SCID) derived tumors from two PI based artificial chromosome (PAC)-transfectants containing the entire LF gene and two LF-cDNA transfectants were analyzed by real time polymerase chain reaction at the DNA and RNA level. Following SCID tumor passage, LF expression was decreased or eclipsed, in all tumors although DNA levels did not change considerably. Promoter methylation and/or rearrangement of the insertion site may be responsible for human LF downregulation in mouse fibrosarcoma derived tumors.

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.

Integration of a short Epstein-Barr virus DNA fragment in a B95-8 virus converted Burkitt lymphoma line expressing Epstein-Barr nuclear antigens EBNA2 and EBNA5

We have analysed the expression of transformation-associated viral antigens, the Epstein-Barr virus (EBV) DNA content and the phenotypic characteristics of two B95-8 virus-converted sublines of the EBV-negative Burkitts lymphoma (BL) line BL28. The converted lines called E95A-BL28 and E95B-BL28, respectively, differed in their EBV gene expression. The E95B convertant expressed virus-encoded nuclear antigens EBNA1 to -6 and the membrane protein LMP1, but only EBNA2 and EBNA5 were detected by immunofluorescence and immunoblotting in the E95A convertant. Only the entire BamHI W, Y and H regions could be detected in the E95A convertant by hybridization of Southern blots with probes covering the BamHI C, W, Y, H, F, E, K and Nhet regions of the EBV genome. EBV episomes were found to be absent in the E95A convertant as seen by Gardella gels. The E95A convertant retained the phenotypic characteristics of the EBV-negative parental line, and remained highly clonable in agarose. In contrast, expression of EBNA1 to -6 and LMP1 was accompanied by a shift towards a more lymphoblastoid cell line-like phenotype and by loss of agarose clonability in the E95B convertant.

Window into the Complexities of Chromosome Interactomes

DNA is folded into increasingly complex yet highly mobile structures to organize the chromosomes. In the interphase nucleus, chromosomes or part of the chromosomes encounter one another preferentially at the boundaries between chromosomal territories. Although this situation implies that the preferred chromosomal neighborhood is a key determinant of interactions between chromosomes, what this means in functional terms is currently not well understood. Using the H19 imprinting control region as a window, it has been demonstrated that epigenetic information of the primary chromatin fiber has dual functions. Thus, epigenetic marks not only influence the proximity between chromatin fibers but also transfer epigenetic states between chromatin fibers both in cis and in trans. High-throughput sequence and DNA fluorescence it situ hybridization (FISH) analyses reveal that these features require chromatin movements that are restricted in space and time. The mechanisms involved in the establishment of chromosome interactomes may provide insight of fundamental importance into pivotal regulatory processes in the nucleus, such as the coordination of transcriptional programs and replication timing.