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.

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.

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.

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.

Horse embryonic stem cell lines from the proliferation of inner cell mass cells.

Inner cell mass (ICM) cells were isolated immunosurgically from day 7-8 horse blastocysts and, after proliferation in vitro for 15-28 passages, three lines of cells were confirmed to be embryonic stem (ES) cells by their continued expression of alkaline phosphatase activity and their ability to bind antisera specific for the recognized stem cell markers, SSEA-1, TRA-1-60, TRA-1-81, and the key embryonic gene Oct-4. When maintained under feeder cell-free conditions in vitro, the three lines of cells differentiated into cells of ectodermal, endodermal, and mesodermal lineages. However, they did not form teratomata when injected into the testes of severe combined immunodeficiency (SCID)/beige immunoincompetent mice, thereby indicating a significant difference in phenotype between ES cells of the horse and those of the mouse and human.

Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system.

An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (<10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.

Organized development from human embryonic stem cells after injection into immunodeficient mice.

Information concerning the development and differentiation of human embryonic stem (hES) cells in vivo is limited. The present study has focused on the in vivo outcome and differentiation of the hESC line HS181, after injection into SCID/beige mice. hES cell-derived teratomas were explored using histological evaluation and by the identification of markers for differentiated cells and tissues. The analyses identified predominant differentiation along a neuronal lineage, the formation of bone/cartilage and epithelia. Fluorescent in situ hybridization (FISH) analysis with a human-specific probe showed the teratomas to be mainly of human origin, with the most organized areas being exclusively human. Importantly, the study revealed interactions between mouse and human tissues, most notably in the formation of vessels. Both mouse and human cells contributed to specific microstructures in which mouse cells could be observed to take on the appropriate histiotypic appearance. Hence, HS181 cells were able to develop into defined mature tissues, supporting the relevant use of this hES cells model for studies of early human development, given the use of appropriate controls for host contribution. Although extensive mitotic activity implicated progenitor cell activity, no detectable multipotent or malignant areas were observed during the observation period. Persisting undifferentiated hESC were not detected.

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.