"Space means Science, unless it's about Star Wars": A qualitative assessment of science communication audience segments.

Scholars of science communication have identified population segments that differ in their perceptions of and attitudes toward science as well as in their patterns of science-related information and media use. So far, however, most of these studies employed quantitative, standardized methods and their descriptions could not go into qualitative detail. This study fills this gap: It delivers an in-depth description of members of four audience segments that were identified in a prior, representative survey in Switzerland. Forty-one of these survey respondents, representing different segments, were asked to note their encounters with science in smartphone-based diaries, and diary entries were discussed in qualitative follow-up interviews. Results show that the segments differ in their criteria for identifying science, expectations toward science, and their reasons to use science communication.

The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation.

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA-DNA-DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.

Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

BACKGROUND: Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome. RESULTS: In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes. CONCLUSIONS: Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

Proof of principle: quality control of therapeutic cell preparations using senescence-associated DNA-methylation changes.

BACKGROUND: Tracking of replicative senescence is of fundamental relevance in cellular therapy. Cell preparations - such as mesenchymal stromal cells (MSCs) - undergo continuous changes during culture expansion, which is reflected by impaired proliferation and loss of differentiation potential. This process is associated with epigenetic modifications: during in vitro culture, cells acquire senescence-associated DNA methylation (SA-DNAm) changes at specific sites in the genome. We have recently described an Epigenetic-Senescence-Signature that facilitates prediction of the state of cellular aging by analysis of DNAm at six CpG sites (associated with the genes GRM7, CASR, PRAMEF2, SELP, CASP14 and KRTAP13-3), but this has not yet been proven over subsequent passages and with MSCs isolated under good manufacturing practice (GMP) conditions. FINDINGS: MSCs were isolated from human bone marrow and GMP-conform expanded for up to 11 passages. Cumulative population doublings (cPDs) and long-term growth curves were calculated based on cell numbers at each passage. Furthermore, 32 cryopreserved aliquots of these cell preparations were retrospectively analyzed using our Epigenetic-Senescence-Signature: DNAm-level was analyzed at six specific CpGs, and the results were used to estimate cPDs, time of culture expansion, and passage numbers. Overall, predicted and real parameters revealed a good correlation, particularly in cPDs. Based on predicted cPDs we could reconstruct long-term growth curves and demonstrated the continuous increase in replicative senescence on molecular level. CONCLUSION: Epigenetic analysis of specific CpG sites in the genome can be used to estimate the state of cellular aging for quality control of therapeutic cell products.

Aging of blood can be tracked by DNA methylation changes at just three CpG sites.

BACKGROUND: Human aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age. RESULTS: We perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs--located in the genes ITGA2B, ASPA and PDE4C--were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita--two diseases associated with progressive bone marrow failure and severe telomere attrition--are predicted to be prematurely aged. CONCLUSIONS: Our epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.

Epigenetic biomarker to determine replicative senescence of cultured cells.

Somatic cells change continuously during culture expansion-long-term culture evokes increasing cell size, declining differentiation potential, and ultimate cell cycle arrest upon senescence. These changes are of particular relevance for cellular therapy which necessitates standardized products and reliable quality control. Recently, replicative senescence has been shown to be associated with highly reproducible epigenetic modifications. Here, we describe a simple method to track the state of senescence in mesenchymal stromal cells (MSCs) or fibroblasts by monitoring continuous DNA methylation (DNAm) changes at specific sites in the genome. Six CpG sites have been identified which reveal either linear hypermethylation or hypomethylation with respect to the number of cumulative population doublings (cPDs). Conversely, the DNAm level at these CpG sites can be analyzed-for example, by pyrosequencing of bisulfite-converted DNA-and then used for linear regression models to predict cPDs. Our method provides an epigenetic biomarker to determine the state of senescence in cell preparations.

Pluripotent stem cells escape from senescence-associated DNA methylation changes.

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.

Induced pluripotent mesenchymal stromal cell clones retain donor-derived differences in DNA methylation profiles.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.

Monitoring of cellular senescence by DNA-methylation at specific CpG sites.

Replicative senescence has fundamental implications on cell morphology, proliferation, and differentiation potential. Here, we describe a simple method to track long-term culture based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic senescence signature can be used as biomarker for various cell types to predict the state of cellular senescence with regard to the number of passages, population doublings, or days of in vitro culture.

Epigenetic-aging-signature to determine age in different tissues.

All tissues of the organism are affected by aging. This process is associated with epigenetic modifications such as methylation changes at specific cytosine residues in the DNA (CpG sites). Here, we have identified an Epigenetic-Aging-Signature which is applicable for many tissues to predict donor age. DNA-methylation profiles of various cell types were retrieved from public data depositories - all using the HumanMethylation27 BeadChip platform which represents 27,578 CpG sites. Five datasets from dermis, epidermis, cervical smear, T-cells and monocytes were used for Pavlidis Template Matching to identify 19 CpG sites that are continuously hypermethylated upon aging (R>0.6; p-value<10-13). Four of these CpG sites (associated with the genes NPTX2, TRIM58, GRIA2 and KCNQ1DN) and an additional hypomethylated CpG site (BIRC4BP) were implemented in a model to predict donor age. This Epigenetic-Aging-Signature was tested on a validation group of eight independent datasets corresponding to several cell types from different tissues. Overall, the five CpG sites revealed age-associated DNA-methylation changes in all tissues. The average absolute difference between predicted and real chronological age was about 11 years. This method can be used to predict donor age in various cell preparations - for example in forensic analysis.

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks.

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colony-forming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled.

Serum after autologous transplantation stimulates proliferation and expansion of human hematopoietic progenitor cells.

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34(+) cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34(+), CD133(+), CD45(-)) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.

Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

Specific age-associated DNA methylation changes in human dermal fibroblasts.

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner.

Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.

BACKGROUND AIMS: Culture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Several studies have demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative for FCS. However, little is known about the variation between different platelet lysates. METHODS: We compared activities of individual HPL on initial fibroblastoid colony-forming units (CFU-F), proliferation, in vitro differentiation and long-term culture. These data were correlated with chemokine profiles of HPL. RESULTS: Isolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype and adipogenic differentiation potential. Osteogenic differentiation was even more pronounced in HPL than FCS. There were significant differences in MSC proliferation with different HPL, but it was always higher in comparison with FCS. Cell growth correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPL facilitated expansion for more than 20 population doublings. CONCLUSIONS: Taken together, reliable long-term expansion was possible with all HPL, although there was some variation in platelet lysates of individual units. Therefore the use of donor recipient-matched or autologous HPL is feasible for therapeutic MSC products.

How to track cellular aging of mesenchymal stromal cells?

Mesenchymal stromal cells (MSC) are currently tested in a large number of clinical trials and raise high hope in regenerative medicine. These cells have to be expanded in vitro before transplantation and several studies demonstrated that long-term culture evokes continuous changes in MSC: proliferation rate decays, the cell size increases, differentiation potential is affected, chromosomal instabilities may arise and molecular changes are acquired. Long-term culture of cell preparations might also have therapeutic consequences, although this has hardly been addressed in ongoing trials so far. Reliable therapeutic regimens necessitate quality control of cellular products. This research perspective summarizes available methods to track cellular aging of MSC. We have demonstrated that gene expression changes and epigenetic modifications are continuously acquired during replicative senescence. Molecular analysis of a suitable panel of genes might provide a robust tool to assess efficiency and safety of long-term expansion.

Landmarks facilitate visual space constancy across saccades and during fixation.

It has been demonstrated that visual objects that are present after saccadic eye movements act as landmarks for the localization of stimuli across saccades, facilitating space constancy (Deubel, 2004). We here study the temporal conditions under which landmark effects occur after saccadic eye movements, and during fixation. Two small objects were presented 6 degrees in the periphery, one above the other. Observers saccaded to the space between them. One of the objects disappeared during the saccade and reappeared with a variable delay during or after the saccade. At the same time either that object or the continuously present one jumped by 1 degrees . The observer's task was to decide which object had moved. The results revealed a strong bias to assign movement to the object that was blanked, regardless of which actually moved. If both objects were blanked, the one that was blanked for a shorter time tended to be seen as stable. The effects were stronger as the onset asynchrony between the stimuli increased. Surprisingly, analogous though weaker effects occurred during visual fixation, suggesting that similar visual mechanisms relying on visual landmarks operate both across saccades and during fixation.

Windei, the Drosophila homolog of mAM/MCAF1, is an essential cofactor of the H3K9 methyl transferase dSETDB1/Eggless in germ line development.

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells.