Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells
TL;DR: It is concluded that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency, which provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.
Abstract: Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.
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TL;DR: Deep insight on Three-dimensional organization of the mammalian nucleus in normal and tumor cells is revealed.
Abstract: Deep insight on Three-dimensional organization of the mammalian nucleus in normal and tumor cells.
01 Jan 2013
TL;DR: Dynamics of Nups were analyzed at different stages of muscle differentiation by live cell imaging and revealed previously unidentified roles of Nup98 and Nup50 in differentiation, which might shed light on the cell-type specific diseases related to Nup mutations.
Abstract: Author(s): Liang, Yun | Abstract: Nuclear pore proteins (Nups) form the only channels on the nuclear envelope that allow macromolecule transport between the cytoplasm and the nucleoplasm. Initial studies in the unicellular organism yeast have confirmed that deletions and/or mutations of multiple Nups result in deficiency in bulk transport between cellular compartments and subsequently lethality. Recent investigations regarding multicellular organisms, however, have revealed that mutations in Nups are often linked to cell-type specific defects and diseases. Therefore, there might be cell-type specific functions of Nups, for example in the context of differentiation. One potential cell-type specific function of Nups could be related to the regulation of genes that are activated in a cell-type dependent manner. Such hypothesis stems from the observation that microscopically, Nups appear to selectively interact with active genomic regions. To test this hypothesis, chromatin immunoprecipitation experiments for Nup98 have been performed in multiple cell types, including human cells at different stages of neural differentiation. It has been found that the patterns of Nup98-genome interaction are highly cell-type specific in the neural differentiation system. Further functional studies have demonstrated a role of Nup98 in regulating the expression of a cohort of genes active during neural development. As an alternative approach to investigate into the potential cell-type dependent behavior of Nups, dynamics of Nups were analyzed at different stages of muscle differentiation by live cell imaging. One Nup, Nup50, was found to exhibit decreased dynamics upon differentiation, serving as the first example of a Nup that has altered dynamics during development. Kifc1, a kinesin-superfamily protein that is predicted to be a nuclear motor, was found to confer the higher dynamics of Nup50 by physical interaction before differentiation. Upon differentiation, Kifc1 is degraded, which could explain the decrease in Nup50 mobility. Knocking down Nup50 decreased efficiency of muscle differentiation, whereas knocking down Kifc1 accelerated muscle differentiation. This suggests that Nup50 is differentially regulated during muscle development and is functionally important for differentiation. Together these studies revealed previously unidentified roles of Nup98 and Nup50 in differentiation, which might shed light on the cell-type specific diseases related to Nup mutations
Cites background from "Distinctive nuclear organisation of..."
...Moreover, on the single-gene level, repositioning of developmental genes and tissue-specific promoters relative to the nuclear periphery during differentiation has been well documented [80-86]....
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Dissertation•
01 Jan 2018
TL;DR: In this paper, a review discusses how important it is to efficiently and sensitively detect human pluripotent stem cells (hPSC) aneuploidies, to understand how these anoeploidy arise, and consider the consequences for the cell, and indeed the individual to whom aneuPLoid cells may be administered.
Abstract: Human pluripotent stem cells (hPSCs) are increasingly used for cell-based regenerative therapies worldwide, with embryonic and induced pluripotent stem cells as potential treatments for debilitating and chronic conditions, such as age-related macular degeneration, Parkinson's disease, spinal cord injuries, and type 1 diabetes. However, with the level of genomic anomalies stem cells generate in culture, their safety may be in question. Specifically, hPSCs frequently acquire chromosomal abnormalities, often with gains or losses of whole chromosomes. This review discusses how important it is to efficiently and sensitively detect hPSC aneuploidies, to understand how these aneuploidies arise, consider the consequences for the cell, and indeed the individual to whom aneuploid cells may be administered.
01 Jan 2008
TL;DR: Combination of controlled differentiation with ground-breaking technologies for the reversal of somatic cells to an ES cell-like state promise the generation of patient-derived pluripotent cell lines for the treatment of disease in the future.
Abstract: Embryonic stem (ES) cells have the capacity to proliferate indefinitely in culture while maintaining the ability to differentiate to form any of the cells of the body. This unique combination of functions suggests that these cells could provide a potentially unlimited source of differentiated cells for the treatment of disease and aging. Understanding the molecular processes that underpin these functions in ES cells will allow us to harness their potential and develop strategies that control their differentiation. Combination of controlled differentiation with ground-breaking technologies for the reversal of somatic cells to an ES cell-like state promise the generation of patient-derived pluripotent cell lines for the treatment of disease in the future.
Cites background from "Distinctive nuclear organisation of..."
...For example, NANOG is localized significantly more centrally in the nuclei of human ES cells compared with a more peripheral location in B cells, and OCT4 loops out from the chromosome territory in human ES cells (Wiblin et al., 2005)....
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TL;DR: In this paper, the authors assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts and used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups.
Abstract: Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.
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TL;DR: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages.
Abstract: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.
15,555 citations
"Distinctive nuclear organisation of..." refers background or methods in this paper
...Human ES cells have been derived from the inner cell mass of blastocysts, and as well as being able to self-renew, they have the ability to differentiate into all three embryonic germ layers when injected into severe combined immunodeficient mice (Thomson et al., 1998)....
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...Human ES cell culture and analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....
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...Germ cells and stem cells in contrast have active telomerase, and robust telomerase activity is detected in hES cells (Thomson et al., 1998)....
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...Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al....
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TL;DR: A successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings and are suitable for scaleup production is demonstrated.
Abstract: Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.
2,092 citations
"Distinctive nuclear organisation of..." refers methods in this paper
...Human ES cell culture and analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....
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..., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....
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TL;DR: The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells and provide a foundation for a more detailed understanding of stem cell biology.
Abstract: The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown. Embryonic and neural stem cells have many similarities at the transcriptional level. These results provide a foundation for a more detailed understanding of stem cell biology.
1,776 citations
"Distinctive nuclear organisation of..." refers background in this paper
..., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1)....
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...In contrast, RCN, which is expressed in both LCLs (Mahy et al., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1)....
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TL;DR: It is suggested that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
Abstract: We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
1,046 citations
"Distinctive nuclear organisation of..." refers background in this paper
...It is interesting to note that recurrent gains of chromosome 12, including iso12p, have been found in human ES cells (Draper et al., 2004)....
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TL;DR: It is demonstrated that the distribution of genomic sequences between chromosomes has implications for nuclear structure and the findings are discussed in relation to a model of the human nucleus that is functionally compartmentalized.
Abstract: Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.
914 citations
"Distinctive nuclear organisation of..." refers background or methods in this paper
...HSA18 is found towards the nuclear periphery in a variety of differentiated cells and HSA19 is in the centre of the nucleus (Croft et al., 1999; Cremer et al., 2003)....
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...Hybridisation was as described previously (Croft et al., 1999) but with the denaturing time reduced to 1....
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...Chromosome paints were labelled with biotin-16-dUTP by nick translation or by PCR amplification (Croft et al., 1999) or obtained commercially (Cambio)....
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...Slides were then subjected to freeze-thaw in 20% glycerol/PBS and FISH was carried out as described previously (Croft et al., 1999)....
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...The radial distribution of CTs was determined in 2D specimens by an erosion script, as previously described (Croft et al., 1999)....
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