scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

01 Sep 2005-Journal of Cell Science (The Company of Biologists Ltd)-Vol. 118, Iss: 17, pp 3861-3868
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.

Content maybe subject to copyright    Report

Citations
More filters
Journal ArticleDOI
TL;DR: The results suggest that oxygen concentrations affect various aspects of HPDLSC physiology and that hypoxia enhances osteogenic differentiation both in vivo and in vitro.
Abstract: Changes in oxygen concentration may influence various innate characteristics of stem cells. The effects of varying oxygen concentration on human periodontal ligament stem cells (HPDLSCs) has not been explored, particularly under hypoxia-related conditions. First, HPDLSCs were cultured from the periodontium of human teeth using the outgrowth method. STRO-1 and CD146 expression of HPDLSCs was investigated by flow cytometry. To detect the multilineage differentiation capacities of HPDLSCs, osteogenic-like and adipogenic-like states were induced in cells. Next, HPDLSCs (passage 3) were exposed to normal oxygen (21% O2) or hypoxia (2% O2) conditions for 7 days and cell proliferation was evaluated. After culture in osteogenic medium for 7 days, osteoblastic differentiation was evaluated by semi-quantitative reverse transcription-polymerase chain reaction analysis to detect 3 osteoblastic markers: core-binding factor a 1/runt-related transcription factor 2, osteocalcin, and osteopontin. In addition, each cell group was incubated with a hydroxyapatite/tricalcium phosphate carrier and transplanted subcutaneously into the back of immunocompromised mice to investigate transplantation differences in vivo. HPDLSCs were isolated, cultured, and successfully identified. After exposure of HPDLSCs to hypoxia for 7 days, the proliferation rate was increased and showed higher osteogenic differentiation potential compared to control cells. After 12 weeks of transplantation, hypoxia-treated HPDLSCs differentiated into osteoblast-like cells that formed bone-like structures. These results suggest that oxygen concentrations affect various aspects of HPDLSC physiology and that hypoxia enhances osteogenic differentiation both in vivo and in vitro. Oxygen concentration may be a critical parameter for HPDLSCs during expansion and differentiation.

25 citations


Cites background from "Distinctive nuclear organisation of..."

  • ...Some studies reported that hypoxia provides favorable culture conditions for promoting proliferation as well as osteogenesis of mesenchymal stem cells through differential growth factor production (Wiblin et al., 2005)....

    [...]

Journal ArticleDOI
TL;DR: A computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization is described, suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts.
Abstract: Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets.

24 citations

Journal ArticleDOI
TL;DR: A change of perspective is a necessary step for developing successful ‘tumour-reversion’ therapeutic strategies and the relationship between the chromatin network organization and gene expression regulation plays a fundamental role.
Abstract: The apparent lack of success in curing cancer that was evidenced in the last four decades of molecular medicine indicates the need for a global re-thinking both its nature and the biological approaches that we are taking in its solution. The reductionist, one gene/one protein method that has served us well until now, and that still dominates in biomedicine, requires complementation with a more systemic/holistic approach, to address the huge problem of cross-talk between more than 20,000 protein-coding genes, about 100,000 protein types, and the multiple layers of biological organization. In this perspective, the relationship between the chromatin network organization and gene expression regulation plays a fundamental role. The elucidation of such a relationship requires a non-linear thermodynamics approach to these biological systems. This change of perspective is a necessary step for developing successful ‘tumour-reversion’ therapeutic strategies.

24 citations

Journal ArticleDOI
TL;DR: Current knowledge about this unique chromosomal region and its relevance for proper cell division, speciation, and disease are reviewed.
Abstract: As the spindle fiber attachment region of the chromosome, the centromere has been investigated in a variety of contexts. Here, we will review current knowledge about this unique chromosomal region and its relevance for proper cell division, speciation, and disease. Understanding the three-dimensional organization of centromeres in normal and tumor cells is just beginning to emerge. Multidisciplinary research will allow for new insights into its normal and aberrant nuclear organization and may allow for new therapeutic interventions that target events linked to centromere function and cell division.

24 citations


Cites background or methods from "Distinctive nuclear organisation of..."

  • ...Such methods have been successfully used [Wiblin et al., 2005]; however, their validity relies on consistency between the inherent assumptions about nuclear geometry and the physical geometry of the samples....

    [...]

  • ...Similarly, human embryonic stem cells have a smaller portion of their centromeres located at the nuclear periphery compared with differentiated cells [Wiblin et al., 2005]....

    [...]

Journal ArticleDOI
TL;DR: An overview of recent findings on the 3D genome organization in PSCs is provided and its putative functional role in regulation of the pluripotent state is discussed.

22 citations


Cites background from "Distinctive nuclear organisation of..."

  • ...by topological re-organization at all structural scales [52,69]....

    [...]

  • ...Microscopy observations support this idea: chromocenters, the visually discernable inactive nuclear compartments that are composed of clustered pericentromeric regions, are more dispersed and randomly positioned in PSCs than in differentiated cells [52,124,125]....

    [...]

  • ...nucleus, while larger and gene-poor chromosomes inhabit more peripheral positions [52,53]....

    [...]

References
More filters
Journal ArticleDOI
06 Nov 1998-Science
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)....

    [...]

  • ...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)....

    [...]

  • ...Germ cells and stem cells in contrast have active telomerase, and robust telomerase activity is detected in hES cells (Thomson et al., 1998)....

    [...]

  • ...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....

    [...]

Journal ArticleDOI
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)....

    [...]

  • ..., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....

    [...]

Journal ArticleDOI
18 Oct 2002-Science
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)....

    [...]

  • ...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)....

    [...]

Journal ArticleDOI
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)....

    [...]

Journal ArticleDOI
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)....

    [...]

  • ...Hybridisation was as described previously (Croft et al., 1999) but with the denaturing time reduced to 1....

    [...]

  • ...Chromosome paints were labelled with biotin-16-dUTP by nick translation or by PCR amplification (Croft et al., 1999) or obtained commercially (Cambio)....

    [...]

  • ...Slides were then subjected to freeze-thaw in 20% glycerol/PBS and FISH was carried out as described previously (Croft et al., 1999)....

    [...]

  • ...The radial distribution of CTs was determined in 2D specimens by an erosion script, as previously described (Croft et al., 1999)....

    [...]

Related Papers (5)